Large scale protein purification of Wt1 ZF(-/-), Wt1 ZF(-/+), and Ciao-1

by

Ami Michele Bitschy

B.Sc., University of Victoria, 2004

A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of

MASTER OF SCIENCE

in the Department of Biochemistry and Microbiology

© Ami Michele Bitschy, 2008 University of Victoria

All rights reserved. This thesis may not be reproduced in whole or in part, by photocopy

or other means, without the permission of the author.

ii

Large scale protein purification of Wt1 ZF(-/-), Wt1 ZF (-/+), and Ciao-1

by

Ami Michele Bitschy B.Sc., University of Victoria, 2004

Supervisory Committee

Dr. Paul Romaniuk, (Department of Biochemistry & Microbiology)_________________ Co-Supervisor Dr. Alistair Boraston, (Department of Biochemistry & Microbiology)________________ Co-Supervisor Dr. Steven Evans, (Department of Biochemistry & Microbiology)___________________ Department Member Dr. Réal Roy, (Department of Biology)________________________________________ Outside Member

iii

Supervisory Committee Dr. Paul Romaniuk, (Department of Biochemistry & Microbiology)_________________ Co-Supervisor Dr. Alistair Boraston, (Department of Biochemistry & Microbiology)________________ Co-Supervisor Dr. Steven Evans, (Department of Biochemistry & Microbiology)___________________ Department Member Dr. Réal Roy, (Department of Biology)________________________________________ Outside Member

ABSTRACT

WT1 has two main isoforms: WT1(-KTS) and WT1(+KTS). Both are known to bind to a DNA consensus sequence with different affinities, and are thus postulated to play overlapping but distinct functional roles in the cell. WT1 is also known to bind to certain RNA moieties as well as to various protein partners (e.g. Ciao-1). This study focuses on the development of large scale protein purification protocols for WT1 zinc finger (ZF) proteins as well as Ciao-1. By using a combination of his-tag affinity and size exclusion chromatography we were able to purify milligram quantities of these proteins. It was also the intention to obtain crystals of the WT1 ZF protein in complex with any one of its known binding partners, in particular the protein Ciao-1 (a WD40 protein) and the 14 mer consensus sequence of DNA (known as WTE). In conjunction with structural studies it was determined that a previously made SELEX RNA library was not selective for the (+KTS) isoform of WT1 ZF, and therefore no RNA candidate could be identified for future structural studies.

iv

Table of Contents

Supervisory Committee ...................................................................................................... ii

Abstract .............................................................................................................................. iii

Table of Contents............................................................................................................... iv

List of Tables ................................................................................................................... viii

List of Figures .................................................................................................................... ix

List of Abbreviations ......................................................................................................... xi

Acknowledgments............................................................................................................. xv

Chapter 1. Introduction ...................................................................................................... 1

1.1. Zinc Finger Proteins.................................................................................................... 2

1.1.1. Cys2His2 Zinc Fingers.................................................................................. 3

1.2. Wilms’ Tumor Suppressor 1........................................................................................ 5

1.2.1. Wilms’ Tumor. Brief history ....................................................................... 5

1.2.2. wt1 The gene ................................................................................................ 5

1.2.3. Cellular Localization.................................................................................... 6

1.2.4. Syndromes associated with WT1................................................................. 8

1.2.5. DNA Binding ............................................................................................. 10

1.2.5.1. Structure of WT1 in complex with DNA.......................................... 14

1.2.5.2. Molecular Basis of WT1-associated disease..................................... 19

1.2.6. WT1 and RNA Binding .............................................................................. 20

1.2.7. WT1 and Protein Binding ........................................................................... 23

1.2.8. WT1 Function ............................................................................................. 26

1.2.8.1. Transcriptional Regulation................................................................. 26

v

1.2.8.2. WT1 and Kidney Development ......................................................... 28

1.2.8.3. Post-transcriptional gene expression.................................................. 30

1.2.9. WT1 and Cancer ......................................................................................... 31

1.3. Ciao-1 ........................................................................................................................ 34

1.3.1. The Protein.................................................................................................. 34

1.3.2. WT1-Ciao-1 Interaction.............................................................................. 34

1.3.2. Function of Ciao-1 ...................................................................................... 35

1.4. WD40 ........................................................................................................................ 36

1.4.1. History and Function.................................................................................. 36

1.4.2. Structure..................................................................................................... 38

1.4.3. Human Disease ................................................................................... 39

1.4.3. Human Disease .......................................................................................... 40

1.5. Thesis Objectives ...................................................................................................... 40

Chapter 2. Materials and Methods ................................................................................... 42

2.1. WT1 plasmid constructs ............................................................................................ 42

2.2. Ciao-1 plasmid constructs.......................................................................................... 42

2.3. Clone Verification...................................................................................................... 42

2.3.1. DH5α Plasmid preparation.......................................................................... 42

2.3.2. Sequencing for Clone Verification ............................................................. 43

2.4. Protein Expression and Purification........................................................................... 43

2.4.1. WT1 ZF(-KTS) and WT1 ZF(+KTS)......................................................... 43

2.4.2. GST-Ciao-1 from pGEX-4T3 ..................................................................... 46

2.4.3. Ciao-1 from pET30a ................................................................................... 47

vi

2.5. Crystallization ............................................................................................................ 49

2.6. WTE and WT1 ZF(-KTS).......................................................................................... 50

2.6.1. DNA preparation......................................................................................... 50

2.6.2. Complexing WT1 ZF(-KTS) with WTE..................................................... 51

2.6.3. Native Gels to Verify WT1 ZF(-KTS) complex with DNA ....................... 51

2.7. Gel Shift Assays......................................................................................................... 51

2.7.1. WT8 Preparation......................................................................................... 51

2.7.2. Gel Shift with M13F Probe......................................................................... 52

2.7.3. Western of Gel Shift ................................................................................... 53

2.8. Filter Binding Assays................................................................................................. 56

2.8.1. Preparation of RNA Selection library......................................................... 56

2.8.2. Preparation of PCR pools from RNA Selection libraries ........................... 56

2.8.3. RNA Preparation. In vitro RNA transcription and radiolabeling by run-off transcription .......................................................................................................... 56

2.8.4. Nitrocellulose filter binding assay .............................................................. 58

Chapter 3. Results ............................................................................................................ 59

3.1. Protein Purification .................................................................................................... 59

3.1.1. WT1 Zinc Finger Domain Peptides ............................................................ 59

3.1.1.1. Refolding of WT1 Zinc Finger Domain Peptides.............................. 63

3.2. Forming a WTE-WT1 Zinc Finger Domain Peptide Complex ................................. 66

3.3. Crystallization of WTE - WT1 ZF Complex ............................................................. 66

3.4. Protein Purification of Ciao-1.................................................................................... 68

3.4.1. Purification of GST-Ciao-1........................................................................ 68

3.4.2. Purification of pET30a Histag-Ciao-1 ....................................................... 68

vii

3.5. Crystallization of Ciao-1........................................................................................... 70

3.6. Co-Crystallization of Ciao-1 and WT1 ZF(-KTS) or (+KTS).................................. 73

3.7. Filter Binding Assay ................................................................................................. 73

3.7.1. Quantitative filter binding of WT1 ZF (-KTS) and (+KTS) with the RNA aptamer Pel22 and 5S rRNA................................................................................. 75

3.7.2. Quantitative filter binding of WT1 ZF (-KTS) and (+KTS) with the SELEX pooled PCR libraries............................................................................... 77

3.7.3. Quantitative filter binding of WT1 ZF (-KTS) and (+KTS) with select aptamers from the 20th round of selection ............................................................ 78

Chapter 4. Discussion ...................................................................................................... 87

4.1. WT1 ........................................................................................................................ 87

4.1.1. Purification of WT1 ZF isoforms .............................................................. 87

4.1.2. Crystallization of WT1 .............................................................................. 87

4.1.2.1. Co-Crystallization of WT1 ZF with Ciao-1...................................... 88

4.1.2.2. Co-Crystallization of WT1 ZF with WTE........................................ 88

4.1.3. Filter Binding Assays................................................................................. 89

4.1.4. Supershift Assay ........................................................................................ 90

4.2. Ciao-1........................................................................................................................ 91

4.2.1. Protein Purification .................................................................................... 91

4.2.2. Crystallization of Ciao-1............................................................................ 91

5.0. Conclusion ............................................................................................................... 93

Literature Cited ................................................................................................................. 94

Appendix………..………………………………………………………………………105

viii

List of Tables

Table 1. Protein binding partners of WT1…………………………………………….. .24

Table 2. WT1 target genes………………………………………………………………27

Table 3. WT1 and human cancers………………………………………………………33

Table 4. WD40 protein functions.………………………………………………………39

Table 5. Primers used in this study .................................................................................. 54

Table 6. WT1 ZF isomer comparison of RNA aptamer binding affinities……………...80

ix

List of Figures

Figure 1. The C2H2 Zinc Finger Structure. ....................................................................... 4

Figure 2. Schematic diagram of the wt1 gene to protein. ................................................. 7

Figure 3. Sequence alignment of WT1 ZF with Sp1 and EGR-1...…………………….11

Figure 4. Crystal structure of WT1 ZF(-/-) in complex with 14bp EGR-1 DNA……....15

Figure 5. Schematic diagram of WT1 ZF (-/-) with DNA……...………………………17

Figure 6. Ribbon diagram of crystal structure of TFIIIA bound to DNA and RNA…...22

Figure 7. wt1 expression in the developing kidney……………………………………..29

Figure 8. WD repeat…………………………………………………………………….37

Figure 9. LICOR 700DX IRDye…...…………………………………………………...55

Figure 10. Expression trial of WT1 ZF (-KTS)…....………..…………………………..60

Figure 11. WT1 ZF (-KTS) purification on Ni-NTA resin……...……………………….61

Figure 12. WT1 ZF (+KTS) purification on Ni-NTA resin……………………...……....62

Figure 13. Gel filtration purification of WT1 ZF (-KTS)……………………………......64

Figure 14. Gel filtration purification of WT1 ZF (-KTS)……...………………………...65

Figure 15. Migration of WT1 ZF (-KTS) refolded with and without WTE DNA……….67

Figure 16. Ni-NTA column purification of Ciao-1 with c-terminal his tag…...…………69

Figure 17. Gel filtration purification of Ciao-1 with c-terminal his tag……………...….71

Figure 18. Ciao-1 histag cleavage…………………………………………………….….72

Figure 19. Filter binding assay controls…………………………………………….……76

Figure 20. Binding assay comparison of the SELEX libraries 15, 17, 19, and 20…... …79

Figure 21. Binding assay for RNA aptamer 3 from the 20th SELEX library………….…81

Figure 22. Binding assay for RNA aptamer 26 from the 20th SELEX library…………...82

x

Figure 23. Binding assay for RNA aptamer 29 from the 20th SELEX library…………...83

Figure 24. Binding assay for RNA aptamer 48 from the 20th SELEX library…………...84

Figure 25. Binding assay for RNA aptamer 58 from the 20th SELEX library…...……....85

Figure 26. Binding assay for RNA aptamer 61 from the 20th SELEX library…………...86

xi

List of Abbreviations

3-D: three dimensional

Ab: antibody

AUC: analytical ultracentrifugation

bp: base pair

BLAST: basic local alignment search tool

BSA: bovine serum albumin

BWS: Beckwith-Wiedemann Syndrome

C2H2: two cysteine two histidine

CIA: cytosolic iron-sulfur (Fe/S) protein assembly machinery

cDNA: complementary deoxynucleic acid

cpm: counts per minute

CS: Cockayne Syndrome

CV: column volume

DDS: Denys-Drash Syndrome

dNTP’s: deoxynucleotide triphosphates:

dATP, deoxyadenosine triphosphate

dCTP, deoxycytidine triphosphate

dGTP, deoxyguanine triphosphate

dTTP, deoxythymidine triphosphate

DNA: deoxyribonucleic acid

DNAse: recombinant human deoxyribonuclease

DSC: differential scanning calorimetry

xii

DTT: dithiothreitol

E.coli: Escherichia coli

EDTA: ethylenediamine-tetraacetic acid

FS: Frasier Syndrome

FSGS: focal and segmental glomerular sclerosis

GST: glutathione S-transferase

HRP: horseradish peroxidase

IH: immunohistochemistry

IP: co-immunoprecipitation

IPTG: isopropyl-β-D-thiogalactopyranosidase

ITC: isothermal titration calorimetry

kb: kilobases

KCl: potassium chloride

KTS: lysine, threonine, serine

LB: Luria-Benton broth

mL: millilitre

mM: millimolar

mRNA: messenger ribonucleic acid

MW: molecular weight

NaOH: sodium hydroxide

ND: not determined

nM: nanomolar

NMR: nuclear magnetic resonance

xiii

Northern: northern blotting

nt: nucleotide

Nuc: nuclear staining

Nucleotide triphosphates:

ATP, adenosine triphosphate

CTP, cytidine triphosphate

GTP, guanine triphosphate

UTP, uridine triphosphate

PAGE: polyacrylamide gel electrophoresis

pI: isoelectric point

pmoles: picomoles

PMSF: phenylmethylsulfonyl fluoride

Q-RT-PCR: quantitative real-time polymerase chain reaction

RNA: ribonucleic acid

rRNA: ribosomal ribonucleic acid

RNP: ribonucleoprotein

RT-PCR: real-time polymerase chain reaction

SACS: small angle X-ray scattering

SDM: site-directed mutagenesis

SDS: sodium dodecyl sulphate

SELEX: systematic evolution of ligands by exponential enrichment

Seq: sequencing

siRNA: small interfering RNA

xiv

TBE: Tris, borate, EDTA

TFIIIA: Transcription Factor III A

Tris-HCl: tris-(hydroxymethyl)-aminomethane hydrochloride

Western: western blotting

WT1: Wilms’ tumor suppressor protein

WTAP: WT1 associating protein

WTE: WT1 responsive element

ZnCl2: zinc chloride

ZF: zinc finger

xv

Acknowledgments

I would like to acknowledge the support of my husband and family as well as the ever-present ear of my fellow graduate students. I would like to thank my two supervisors; Dr. Paul Romaniuk and Dr. Alisdair Boraston, who were gracious to offer their financial and intellectual support. Last, but not least, I would like to thank my son, Colwyn, who may have slowed me down, but gave me the courage to finish.

1

Chapter 1. Introduction

Ever since the first protein structure of myoglobin was solved in 1958 (Kendrew

et al.,1958) huge strides have been made in the area of protein crystallography. While

crystallography was first used as a structure determinant, it now plays a large role in

understanding the intricacies of protein-protein interactions. Other

biochemical/biophysical methods such as Isothermal Titration Calorimetry (ITC), site

directed mutagenesis (SDM), differential scanning calorimetry (DSC), and analytical

ultracentrifugation (AUC), have, and are still, being used to predict or verify tertiary and

quaternary structure. However, none display, with the same detail or accuracy, the

organization of a protein with or without its binding partner (Dauter et al.,1997). Protein

crystallization has the advantage of offering a comprehensive 3-D visual model of the

exact orientation and position of each atom. This advantage, however, does come with

some drawbacks and challenges. There is no exact science or protocol for the

crystallization of a protein; rather, this is a hit or miss process. There are many

commercially available crystallization kits, but protein crystals are never guaranteed. For

crystallization to occur there must be a large proportion of order in the protein structure.

Therefore proteins with numerous modules or large mobile sections are less likely to

crystallize. In addition, large quantities of very pure, soluble protein are required.

In the study of protein-DNA complexes it has been quite difficult to produce good

crystals for diffraction (Pavletich and Pabo,1991; Fairall et al.,1993; Pavletich and

Pabo,1993; Elrod-Erickson et al.,1996; Omichinski et al.,1997; Nolte et al.,1998; Segal

et al.,2006). Due to this difficulty, which is thought to be a result of the inherent mobility

of nucleic acids, other biophysical methods are more widely used to obtain structural or

2

functional data on protein-DNA complexes. One of the preferred functional methods is

filter binding assays where one can take advantage of the inability of RNA/DNA to bind

nitrocellulose except when already bound to a protein partner. Filter binding, when

paired with mutagenesis, can allow for the identification of key residues, but does not

yield structural information.

In this study, the crystallization of WT1 with its DNA partner, as well as with

Ciao-1 (one of its many protein binding partners) was attempted. WT1 has the unique

ability to bind to all three components of the central dogma, DNA, RNA, and protein. As

of yet WT1 has only been solved in complex with DNA. The interaction of WT1 ZF

isomers with various RNA aptamers from a selection library was also investigated.

1.1. Zinc Finger Proteins

There are a number of different families of zinc finger proteins which contain

multiple cysteine and/or histidine residues to stabilize their fold. First identified as the

DNA binding portion of transcription factors (Miller et al.,1985), they appear in tandem

repeats of no more than 16 and no less than 2. The average transcription factor contains 3

to 4 repeating zinc finger domains. In general it is said that the more zinc finger domains

the higher the affinity the protein has for its DNA binding partner. The most common

and well known zinc finger proteins are the Cys2His2 zinc fingers; in fact these were the

first of this superfamily to be identified (Miller et al.,1985).

3

1.1.1. Cys2His2 Zinc Fingers

The C2H2 zinc fingers were first noted as the repeating unit of the eukaryotic

transcription factor TFIIIA in 1986 (Miller et al.,1985; Schuh et al.,1986). Proteins

containing the C2H2 zinc fingers are the most common transcription factors in eukaryotes

and they make up almost 3% (Lander et al.,2001; Venter et al.,2001) of the human

genome. Each finger binds a single zinc ion that is sandwiched between the two-stranded

antiparallel β-sheets and an α-helix (Figure 1).

The zinc finger domain is made up of ~30 amino acids, the consensus sequence is

as follows: (F/Y)-X-C-X2-5-C-X3-(F/Y)-X5-ψ-X2-H-X3-5-H, where F is phenylalanine, ψ

is a hydrophobic residue, and X is any residue (Bouhouche et al.,2000; Wolfe et

al.,2000). The linker between the tandem repeats is also a conserved sequence and

consists of the following five amino acids: TGEKP (Laity et al.,2001). The size of this

linker is believed to optimize the distance between fingers so that they are properly

stacked around the turning DNA double helix, so that the α-helix is within the major

groove.

All pertinent DNA-protein interactions occur in the α-helix of the zinc finger fold.

In particular, the most important residues are -1, 2, 3, and 6, as these have been shown in

crystallographic as well as in filter binding studies (Pavletich and Pabo,1991; Wolfe et

al.,2000) to provide the DNA binding specificity to the zinc finger.

A number of C2H2 proteins have also been identified which bind to RNA as well,

although their biological significances have yet to be elucidated. One such protein, WT1,

was initially characterized as a DNA binding protein, but now appears also to function at

the RNA level of gene regulation.

4

Figure 1. The C2H2 zinc finger structure. Ribbon diagram of the ββα motif representative of a common C2H2 zinc finger (ZF 2 of Zif268). Shown are the two cysteines (rose) and two histidines (green) residues of the zinc finger involved in coordination of the zinc (blue) atom. Reprinted, with permission, from the Annual Review of Biophysics and Biomolecular Structure, Volume 3 (c) 2000 by Annual Reviews www.annualreviews.org (Wolfe et al.,2000).

5

1.2. Wilms’ Tumor Suppressor 1

1.2.1. Wilms’ Tumor. Brief history

A nephroblastoma or kidney tumor that was first described by Max Wilms in

1899, Wilms’ tumor is one of the most common childhood malignancies affecting 1 in

every 10,000 children. Most commonly, these children are under the age of 5. New

developments in surgical techniques in the 20th century improved the prognosis for this

previously lethal malignancy. Surgical intervention combined with radiotherapy and

chemotherapy has dramatically improved survival rates. Most patients presenting with

such a nephroblastoma can be successfully treated with chemotherapy and nephrectomy.

Today the overall survival rate of Wilms’ tumor patients is 90%, a great success story for

cancer and modern medicine. The Wilms’ tumor suppressor 1 gene (wt1) is the only

gene involved in the development of Wilms’ tumor that has been cloned and

subsequently classified as a tumor suppressor gene. The wt1 gene is mutated in 5-10% of

all Wilms’ tumors (Metzger and Dome,2005).

1.2.2. wt1 The gene

In 1990 Call and colleagues described the utilization of positional cloning studies

to identify the chromosomal location of the wt1 gene. The results portrayed a gene of 50

kilobases at 11p13. Consequently, it was determined that this 50kb region contained ten

exons of varying length (Call et al.,1990), the first of which encodes a proline glutamine

rich regulatory polypeptide, while exons seven to ten each encode an individual zinc

finger of the C2H2 variety (Figure 2). Exon 5 contains an alternative splice site that

controls the inclusion or exclusion of a 17 amino acid stretch within the regulatory

6

region. This splicing event is only conserved in mammals. Exon 9 contains a second

alternative splice site, the use of which results in the inclusion or exclusion of three

amino acids (lysine, threonine, serine) in between zinc fingers three and four. Thus,

alternative splicing yields 4 main isoforms of WT1.

These four main isoforms are referred to in various ways but in this study we will

refer to them as follows: WT1 (-KTS), excluding both the KTS and the 17mer fragment;

WT1 (+KTS), excluding only the 17mer fragment; WT1(+/-), excluding only the KTS

fragment; and WT1 (+/+), containing both the KTS and the 17mer fragments.

Five years after the two alternative splice sites were discovered, Pelletier and

Bruening determined the location of another translation start site 219 base pairs (bp)

upstream. This CTG codon gave rise to four more WT1 isoforms (Bruening and

Pelletier,1996). Subsequent studies demonstrated that RNA editing at codon 280 can

code for a leucine or a proline. As well another downstream translation start codon was

discovered at bp 378, bringing the running total of WT1 isoforms to 24. In 2004 Dallaso

and colleagues identified the AWT1 transcript which resulted from an alternative exon 1

with its own promoter sequences (Dallosso et al.,2004). What was thought to be one

protein in 1990 has now shown itself to contain no less than 32 isoforms (Figure 2).

1.2.3. Cellular Localization

Different WT1 isoforms localize to distinct compartments of the nucleus. WT1(-

KTS) isoforms display a parallel distribution to that of classic transcription factors such

as Sp1 and TFIIB. These isoforms are found most often associated with transcriptional

machinery. The WT1(+KTS) isoforms are now known to be preferentially co-localized

7

Figure 2. Schematic diagram of the wt1 gene to protein. The four start sites are noted in the wt1 gene. The alternative splice sites (exon 5 and KTS) and RNA editing location are shown within the mRNA. * denote possible phosphorylation sites. Adapted by permission from MacMillan Publishers Ltd: [Leukemia] (Yang et al.,2007).

8

with interchromatin granules and coiled bodies (Larsson et al.,1995; Bruening et

al.,1996).

1.2.4. Syndromes associated with WT1

As mentioned above there is an abundance of WT1 isoforms, but what is the

functional role of each of these proteins? While the exact functions of many of these

isoforms are not known, some insights have been gained from individuals that possess

mutations in their wt1 gene. While it is known that certain mutations can give rise to

Wilms’ tumors, mutations can also give rise to various developmental diseases,

sometimes in conjunction to Wilms’ tumor. Some disorders known to be caused by wt1

mutations include WAGR (congenital abnormality, Wilms’ tumor, aniridia, genitourinary

abnormalities, mental retardation), Beckwith-Wiedemann, Denys-Drash and Frasier

Syndromes.

WAGR facilitated the mapping of the minimal critical region on chromosome

11p13 for Wilms’ tumor. It was first identified by Miller and colleagues in 1964 (Miller

et al.,1964). Most patients have a de novo deletion in the distal band of 11p13, thus

WAGR is a contiguous gene deletion syndrome. Children born with WAGR can present

immediately with sporadic aniridia. Genital abnormalities more commonly occur in

males. There is also a high risk of renal failure as a result of focal and segmental

glomerulosclerosis (FSGS). If the syndrome is suspected, a combination of lymphocyte

high resolution chromosome study and molecular cytogenetic fluorescence in situ

hybridization is recommended to demonstrate the characteristic deletion and thus confirm

the diagnosis (Fischbach et al.,2005). A fast and early diagnosis can do much to improve

9

survival and quality of life. The risk of Wilms’ tumor in children with WAGR is ~45%

(Muto et al.,2002).

Beckwith-Wiedemann Syndrome (BWS) is characterized by asymmetric

organomegaly, umbilical hernia, hypoglycemia, and a modestly increased risk for various

cancers, including adrenal carcinoma, hepatoblastoma and Wilms’ tumor

(Wiedemann,1964; Beckwith,1969). Denys-Drash (DDS) and Frasier Syndrome (FS) are

two related conditions caused by substitution mutations, rather than deletions, of the wt1

gene. Both syndromes are characterized by male pseudohermaphroditism, progressive

glomerulopathy, and the development of genitourinary tumors. DDS demonstrates

diffuse mesangial sclerosis (DMS) while FS demonstrates focal and segmental

glomerulosclerosis (FSGS). DDS usually presents itself in newborns with ambiguous

genitalia, and nephropathy commonly appears in the first year of life. The most common

mutation of wt1 that causes DDS is an arginine to trytophan mutation at codon 394

(McTaggart et al.,2001). This region is within the third zinc finger, although some other

common mutations occur within the second finger as well. The presence of rare DDS

cases due to amino terminal truncations of WT1 suggest a loss-of-function mechanism.

An intronic mutation in the alternative splice site that gives rise to the +KTS isoforms

leads to a decrease in the +KTS/-KTS isoform ratio. This shift in the ratio of WT1

isoforms appears to be the main cause of FS.

As there is so much overlap between the BWS and FS it has been suggested that

all patients should be classified as part of the same syndrome whose basic abnormality is

a mutation of the wt1 gene. This classification should show the following characteristics:

combination of intersex abnormality, nephropathy, and genitourinary tumors.

10

1.2.5. DNA Binding

Rauscher and colleagues (Rauscher et al.,1990) used a WT1 ZF affinity column to

bind any double stranded oligonucleotide from a completely degenerate library to

determine possible DNA binding partners for WT1. The oligonucleotides that were

found to bind to the column were eluted and cloned. Most of these were GC-rich and

contained stretches of 4 to 5 cytosine (C) residues. These DNA sequences were very

similar to the consensus binding site of EGR-1 (Knox-1, zif268, TIS-8, NGF1-A). The

EGR-1 protein is a serum inducible, nuclear phosphoprotein that has three zinc fingers.

WT1 was shown to have a 51 % amino acid similarity to the EGR-1 zinc finger region

(Figure 3). When the WT1 zinc finger (ZF) region was tested in gel shift assays against

the EGR-1 binding site, clear and strong binding was seen. The EGR-1 consensus

sequence is as follows; 5’-GCG GGG GCG -3’. The +KTS variant of WT1, as well as a

naturally occurring mutation that is known to occur in some cases of Wilms’ tumor (WT)

where the third zinc finger is deleted, showed negligible binding to the EGR-1 sequence

(Bickmore et al.,1992).

The zinc fingers of WT1(–KTS) bind DNA in a very similar fashion to EGR-1.

Each zinc finger contacts a three base pair (bp) subsite of DNA anti-parallel to the

guanine rich strand of the double helix, in this way ZF1 contacts the 5’end of the binding

site. The amino acids in the amino-terminal portion of the α-helix of each finger contact

a guanine residue in the DNA. Amino acid -1 (the amino acid preceding the α-helix of

the zinc finger) contacts the third base of the subsite (- - G). The third residue of the α-

helix (+3) contacts the second base (- G -), and the sixth residue of the α-helix (+6)

11

WT1 (1) SEKRPFM CAYPGC NKRYF K LS H LQ M HSRKHSP1 (1) GKKKQHI CHIQGC GKVYG K TS H LR A HLRWH

WT1 (2) TGEKPYQ CDFKDC ERRFS R SD Q LK R HQRRHSP1 (2) TGERPFM CTWSYC GKRFT R SD E LQ R HKRTHEGR1(1) PHERPYA CPVESC DRRFS R SD E LT R HIRIH

WT1 (3) TGVKPFQ C-KT-C QRKFS R SD H LK T HTRTHSP1 (3) TGEKKFA C-PE-C PKRFM R SD H LS K HIKTHEGR1(2) TGQKPFQ C-RI-C MRNFS R SD H LT T HIRTH

WT1 (4) TGEKPFS CRWPSC QKKFA R SD E LV R HHNMHEGR1(3) TGEKPFA C-DI-C GRKFA R SD E RK R HTKIH

KTS

β-hairpin α-helix

WT1 (1) SEKRPFM CAYPGC NKRYF K LS H LQ M HSRKHSP1 (1) GKKKQHI CHIQGC GKVYG K TS H LR A HLRWH

WT1 (2) TGEKPYQ CDFKDC ERRFS R SD Q LK R HQRRHSP1 (2) TGERPFM CTWSYC GKRFT R SD E LQ R HKRTHEGR1(1) PHERPYA CPVESC DRRFS R SD E LT R HIRIH

WT1 (3) TGVKPFQ C-KT-C QRKFS R SD H LK T HTRTHSP1 (3) TGEKKFA C-PE-C PKRFM R SD H LS K HIKTHEGR1(2) TGQKPFQ C-RI-C MRNFS R SD H LT T HIRTH

WT1 (4) TGEKPFS CRWPSC QKKFA R SD E LV R HHNMHEGR1(3) TGEKPFA C-DI-C GRKFA R SD E RK R HTKIH

KTS

β-hairpin α-helix

Figure 3. Sequence alignment of WT1 ZF with Sp1 and EGR1 Amino acid sequence alignment of selected zinc fingers. Structure is indicated by the blue arrows (indicating beta sheets) and by the pink alpha-helices. Ligans cysteines and histidines are in bold. Amino acids of each zinc finger that are hypothesized to interact with the DNA are shown in red. The alternate splice site where the seqence KTS is inserted in the WT1 (+KTS) isoform is indicated between residues 407 and 408.

12

contacts the first base (G - -). This binding pattern is conserved among most C2H2 zinc

fingers proteins (Drummond et al.,1994; Nakagama et al.,1995; Elrod-Erickson et

al.,1996; Elrod-Erickson et al.,1998). However, DNA binding is not always clear for

only two of the three possible interacting amino acids actually contact DNA for any one

zinc finger.

Common structural features of ZF proteins have been elucidated with the help of

DNA-C2H2 ZF crystallographic studies of EGR-1 (zif268), Tramtrack and GL1

(Pavletich and Pabo,1991; Fairall et al.,1993; Pavletich and Pabo,1993). In general these

features include 1) ZFs bind in the major groove of DNA, with successive fingers

wrapping around the double helix and 2) critical nucleotide contacts are most commonly

made by defined amino acid positions located near the amino-terminus of the ZFs α-

helix. For WT1 the amino acid residues occupying these putative DNA-interacting sites

in the carboxy-to-amino direction are 4RER 3THR 2RQR 1MHK (fingers 4 to 1) (Call et

al.,1990). There is some debate as other groups have published the interacting amino

acids to be 4RDR 3HDR 2RQR 1MH/SK (fingers 4 to 1) (Hamilton et al.,1998). The EGR-

1 has a nearly identical amino acid composition in its three ZFs 3RER 2THR 1RER

(fingers 3 to 1), which would help to explain why WT1 and EGR-1 have very similar

DNA consensus sequences (Figure 3). This also helps us to understand why WT1 with a

mutated ZF 1 can still bind to the consensus sequence whereas WT1 (+KTS) has a

decreased affinity.

It was also shown that WT1 could bind a recognition element (GGG GCG GGC)

that was very similar to the Sp1 consensus sequence (GGG GCG GGG). The amino acid

composition in Sp1s ZFs 3KHR 2RER 1AHK (fingers 3 to 1) closely resembles the amino

13

acids in ZFs 3 to 1 and thus they are able to bind similar recognition sites (Kriwacki et

al.,1992) (Figure 3). Drummond noted that WT1 ZF 1 can tolerate some degree of

sequence diversity, as can the first ZF of Sp1. This may imply that the degree of

conformational flexibility for amino acid side chains in an MHK/AHK vs. a RER ZF can

tolerate a greater range of nucleotide contacts. This may also imply that the contribution

of zinc finger 1 to DNA binding is less important than the last three fingers.

In 1993, Wang and colleagues discovered another WT1 binding site that was

equally effective, a TCC repeat motif 5’-(TCC)n-3’. This is found in at least five growth-

related gene promoters that are proposed to bind WT1 (Wang et al.,1993). The fact that

WT1 can bind to more than one type of consensus sequence implies that there is some

degree of flexibility in the WT1-DNA interaction; this is not uncommon for most zinc

finger transcription factors.

Drummond examined binding site discrimination of the WT1 ZF splice variants

(Drummond et al.,1994). Their results demonstrated that alternative splicing of the ZF

domain, whether by RNA alternative splicing events or through mutation, resulted in

altered binding specificities. Based on their data it was hypothesized that the insertion of

+KTS in the third ZF of WT1 does not displace the finger from its DNA binding triplet

but rather changes its alignment to the DNA therefore weakening the stability of the

nucleotide-amino acid hydrogen bonds.

Nakagami and colleagues used whole-genome PCR to identify a genomic

fragment with a higher affinity for WT1 than the EGR-1 consensus sequence.

Footprinting analyses revealed protection within a 10 nucleotide (nt) sequence (GCG

14

TGG GAG T), known as WTE (WT1 responsive element) (Nakagama et al.,1995).

Analysis by scatchard plot revealed a 20 fold increase in affinity over the EGR-1

sequence. Mobility shift experiments with various ZF mutations showed that any

integrity damage to ZFs 2-4 completely abolished binding. This would seem to insinuate

that finger 1 was not involved in binding to the WTE fragment. However, Hamilton and

colleagues (Hamilton et al.,1995) later showed that the first ZF of WT1 bound to a 3 bp

subsite within a 12 bp consensus binding site. According to their study this subsite can

be composed of the following bp triplet T/G G/A

/T G/

T . If the first ZF does interact with the

same strand of DNA as the other three ZFs it would appear to prefer binding to keto

substituents in the major groove.

1.2.5.1. Structure of WT1 in complex with DNA

Recently, the crystal structure of WT1 ZF (-KTS) in complex with both a 14 bp

(3’-G TCT GCG GGG GCG C-5‘) and a 17 bp (3’-C GCG TCT GCG GGG GCG C-

5‘) oligonucleotide of DNA was solved (Stoll et al.,2007) (Figure 5) . The 14 bp

sequence was selected to give high binding affinity for the +KTS isoform, based on

observed sequence preferences for the EGR-1 consensus sequence and DNA interactions

with ZF 1(Drummond et al.,1994; Hamilton et al.,1998; Laity et al.,2000). While

investigating the structure of WT1 ZF (-KTS) complexed to DNA using NMR, they

discovered diffraction-grade crystals in the NMR tubes. The crystals were grown at 4°C

over the period of 4 weeks in 10 mM argon-saturated dII-Tris-HCl (pH 6.7) in a mixture

of 90 % H2O/ 10 % D2O containing 20 mM KCl, 5 µM ZnSO4, and 2 mM NaN3. The

sample concentration of WT1 ZF (-KTS) was 0.6 mM (approximately 10 mg/mL).

15

Figure 4. Crystal structure of WT1 ZF in complex with the 14bp EGR-1 DNA Protein backbone is shown as ribbon diagram and the individual zinc fingers are identified using different colors. Coding strand of DNA is shown in gold, non-coding strand is shown in orange. Reprinted from JMB, 372/5, Stoll et al., Structure of the Wilms Tumor Suppressor Protein Zinc Finger Domain Bound to DNA, pg 1227-1245, Copyright (2007), with permission from Elsevier. (Stoll et al.,2007)

16

The DNA-ZF complexes were prepared by titrating protein aliquots into the DNA

solution to a final stoichiometry of 1:1.

The crystallographic resolution obtained was modest at 3.15 Å but they were able

to combine this with their NMR spectra to provide a more detailed picture of the

complex. This structure is of importance because it can provide new insights into

sequence-specific recognition of DNA by WT1. In this way it may provide a framework

for understanding the structural and mechanistic effects of disease-causing mutations.

The ZF contacts with DNA are almost identical to those seen in the Zif268/DNA

complex (Elrod-Erickson et al.,1996; Elrod-Erickson et al.,1998) (Figure 6). All amino

acid residues involved in base recognition by WT1 ZFs 2,3, and 4 are identical to those of

ZFs 1,2, and 3, respectively, of Zif268. This evidence would suggest that similar

interactions are involved in recognition of the EGR-1 sequence by both WT1 and Zif268.

As seen with many other DNA/ ZF protein complexes the majority of the base specific

contacts are with the purine-rich coding strand (Fairall et al.,1993; Pavletich and

Pabo,1993; Omichinski et al.,1997; Elrod-Erickson et al.,1998; Nolte et al.,1998; Segal

et al.,2006).

The role of ZF 1 in DNA binding was determined unequivocally. The structure

obtained by Stoll et al. demonstrated that ZFs 2-4 make base-specific contacts in the

major groove of the DNA duplex, while ZF 1 interacts only with the phosphodiester

DNA backbone (Figure 5). The NMR spectra clearly showed that ZF 1 lies in the major

groove of both the 14bp and the longer 17bp oligonucleotide, but interacts less

extensively and makes no base-specific contacts. The α-helix of ZF 1 was placed in the

major groove of the DNA, but unlike Zfs 2-4, the fingertip points away from the DNA,

17

Figure 5. Schematic diagram of WT1 ZF with DNA Schematic diagram of zf1-4 as deduced by the crystal and NMR structures of the Wt1 (-KTS) complexed with 14bp EGR-1 DNA. Contact amino acids are colored green, pink and red according to the zinc finger (2, 3, and 4, respectively). The contacts of finger 1 inferred from the X-ray/NMR structure of the 17bp duplex are shown as circles, with amino acids labelled in blue. Dotted circles indicate that the amino acid side-chains show a wide variety of interactions in the ensemble of 20 structures. Reprinted from JMB, 372/5, Stoll et al., Structure of the Wilms Tumor Suppressor Protein Zinc Finger Domain Bound to DNA, pg 1227-1245, Copyright (2007), with permission from Elsevier. (Stoll et al.,2007)

18

so that the side-chains commonly involved in base recognition cannot contact the DNA.

This structural visualization is consistent with binding site selection experiments, which

suggested a weak base specific sequence for ZF 1 (T/G G/A

/T G/

T) (Hamilton et al.,1995;

Hamilton et al.,1998). The 3 bp DNA sequence shown to interact with ZF 1 in the

structure was TCT. As postulated by Hamilton and Call one of the most important amino

acids of ZF 1 DNA contacts was M342, however the proposed H339/S248 or K336 were

not found to be vital from the structure (Call et al.,1990; Hamilton et al.,1998; Stoll et

al.,2007).

The main ZF contacts of ZF 2 are as follows; the guanadinium groups of R366

and R372 make contacts with the major groove edges of two guanine bases. This is also

in agreement with Call and Hamilton, although they had also shown Q397 to be a key

contact and this was not the case (Call et al.,1990; Hamilton et al.,1998). ZF 2 also

makes some contacts with the phosphate backbone, the Nδ1 of the zinc ligand H373 is

close to an oxygen of the phosphodiester moiety between two guanine bases, and one can

conclude, forms a hydrogen bond. S367 of ZF 2 is responsible for a contact with the

phosphate backbone of the non-coding strand. Hamilton was correct in that ZF 3 makes

key DNA contacts through R394, D396, and H397(Hamilton et al.,1998). R394 and

D396 interact with a guanine and a cytosine base, respectively. H397 interacts with the

major groove edge of guanine base. Potential hydrogen bonding with the phosphate

backbone can be seen with the side chains of S393, K399, and the zinc ligand H401. The

fourth ZF, like ZF 2, contacts the edges of two guanine bases with the guanadinium

groups of R424 and R430. Both Call and Hamilton were correct in that R424 and R430

were key residues for DNA binding, however the third amino acid E427 (Call) or D426

19

(Hamilton) were not shown to play a key role in the structure (Call et al.,1990; Hamilton

et al.,1998).

1.2.5.2. Molecular Basis of WT1-associated disease

Almost 95% of the mutations seen in Denys-Drash syndrome occur within zinc

fingers two and three. These mutations fall into two categories (1) mutations that

destabilize the zinc finger structure; and (2) mutations that replace key base contact

residues in zf2 or zf3. Perhaps the structurally obvious class 1 mutations involve those

that occur in the ligand binding residues (the cysteines or the histidines), as these would

most definitely result in improperly folded fingers. Common class 2 mutations involve

Arg394 of zinc finger 3, which contacts GUA7, by Gln, Leu, Pro, or Trp; all of these

would disrupt the zf3-DNA interaction

It has been known for some time where the distinctive mutations within such

syndromes as Denys-Drash and Frasier are located. In 1996, Borel and colleagues (Borel

et al.,1996) published a paper on the effects of disease causing mutations on the DNA

binding capacity of the WT1 ZF protein. They demonstrated without a doubt that many

of the ZF 2 and ZF 3 mutations known to cause disease were also responsible for key

DNA contacts. FBAs showed anywhere from a 6-10 fold increase in the disassociation

constant over wt binding. By solving the WT1 ZF protein/DNA complex, Stoll was able

to confirm and further validate these earlier findings (Stoll et al.,2007). This WT1-DNA

complex allowed us to visualize and thus better understand the function these mutations

play.

20

1.2.5.3. Phosphorylation of WT1

The phosphorylation of Ser367, and Ser393 of WT1 is known to inhibit DNA

binding and transcriptional repression activity (Ye et al.,1996; Sakamoto et al.,1997; Kim

et al.,1999). The structure of the WT1-DNA complex allows for a simple explanation, in

that Ser393 of ZF 3 is in direct contact with the phosphate backbone of the coding strand

and thus any phosphorylation of this residue would most certainly result in a steric/charge

clash and impair potential binding. The Ser367 of ZF 2 is close to the DNA backbone,

inhibition of DNA binding is most-likely due to charge repulsion.

1.2.6. WT1 and RNA Binding

Bardeesy and colleagues described and identified RNA ligands for WT1(-KTS)

using systematic evolution of ligands by exponential enrichment (SELEX) (Bardeesy and

Pelletier,1998). Using competition experiments it was determined that the WT1 protein

could indeed bind to both DNA and RNA, however binding to both simultaneously was

unlikely as the DNA and RNA binding sites were found to be overlapping. They

identified 3 families of RNA ligands which showed specific binding to WT1 (-KTS) with

affinities comparable to that seen for binding of this protein to DNA.

To study structural requirements for RNA-WT1 binding, efforts were focused on

the RNA aptamer pel22. From deletion mutants it was noted that WT1 without zinc

finger 1 was still capable of binding pel22 while WT1 without zinc finger 4 reduced

RNA recognition. Bardeesy concluded that residues within WT1 zinc fingers 2-4 are

sufficient and necessary for RNA binding. It should be noted that other literature stated

the opposite in that ZF1 was necessary for RNA binding (Caricasole et al.,1996).

21

However in unpublished data it was clearly shown that indeed zinc finger 4 is very

important for RNA binding while zinc finger 1 is not (Foster,2006).

BLAST searches with the SELEX ligands to identify naturally occurring RNAs

that could potentially be in vivo targets for WT1 (-KTS) only produced sparse results and

none were from pel22.

Zhai and colleagues (Zhai et al.,2001) went a step further and determined the

apparent dissociation constants for the WT1- pel22 complex. This was done using

nitrocellulose filter binding assays. They were also able to demonstrate the effects of

particular buffer components on WT1-RNA complex formation such as pH, Mg

concentration, monovalent salt and temperature dependence. RNA binding was

significantly less dependant upon ionic interactions than DNA binding. Mutational

analyses of the pel22 RNA demonstrated that nucleotides outside the consensus sequence

are also critical for binding to WT1 ZF, the overall structure of the RNA ligand would

appear to be a major determinant of binding affinity.

The most studied RNA-C2H2 zinc finger interaction is that of TFIIIA in Xenopus

oocytes which binds the 5S rRNA to form the 7S ribonucleoprotein particle. This

particle stabilizes the RNA until it is required for ribosome assembly and facilitates

nuclear export of the 5S rRNA. The crystal structure of zinc fingers 4-6 of TFIIIA in

complex with a minimal 5S rRNA provided some remarkable insights into how C2H2 ZF

proteins bind to RNA (Lu et al.,2003) (Figure 6). In particular, it was interesting to see

how the same ZF could be used to bind to both RNA and DNA. ZF 5 of TFIIIA binds to

DNA in the typical C2H2 ZF fashion, with side chains from the α-helix binding to bases

and the backbone atoms in the major groove of DNA. In RNA, ZF 5 is not so interwoven

22

F5

F6

F4

F5

F6(a) (b)

Figure 6. Ribbon diagram of the crystal structure of TFIIIA bound to DNA or RNA (a) TFIIIA ZF 4 to 6 bound to DNA (Nolte, Conlin et al. 1998) (b) TFIIIA ZF 4 to 6 bound to RNA (Lu, Searles et al. 2003) Figure was made using PyMOL

23

with the nucleic acid. ZF 5 does not contact the bases even though the equivalent nucleic

acid sequence is present. In contrast, it uses the side chains from the α-helix to bind the

major groove backbone of a double helical region of RNA. The side chains used in RNA

recognition are many of the same as those used in DNA recognition. Therefore one can

deem TFIIIA ZF 5 a bifunctional DNA/RNA binding domain, which utilizes overlapping

sets of side chains for nucleic acid recognition. TFIIIA ZF 4 and 6 both function as

spacer regions in DNA binding but they recognize focused elements in loop regions of

the RNA using the amino-terminal end of their α-helices. Many of the same residues of

the α-helix (-1, -2, +1, +2) are making contacts, but the orientation of the α-helix towards

the nucleic acid is not the same. For DNA binding the side of the α-helix is typically

directed towards the nucleic acid, but in the case of ZF 4 and 6 of TFIIIA in complex

with 5S rRNA the amino-terminal end of the α-helix is directed toward the RNA.

1.2.7. WT1 and Protein Binding

Over the years many methods have been used to determine protein binding

partners for WT1, some of these include the yeast-2 hybrid system and

coimmunoprecipitation. In the last two decades there have been a great number of

binding partners identified. Yet the functional importance of many of these associations

are still unknown. Table 1 demonstrates some of the investigated WT1 protein binding

partners.

The CREB binding protein (CBP) is probably the most understood protein

partner, it functions as a coactivator. It was shown that CBP and WT1 interact directly,

mediated by the first two ZFs of WT1 and the E1A binding domain of CBP. Association

24

Table 1. Protein binding partners of WT1 Protein Binding Partners of WT1. Adapted by permission from MacMillan Publishers Ltd: [Leukemia] (Yang et al.,2007).

Gene Effect WT1 region required for

binding (amino-acid residue)

Evidence of interaction

Signal transducers and activators of transcription 3 (STAT3)

Synergistic, activator of survival function 1-281 Yeast two-hybrid, IP

Brain acidic soluble protein 1 (BASP 1) Repressor 71-101 Affinity chromatography,

IP Creb binding protein (CBP) Activator ZF 1 and 2 IP P53 Repressor ZF 1 and 2 IP Par4 Repressor ZF 1-4 Yeast two-hybrid, IP Activator 245-297 protein affinity, IP P73 Repressor ZF 1-4 IP

Bone Marrow Zinc finger 2 Repressor ZF 1-4 Affinity chromatography, IP

Ciao-1 Repressor ZF 1-4 Yeast two-hybrid, IP Hsp-70 Activator 6-180 IP E1B 55K Repressor ZF 1 and 2 IP Human ubiquitin conjugating enzyme 9 (hUCE9) Repressor 85-179 Yeast two-hybrid, IP

U2AF65 RNA processing ZF 1-4 Yeast two-hybrid, IP Orphan nuclear receptor SF-1 Synergistic activation ND Yeast two-hybrid, IP Four-and-half LIM-domain FHL2 Activator 182-298 Yeast two-hybrid, GST

pull-down, IP

WT1-associating protein (WTAP) ND C-terminus KTS(-) Yeast two-hybrid, GST

pull-down, IP

WT1 Repressor N-terminus Yeast two-hybrid, GST pull-down, IP

25

between the two seems to enhance the transcriptional activation of WT1 target genes, in

particular, Amphiregulin (Wang et al.,2001). The WT1 target site in the Amphiregulin

promoter is within 10 nt of a consensus sequence for CRE binding proteins (CREB) (Lee

et al.,1999). Both isoforms (-KTS and +KTS) of WT1 are capable of binding CBP,

insinuating that both have the potential of regulating transcription, even in the absence of

DNA binding. CBP is a chromatin structure modifier and its association with WT1 was

the first demonstration of WT1-mediated transcriptional regulation in a native chromatin

context.

Another important binding partner of WT1 is the tumor suppressor protein p53

which also associates with the first two ZFs of WT1. WT1 co-expression appears to

increase the stability and the half life of p53. p53 and WT1 would seem to reciprocally

modulate each others transactivational properties (Maheswaran et al.,1993; Maheswaran

et al.,1995). Unlike p53, ectopically expressed Par4 (prostate apoptosis response)

appears to repress the transcriptional activation of WT1 when binding to the ZF region

(Johnstone et al.,1996). A later study showed that Par4 could also bind the 17 amino acid

alternative splice domain of WT1, within the proline-glutamine rich region (Richard et

al.,2001). WTAP (WT1 associating protein) encodes a ubiquitously expressed protein of

unknown function which binds the carboxy terminal ZF domain of WT1 (Little et

al.,2000). U2AF65 is a ubiquitous splicing factor that has also been shown to interact

with WT1 but with a slightly higher affinity for the +KTS isoform (Davies et al.,1998).

In fact, two WT1 domains appear to be required for U2AF65 binding the region just prior

to the ZF domain and the ZF domain itself (excluding ZF 1). The functional implications

of this interaction on either protein (WT1 or U2AF65), has yet to be elucidated, but RNA

26

processing is the best contender. WT1 has also been shown to interact with a novel

WD40 protein named Ciao-1, this will be discussed later in detail.

1.2.8. WT1 Function

1.2.8.1. Transcriptional Regulation

WT1 encodes a proline-glutamine rich region and a DNA binding domain

characteristic of a transcription factor (Rauscher et al.,1990). Depending on the promoter

and cellular circumstances this factor is either an activator or a repressor of gene

transcription (Table 2). WT1 is also an autoregulator in that it is capable of repressing its

own promoter (Malik et al.,1994; Rupprecht et al.,1994; Hewitt et al.,1996). Other genes

that are known to be repressed by WT1 include c-myc (Hewitt et al.,1995), bcl-2 (Hewitt

et al.,1995; Heckman et al.,1997), EGR1 (Madden et al.,1991) and tgfβ (Dey et al.,1994)

as well as many others. Two WT1 binding sites are required for repression of

transcription, possibly by preventing the formation or stabilization of the initiation

complex.

WT1 is a member of the EGR1 family (early growth factor response), this family

is defined by the recognition of a common DNA sequence. Many EGR proteins are

growth factor inducible, short-lived transcription factors (activators) capable of initiating

certain patterns of genetic expression. It is hypothesized that this pattern may be altered

by developmentally regulated, tissue-specific transcription factors that bind to the same

promoters. Of all the EGR proteins, WT1 is the only repressor and may function as a

developmental marker by inhibiting cellular proliferation and initiating a tissue specific

program of gene expression (Reizner et al.,2005). It should be noted that WT1 isoforms

27

Table 2. WT1 target genes Proposed WT1 Target Genes. Adapted from Exp. Cell Res., 264/1, Lee and Haber, Wilms Tumor and the WT1 Gene, pg 74-99., Copyright (2001), with permission from Elsevier. (Lee and Haber,2001)

Genes Promoter-reporter assays Target Sequence

EGR-1 Repression/Activation EGR-1 concensus PDGF-A Repression/Activation EGR-1 concensus, TCC repeats EGFR Repression TCC repeats IFG-II Repression EGR-1 concensus IGF-1R Repression EGR-1 concensus Midkine Repression EGR-1 concensus C-myc Repression EGR-1 concensus N-myc Repression EGR-1 concensus WT1 Repression EGR-1 concensus PAX2 Repression EGR-1 concensus TGF-β Repression EGR-1 concensus RAR-α Repression EGR-1 concensus MDR-1 Repression EGR-1 concensus NOVH Repression ND SRY Activation EGR-1 concensus Androgen receptor Repression EGR-1 concensus CTGF Repression ND p21Cip1 Activation ND Amphiregulin Activation WTE Syndecan-1 Activation EGR-1 concensus E-cadherin Activation EGR-1 concensus BCL-2 Repression/Activation WTE DAX-1 Activation EGR-1 concensus MIS Activation SF-1 site (indirect) CSF-1 Repression/Activation EGR-1 concensus RbAp46 Activation ND VDR Activation WTE ODO Repression EGR-1 concensus TERT

Repression

EGR-1 concensus

28

can bind to two DNA motifs (1) GC-rich sequences with the conserved consensus

sequence 5’-GG/YGGGGGAG/

C-3’ (EGR) or (2) (TCC)n-containing sequences.

1.2.8.2. WT1 and Kidney Development

WT1 is expressed in all stages of kidney development (Figure 7). The

metanephric kidney is formed through reciprocal inductive signals between the

mesodermal mesenchyme and the ureteric bud (an outgrowth of the Wolffian duct)

(Saxen and Sariola,1987). Initially, the wt1 expression is low but detectable. The

proliferating mesenchyme condenses around the ureteric bud and induces the bud

branching necessary for nephrogenesis. At this point wt1 expression increases

dramatically. Metanephric kidney development proceeds through transitory structures

known as comma and S-shaped bodies into the mature nephrons where WT1 protein

production is restricted to the podocytes (Armstrong et al.,1993; Bard et al.,1993).

Podocytes form a special layer of epithelial cells in the glomerulus, this layer surrounds

the blood vessels and is involved in the ultra-filtration of the primary urine. During

nephrogenesis, the metanephric mesenchyme differentiates into epithelial components of

the nephrons- this process is referred to as mesenchyme to epithelium transition. Wilms’

tumors are known as triphasic because they consist of undifferentiated mesenchyme,

stromal and epithelial cells. The tumors are thought to ascend from the condensing

metanephric mesenchyme that failed to undergo mesenchyme to epithelium transition and

continued to proliferate (Machin and McCaughey,1984; Hastie,1994).

29

Figure 7. wt1 expression in the developing kidney. WT1 (green), Laminin (red). “(A) A control kidney shows a typical pattern of Wt1

expression; Wt1 is absent from the ureteric bud (‘ub’), present at a low level in condensing mesenchyme (‘cm’) and upregulated strongly in the presumptive glomeruli (‘g’). (B) Kidney with wt1 suppressed. There is some low level expression of wt1 that remains in the mesenchyme but epithelial nephrons are absent and only a few scattered cells express high levels of WT1. Inhibition of nephrogenesis can be seen clearly by comparing (C), a low-power view of a control kidney, in which there are many nephrons (arrows show examples) and (D) a kidney cultured in Wt1 siRNA in which no nephrons are present”. Davies JA, Development of an siRNA-based method for repressing specific genes in renal organ culture and its use to show that the Wt1 tumor suppressor is required for nephron differentiation, Human Molecular Genetics, 2004, 13, 2, pg 235-246, by permission of Oxford University Press. ((Davies et al.,2004))

30

The essential need for WT1 for proper kidney development is best demonstrated

by the use of knockout mice. Studies of mice that exclusively express either WT1(-KTS)

or WT1(+KTS) isoforms have demonstrated that both play a role in early nephrogenesis,

but the (-KTS) isoforms were found to play a more critical role in the development of the

podocytes (Hammes et al.,2001). One can thus make the assumption that the

transcriptional function of WT1 has a major effect on kidney development and, in

particular, podocyte formation. Although hemizygous inactivation does not appear to

have any significant phonotype, wt1-null mice die at a very early stage of development in

utero. wt1-null mice have no kidney (the metanephric mesenchyme undergoes apoptosis)

but the cause of death is most likely due to the malformed heart and diaphragm structures

which are lined by wt1-expressing mesothelial surfaces (Kreidberg et al.,1993). This

shows how necessary WT1 is for normal embryonic development, as it plays major roles

in the development of many organs including the heart, lungs, spleen, gonads and the

kidneys.

1.2.8.3. Post-transcriptional gene expression

The first indication that WT1 may have a role in post-transcriptional function

came when it was determined that WT1(+KTS) co-localized with nuclear speckles

(Larsson et al.,1995). A subsequent study demonstrated an interaction between

WT1(+KTS) and the ubiquitous splicing factor U2AF65, as well as showing that WT1

associated with large molecular weight complexes associated with pre-mRNA (Davies et

al.,1998). It was also shown that WT1 is present in nuclear poly(A)+ RNP particles and

associated with the splicing machinery (Davies et al.,1998).

31

Very recently the WT1(+KTS) isoform was shown to be involved in promoting

gene expression at the post transcriptional level (Bor et al.,2006). A known specific viral

element capable of facilitating the export of mRNAs with retained introns into the

cytoplasm is derived from Mason-Pfizer Monkey Virus (MPMV) and is known as the

constitutive transport element (CTE) (Hammarskjold,2001). The CTE element is

essential for the export of the genomic, unspliced RNA that is expressed in MPMV-

infected cells. CTE functions by interacting with the host cell machinery, in particular,

Tap (NXF1) a proposed cellular mRNA export receptor (Coyle et al.,2003; Jin et

al.,2003). Tap forms a heterodimer with the cellular NXT1 protein and both have been

demonstrated as being important for CTE function. It was recently demonstrated that

along with mRNA export the Tap-NXT1 heterodimer promotes translation of the CTE-

mRNA in the cytoplasm. Tap appears to associate with polyribosomes.

Yeou Bor et al. have now discovered the existence of a cellular CTE which shows

strong homology with an RNA that coimmunoprecipitates with epitope tagged

WT1(+KTS) protein. They were able to show that WT1(+KTS), in contrast to WT1(-

KTS), was able to strongly enhance expression from reporter constructs containing the

MPMV CTE. As well, the expression of WT1(+KTS) promotes the polysome

association of CTE-containing mRNA (Bor et al.,2006). This initial observation seems

to indicate a role for WT1(+KTS) in translational regulation of target mRNAs.

1.2.9. WT1 and Cancer

wt1 is a tumor suppressor gene that plays a significant role in the carcinogenesis

of Wilms’ tumor. In regards to Wilms’ tumor the WT1 protein functions as a repressor

32

of transcription of the insulin-like growth factor II (IGF-2) gene. IFG-2 functions as an

autocrine growth factor, stimulating cell growth (Hewitt et al.,1995). When wt1 is

mutated the tumors grow unchecked due to lack of repression of IGF-2. Over the last

few years it has been noted that wt1 is expressed in a number of adult tumors from

different origins including colorectal, breast, desmoid, and brain tumors (Table 3).

Evidence shows that for these cancers, wt1 is required for proliferation, not suppression,

as it appears to inhibit apoptosis of tumor cells in culture.

In 1993, Park and colleagues demonstrated that the wt1 gene was mutated

in a human mesothelioma thus indicating that wt1 contributes to other malignancies (Park

et al.,1993). Soon after, the expression of wt1 in human hematopoietic malignancies was

examined. There was a clear correlation observed between the levels of wt1 gene

expression and poor prognosis of minimal residual disease in acute leukemia. Significant

levels of wt1 gene expression were observed in all leukemia patients associated with

immature leukemia (or tumor) cells. For many types of acute leukemia, patients with

relatively low levels of wt1 mRNA expression generally had a better prognosis than

patients with higher levels (Inoue et al.,1994; Bergmann et al.,1997).

Yamagami et al showed a reduction in WT1 protein production via wt1 antisense

experiments. This reduction resulted in inhibited leukemic growth, indicating a link

between leukemic growth and high levels of wt1 expression (Yamagami et al.,1996).

Therefore, it was argued that WT1 might be a possible target for cancer treatment, as it is

known that WT1 is only expressed in adult kidney podocytes. In this way WT1 could be

a plausible tumor-specific target for therapeutic intervention. A recent publication in

33

Table 3. WT1 and human cancers Expression of wild-type WT1 in various human cancers. Adapted by permission from MacMillan Publishers Ltd: [Leukemia] (Yang et al.,2007).

Type of Cancer Frequency of detection

Astrocytic tumors 23/25 Bone and soft-tissue sarcomas 28/36 Brain tumor 23/26 Breast cancer 27/31 Colorectal adenocarcinoma 20/28 De novo lung cancer 41/46 54/56 Desmoid tumor 5/5 Esophogeal squamous cell carcinoma 12/12 36/38 Head and neck squamous carcinoma 42/56 Leukemia 30/59 68/86 Malignant methelioma 54/56 50/67 nuc Melanocytes 7/9 Neuronal tumors: brain 16/36 Neuroblastomas 6/18 Ovarian carcinomas Serous 28/28 Endometrial 34/130 10/29 Epithelial 78/100 Pancreatic ductual adenocarcinoma 30/40 Primary thyroid cancer 33/34 Renal cell carcinoma

4/5

34

2004 by Oka et al showed promising results using a WT1 peptide in trials against

leukemia, breast, and lung cancer (Oka et al.,2004).

1.3. Ciao-1

1.3.1. The Protein

Ciao-1 is a 339 amino acid protein; it is a WD40 protein that characteristically

consists of repeating domains. From the amino acid sequence it was determined that

Ciao-1 consists of seven β-transducin repeats. When looking at human tissues it was

determined that two major species of mRNA existed at 4.4 and 1.5kb (Johnstone et

al.,1999). These two species were detected using hybridization with the Ciao-1 clone as

a probe in all tissues. It was determined that Ciao-1 is ubiquitously expressed in all the

tissues tested (heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen,

thymus, prostrate, testis, ovary, small intestine, colon, peripheral blood leukocytes). The

Ciao-1 gene was mapped to chromosome 2q11.2, and while this chromosomal location

had not previously been described as a loci associated with Wilms’ tumor, it should be

noted that genetic abnormalities in this region have been demonstrated in acute non-

lymphocytic leukemia. Analysis of the Ciao-1 gene also provided evidence for

evolutionary conservation, as there were cDNA homologues identified in mouse, rat, fly,

worm, slime mould, and yeast.

1.3.2. WT1-Ciao-1 Interaction

Johnstone and colleagues (Johnstone et al.,1998; Johnstone et al.,1999) stated that

339 aa protein, Ciao-1, was able to bind all four main isoforms of WT1 both in vivo

35

(mammalian cells and yeast) and in vitro using GST pulldown assays. They were able to

demonstrate a change in the mobility of a WT1-DNA complex in vitro, thus indicating

separate binding sites for Ciao-1 and DNA. When they increased Ciao-1 production a

dose-dependant decrease was seen in WT1-mediated transcriptional activation. Ciao-1

by itself did not appear to possess intrinsic transcriptional repression activity (Johnstone

et al.,1999). It was postulated that for Ciao-1 to inhibit WT1-mediated transcription it

must either prevent WT1 from binding its consensus DNA sequence (which they showed

it did not), cause a conformational change in WT1 which masks its activation function, or

negatively interfere with the communication between the activation domain of WT1 and

the basal transcriptional machinery. By producing different lengths of WT1 constructs

and performing GST pull downs it was noted that Ciao-1 requires the carboxy-terminus

of WT1 to bind. Specifically, the last l22 amino acid sequence that contains the full ZF

domain of WT1, was sufficient for binding (Johnstone et al.,1998). These findings

indicate that Ciao-1 may be responsible for the regulation of WT1 transcriptional and

physiological functions.

1.3.2. Function of Ciao-1

The Cia1 structure (Figure 8b), a yeast homologue of Ciao-1, has recently been

solved to 1.7Å (Srinivasan et al.,2007). In addition to solving the 3-D structure, the

group also clearly identified one of the functions of Cia1 as an essential, conserved

member of the cytosolic iron-sulfur (Fe/S) protein assembly (CIA) machinery in

eukaryotes. Cia1 is involved in the nuclear and cytosolic maturation of Fe/S proteins.

36

They were also able to show that Ciao-1 was able to functionally replace Cia1, and

support cytosolic Fe/S protein biogenesis.

Cia1 was shown to localize mainly in the nucleus and had a cellular concentration

of at least 10-fold higher than that of the other CIA proteins (Cfd1, Nbp35, and Nar1).

The other three members of this CIA family are predominantly found within the cytosol.

These finding would suggest an additional function for Cia1/Ciao-1 in the nucleus, where

the affinity for WT1 may come into play.

Of interest they mention that the CIA machinery is responsible for the

incorporation of FE/S clusters at the N-terminus of Rli1, which is an essential protein that

plays a crucial role in the export of ribosomal subunits from the nucleus and in translation

initiation (Dong et al.,2004; Kispal et al.,2005; Yarunin et al.,2005). It would be

interesting to see if the human homologue of Rli1 is able to interact or coexist with WT1,

as this may link Ciao-1 and WT1 with the production/transportation and processing of

RNA within the nucleus.

1.4. WD40

1.4.1. History and Function

The WD repeat was first discovered in 1986 (Fong et al.,1986) in the β-subunit of

the GTP binding protein, transducin, and is referred to as the transducin repeat, the GH-

WD repeat, or the WD-40 repeat. In general, most WD40 proteins have seven repeat

clusters, although some have as many as 16 and some have as few as four. Just as the

number of repeats varies, so do the functions of this fast-expanding family of proteins.

Only these functions have been ascertained: signal transduction, RNA synthesis and

37

TOP

BOTTOM

TOP

BOTTOM

(b)

(a)

Figure 8. WD Repeat (a) “Structural elements within a single repeat. The alternative amino acids for each position are listed in approximate order of their frequency of occurrence.” Reprinted from Publication Title, 24/5, Smith et al., The WD repeat: a common architecture for diverse functions, pg 181-185, Copyright (1999), with persmission from Elsevier. (Smith et al.,1999). (b) Ribbon diagram of the S. cerevisiae WD40 protein Cia-1. The black ball represents the calcium ion found within the crystal structure, the structure is made up of seven WD40 units. Reprinted from Cell, 15, Srinivasan V et al., Structure of the Yeast WD40 Domain, A Component Acting Late in Iron-Sulfur Protein Biogeneis, pg 1246 - 1257, Copyright 2007, with permission from Elsevier. Polar, polar residue.

38

processing, chromatin assembly, regulation of vascular trafficking, cytoskeletal assembly,

cell cycle regulation, and apoptosis (Table 4). The underlying common function of all

WD repeat family members is their ability to coordinate multiprotein complex

assemblies.

1.4.2. Structure

A WD40 protein is defined by the presence of four or more repeating units known

as WD repeats or β-transducin repeats. These repeats have conserved domains of ~40-60

amino acids initiated by a glycine-histidine rich (GH) dipeptide within the first 11 to 24

amino acid residues from the amino terminus and ending with a tryptophan aspartic acid

(WD) dipeptide at the carboxy-terminus. Between these two extremes is a conserved ~40

amino acid sequence (Figure 8a).

The repeat adopts a β-propeller fold, a highly symmetrical structure. Each repeat

comprises a four-stranded anti-parallel β-sheet. The β-propeller however, is comprised of

the last strand of the previous repeat and the first three strands of the subsequent repeat.

This strand sharing between two blades of the β-propeller is thought to stabilize the

molecule. It has been proposed that most stable structure is one of at least seven blades

requiring a minimum of seven WD repeats (Murzin,1992).

The propeller structure provides extensive surface exposure of three surfaces; top,

bottom, and circumference. These three surfaces are ideal for several simultaneous

protein-protein interactions (Figure 8b).

39

Table 4. WD40 protein functions Diverse biological functions of WD-repeat proteins. Adapted with kind permission from Springer Science + Business Media; Cellular and Molecular Life Sciences, 58, 2001, pg 2085-2097, Li and Roberts, Table 1.(Li and Roberts,2001) Biological Function Representative of WD-repeat proteins involved

Signal transduction Gβ protein, RACK1, Stratin, STE4, LIS1, MSI1, PR55, PLAP, RbAp-48

RNA synthesis and processing TATA box-binding protein (TBP) associated factors (TAFs),I, cleavage stimulation factor (CstF)

Chromatin assembly chromatin assembly factor-1 (CAF-1), HIR1 and HIR2 transcriptional repressors of S.cerevisiae

Vesicular trafficking Golgi coat promoter or 'coatomer', most of α and β COP proteins, yeast SEC13

Cytoskeletal assembly microtubule-associated protein (MAP), intermediate chain of cytoplasmic dynein, actin-related proteins Arp2 and Arp3

Cell cycle regulation cell division control protein 4 (CDC4), CDC20, CDC40, p55 (CDC), Coronin, the spindle checkpoint protein Mad2

Programmed cell death apoptotic protease activating factor-1 (Apaf-1), cell-death proteins, Dark, which encodes a Drosophila homologue of mammalian Apaf-1 and C.elegans CED-4

Fe/S protein assembly Cia-1, Ciao-1

40

1.4.3. Human Disease

Knowing that a gene can contribute to a particular disease can provide very

important information about the function of that gene, as well as provide possible

avenues for disease treatment. For genes with no known function it is particularly

interesting as any indication of function can be greatly revealing.

With so many WD40 proteins responsible for so many functions, it is of no

surprise some cause human disease. One such WD40 gene produces the protein CSA

(Henning et al.,1995). CSA interacts with CSB (a DNA unwinding protein) and p44 (a

subunit of the human RNA polymerase II transcription factor IIH). From these known

interactions it is reasonable to postulate that something goes awry in the transcription

mechanism when CSA is mutated. The mutation was discovered in patients with

Cockayne Syndrome (CS), an autosomal recessive disease characterized by abnormal and

slow development evident within the first few years of birth. CS characteristics include;

dwarfism, precociously senile appearance, pigmentary retinal degeneration, optic

atrophy, deafness, marble epiphyses in some digits, sensitivity to sunlight, mental

retardation, and some unusual skeletal features such as disproportionably long limbs with

large hands and feet, and flexion contractures of joints.

1.5. Thesis Objectives

If the 3-D structure of WT1 could be elucidated then perhaps many of the

questions pertaining to the function of this protein could be answered. The primary

objective of this study was to produce large enough concentrations of pure WT1 and

41

Ciao-1 protein in order to enable the crystallization and subsequent determination of

these structures by themselves and in complex with each other.

By utilizing various purification methods and differing growth conditions we

were able to produce the pure protein that was required for crystallization trials. Though

diffraction quality crystals were not obtained these methods may be applied to further

crystallographic studies.

A second objective of this thesis was to investigate a selective library of RNA

aptamers with a specificity for the WT1(+KTS) isoform over the WT1(-KTS) isoform.

The affinities of the pooled PCR libraries of each of the last four selection rounds as well

as six of the RNA aptamers from the last round of amplification were investigated to

determine specificity.

42

Chapter 2. Materials and Methods

2.1. WT1 plasmid constructs

cDNA encoding zinc fingers I-IV of WT1(-KTS), as well as cDNA encoding zinc

fingers I-IV of WT1(+KTS), cloned into pET16b (Novagen) were cloned previously by

Dr. Paul Romaniuk. pET16b encodes an N-terminal 6X His fusion tag.

2.2. Ciao-1 plasmid constructs

cDNA encoding full length Ciao-1 cloned into the expression vector pGEX-4T3 (

GE Healthcare) and the expression vector pET30a (Novagen) were cloned previously by

Dr. Paul Romaniuk. pGEX-4T3 encodes an N-terminal GST Fusion tag while pET30a

encodes a C-terminal 6X His fusion tag.

2.3. Clone Verification

2.3.1. DH5α Plasmid preparation

All uncharacterized clones were transformed into Escherichia coli DH5α using 1

to 50 ng of clone DNA. Competent DH5α cells (25 µL) were then mixed with 1 to 50 ng

of clone DNA aseptically, incubated on ice for 20 to 60 minutes, then the cell mixture

was heat shocked at 42 ºC for 90 seconds. This mixture was flash cooled on ice for one

to two minutes and then ten times the mixture volume of Luria Bertani (LB) broth was

aseptically added (LB: 10 g Tryptone, 5 g Yeast Extract, 10 g NaCl, 1 L dH2O ) (For 30

µL reaction add 300 µL of LB). The mixture was then allowed to recover for one hour at

37 ºC with shaking at 250 rpm. A volume of 150 to 200 µL of the transformed reaction

43

mixture was then plated onto a LB agar plate containing the appropriate antibiotic (100

µg/mL ampicillin or 50 µg/mL kanamycin) and the plates were incubated at 37 ºC

overnight.

One single colony was added to 5 mL of LB media with antibiotic for overnight

incubation at 37 ºC with 250 rpm shaking. Plasmids were then extracted and purified

using Qiaprep mini prep spin columns following the procedure outlined in the QIAquick

Miniprep Handbook (Qiagen,2002).

2.3.2. Sequencing for Clone Verification

All clones were verified by direct di-deoxy DNA sequencing by the Centre for

Biomedical Research sequencing facility at the University of Victoria.

2.4. Protein Expression and Purification

2.4.1 WT1 ZF(-KTS) and WT1 ZF(+KTS)

Plasmids were transformed into the E.coli strand BL21 DE3* as follows: 1 to 10

ng of WT1 ZF(-KTS) or WT1 ZF(+KTS) in pET16b was incubated with 25 µL of

competent E.coli BL21 DE3* cells on ice for 20-40 minutes. The mixture was then heat

shocked at 42 ºC for one to two minutes. A volume of 250 µL of LB media was added

aseptically prior to recovery at 37 ºC for one hour with agitation at 250 rpm.

The recovered mixture (100-200 µL) was plated onto LB agar with 100 µg/mL

ampicillin plates, which were then incubated for no less than 16 hours at 37 ºC. To begin

a culture, one of two methods was used: single colony or multiple colony expression. For

44

the single colony expression on the following day an overnight culture was started by

inoculating 20-100 mL of LB media (100 µg/mL ampicillin) with one colony, which was

subsequently incubated overnight at 37 ºC at 250 rpm. On the following morning, 1.5 to

6 L of LB media with 100 µg/mL ampicillin was inoculated with the overnight culture at

1/100 of the final volume (15 mL into 1.5 L).

In the case of multicolony expression, no overnight culture was required. Instead,

half to all of one freshly transformed plate of cells was used to inoculate a 1.5 L volume

of LB media with 100 µg/mL ampicillin. From this point on, the two methods converge.

Cultures were grown in 4 L flasks (maximum of 1.5 L / 4 L flask) to allow proper

aeration at 37 ºC with shaking at 250 rpm. Once the cultures obtained an O.D.600 of 1.0,

they were induced with a final concentration of 1 mM isopropyl-β-D-

thiogalactopyranoside (IPTG). After induction cells continued to incubate at 37 ºC with

shaking at 250 rpm for four hours, at which time the cells were harvested by

centrifugation at 5,000 rpm for ten minutes in a Beckman JA-16.250 rotor. Pellets were

pooled and resuspended in approximately 40 mL of Buffer A (10 mM TrisHCl pH 7.5, 5

mM MgCl2, 250 mM NaCl, 1 mM Dithiothreitol (DTT)) and then french pressed three

times to lyse the cells. After lysis cell debris was pelleted by centrifugation at 15,000

rpm in a Beckman JA-20 rotor for 45 minutes. The supernatant was discarded and the

pellet was purified using an inclusion body preparation.

The inclusion body preparation required the use of three buffers: Buffer A, Buffer

B (Buffer A with 0.1 % v/v Triton X-100) and Buffer C (BufferA with 6 M Urea and 10

mM Imidazole, no DTT). To begin the pellet was resuspended in ice cold Buffer B

(approximately 40 mL) then spun down at 15,000 rpm for ten minutes, the supernatant

45

was discarded, this wash step was repeated another two times. The pellet was washed

twice in 40 mL of Buffer A, again each wash was pelleted by centrifugation at 15,000

rpm for ten minutes and the supernatant was discarded. The washed pellet was then

resuspended in approximately 100 mL of Buffer C, this was placed in a larger flask with

a magnetic stir bar and allowed to stir overnight at room temperature.

The inclusion body prep was then pelleted at 15,000 rpm for 45 minutes, the

supernatant was retained and the pellet was discarded. The supernatant was divided into

two 50 mL falcon tubes with 2.5 mL of His-Select High Affinity matrix (Sigma) in each.

These mixtures were rotated at room temperature for one hour then applied to a 1.5 mm

column (Biorad). Once the resin had settled and the supernatant was collected, the resin

was washed with ten column volumes (CV) or 50 mL of Buffer C. The protein was

subsequently eluted off in 15 mL aliquots of a stepwise imidazole gradient, ranging from

15 to 500 mM imidazole made up in Buffer C (15 mM, 25 mM, 50 mM, 75 mM, 100

mM, 150 mM, 200 mM, 500mM imidazole). Each fraction was checked for purity and

size using 15 % Sodium-dodecyl-sulfate (SDS) polyacrylamide gel electrophoresis

(PAGE).

Fractions were pooled together and concentrated to approximately 4 mL using a

5,000 molecular weight (MW) cutoff membrane filter in a 100 mL Amicon device

(Millipore, USA) under 50 mPa of N2 gas pressure. Ethylenediamine tetraacetic acid

(EDTA) was added to the concentrated protein to a final concentration of 50 mM,

followed by incubation at room temperature for 30 minutes. The protein was then either

spun down at 13,500 rpm for five minutes in the microcentrifuge (Microlite RF, Thermo

IEC) or filtered using 0.2 µm filter to remove any precipitated protein. The size

46

exclusion column (SEC) (High Prep 16/60 Sephacryl S-200) was equilibrated with 1.5

CV of Buffer D (5 M Urea, 200 mM NaCl, 20 mM TrisHCl pH 7.5, 1 mM DTT). A

volume of 2 mL of the concentrated and filtered protein was loaded into a 2 mL sample

loop. Size exclusion chromatography was performed using the High Prep 16/60

Sephacryl S-200 column. The SEC was programmed as follows; 0.5 mL/min flow rate,

1.5 mL fractions, 3 mL injection for load, 150mL of Buffer D to elute. This program

took approximately two hours at room temperature. The size and purity of each eluted

fraction was determined using 15 % SDS PAGE gels, along with elution chromatograms.

Fractions were pooled and stored at 4 ºC until needed. Protein concentration was

determined by UV spectroscopy in 5M Urea at O.D.280 (NanoDrop ND-1000, NanoDrop

Technologies,USA)

2.4.2. GST-Ciao-1 from pGEX-4T3

Plasmid transformation was done as previously mentioned using a concentration

of 100 µg/mL ampicillin. A volume of 1 L of 2X YTA (16 g Tryptone, 10 g Yeast, 5 g

NaCl pH’d to 7.0 with NaOH) media was prepared and autoclaved before use. One

colony was picked off a freshly transformed E.coli BL21 DE3* plate and used to

inoculate a 10 mL volume of 2X YTA media with 100 µg/mL ampicillin for an overnight

culture. In the morning, two 500 mL volumes of 2X YTA media with 100 µg/mL

ampicillin in 2 L flasks was inoculated with 5 mL each of the overnight culture. Cultures

grew at 37 ºC at 250 rpm until an O.D.600 of 0.8-1.0 was achieved, at which time the

temperature was lowered to 25 ºC and the cultures were induced with 1 mM IPTG. The

cultures were induced for four to 16 hours and then harvested as stated previously. After

47

harvesting, cells were pooled and resuspended in 40 mL of phosphate buffered saline

solution (PBS) (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 pH 7.3)

and protease inhibitor cocktail (Roche Complete Mini Protease Inhibitor tablet).

Using a french pressure cell, cells were lysed and the lysate was incubated for 40

minutes at 10 ºC with slight rotation after the addition of 1 % v/v Triton X-100. The

lysate was then pelleted at 15,000 rpm for 45 minutes and the supernatant was retained.

This was incubated for one hour at 10 ºC with very little rotation (60 rpm) with 5 mL of

Sepharose/Glutathione resin (Amersham). After incubation the mixture was decanted

into a 1 cm diameter column (Biorad) and the supernatant was collected. The resin was

washed with approximately ten CV of PBS and the protein was eluted using 20 mL of 20

mM Glutathione solution (20 mM reduced glutathione, 50 mM Tris pH 8.0). The elution

fraction was concentrated using the 100 mL Amicon with a 5,000 MW cutoff filter

membrane (Millipore, USA) to approximately 2 mL, which was subsequently loaded

onto the SEC (High Prep 16/60 Sephacryl S-200 column, GE Healthcare) with a flow rate

of 0.5 mL/min and a fraction size of 1.5 mL.

2.4.3. Ciao-1 from pET30a

Plasmid transformations into E.coli BL21 DE3* (Invitrogen) were done the same

as previously mentioned for WT1, however, kanamycin was used in lieu of ampicillin in

the LB agar plates at a concentration of 50 µg/mL. Half a freshly transformed plate was

added to each 1.5 L of LB media with two times the yeast and 50 µg/mL kanamycin.

This was then agitated at 250 rpm at 37 ºC until an O.D.600 of 0.8 was obtained, at which

time the cultures were induced with 1 mM IPTG. After induction the temperature was

48

decreased to 25 ºC and the culture was allowed to grow for no longer than 16 hours with

agitation at 250 rpm. The culture was then harvested in the same manner as previously

mentioned and the pellets were pooled and resuspended in approximately 40 mL of

Binding Buffer (20 mM TrisBase pH 8.0, 500 mM NaCl).

Cells were lysed using one of two methods; pressure cell or chemical lysis. In the

case of chemical lysis, the pelleted cells were resuspended in 30 mL of a sucrose solution

(50 mM Tris Base pH 8, 25 % w/v sucrose) to this was added 10 mg of Lysozyme. The

solution was stirred until complete lysis was seen. Once all the cells were lysed 60 ml

(always two times the volume of the sucrose solution) of deoxycholate solution (1 % w/v

Sodium Deoxycholate, 1% v/v Triton X-100, 20 mM Tris pH 7.5, 100 mM NaCl) was

added. The solution continued to stir for ten minutes, at which time a final concentration

of 5 mM MgCl2 was added followed by 0.2 mg of DNAse I (Bovine Pancreas, Sigma).

This mixture was stirred until smooth and centrifuged at 15,000 rpm for 45 minutes to

pellet all cell debris.

The supernatant was loaded onto 1.5 mL of Ni-NTA matrix (Amersham) using a

1.0 mm flow adaptor/column (Biorad). Both the chemical and the mechanical lysis

protocols for the Ni-NTA resin were the same. The flow through was collected and the

resin was washed with approximately 33 CV (50 mL) of Binding Buffer. The protein

was eluted off using 15 mL aliquots of a stepwise imidazole gradient (5 mM, 40 mM, 75

mM, 150 mM, 300 mM imidazole). To determine size and purity of the fractions 10 %

SDS PAGE gels were run. Fractions from the column were pooled and fresh protease

inhibitor cocktail (Roche) was added.

49

The pooled fractions were concentrated to 2 mL using the Amicon with a 5,000

MW cutoff filter membrane (Millipore,USA). The SEC column (High Prep 16/60

Sephacryl S-200 column) was equilibrated with 20 mM Tris pH 8.0. The column was

run with a flow rate of 0.5 mL/min and a fraction volume of 1.5 mL. The fractions

containing UV absorbing material were run on a 10 % SDS PAGE gel and those that

showed the most purity were pooled and kept at 4ºC until needed. The concentration was

obtained using UV spectroscopy at an O.D.280.

2.5. Crystallization

Once protein was purified to as great an extent as possible, the protein would be

concentrated to somewhere between 5 and 20 mg/mL. The crystal drops were set up as

follows; 1 µL concentrated protein, 1 µL of well solution or 2 µL of each were added to a

cover slip and hung upside down over 500 µL of well solution. The well solution comes

from the crystal screens and always consists of a combination of the following three

components; salt, precipitant, and buffer. Each drop was sealed using vacuum grease.

Sitting drops were also used with the same ratios mentioned above, but in this case drops

were placed on a platform over 500 µL of well solution and sealed with crystal clear tape

(Hampton Research).

In general, all proteins were tested with three different screens: Crystal Screen I,

Crystal Screen II (commercially available through Hampton Research) and the PEG/ION

screen. The PEG/ION screen was made in the lab from chemicals obtained from Sigma

and based on the Hampton commercially available screen. Contents of each commercial

screen are shown in Tables i to iii of the appendix. If there was promising precipitate

50

(granular) or possible crystals obtained within the first screens, then one would note the

well solutions that produced these results and expand upon them. For example if there

was a hit with the following well solution; 50 % PEG 8000, 100 mM Tris pH 7.5, 200

mM CaCl2, then we would expand the PEG 8000 concentrations, exploit the pH of the

Tris buffer and/or the molarity, and lastly we would expand the CaCl2 concentrations. If

this resulted in better crystals, that were not quite diffraction grade, we would take the

best well solutions from the latest screens and include additives. Additives can be sugars,

alcohols, divalent metals or salts. They are known to perform a variety of functions such

as stabilization, solubilization, precipitation, cryo, and etcetera.

2.6. WTE and WT1 ZF(-KTS)

2.6.1. DNA preparation

Sequence of WTE DNA are as follows; WTE 5’ AGCG TGG GAG TGTT ,

WTE 3’ CGC ACC CTC ACA AT. AT overhangs are said to help stabilize the

complex. The above sequences were ordered (Operon) and once obtained were

resuspended in either 25 µL of dH2O or 1.1 X TMK buffer. Once resuspended, the

concentration of WTE5’was 92.06 mg/mL (21.04 mmol) and WTE3’was 95.46 mg/mL

(22.96 mmol). Both WTE5’ and WTE3’ were mixed together in equimolar amounts

and heated to 94 ºC for five minutes. The mixture was allowed to cool slowly at room

temperature.

51

2.6.2. Complexing WT1 ZF(-KTS) with WTE

For the addition of DNA to WT1 ZF (-KTS), the WT1 must be unfolded. WT1

ZF (-KTS) in Buffer D had a final concentration of 50 mM DTT added. An equimolar

ratio of DNA was added to 0.5 mg of WT1 ZF (-KTS). This mixture was placed in 1,000

MW cutoff dialysis tubing (Spectro/Por, FISHER) and slowly dialyzed until refolded into

Buffer E (5 mM DTT, 500 mM NaCl, 0.005 % v/v TWEEN 20, 10 % Glycerol, 5 mM

MgCl2, 0.5 mM ZnCl2, 50 mM Tris pH 7.5).

2.6.3. Native Gels to Verify WT1 ZF(-KTS) complex with DNA

Two 12.5 % Native gels were prepared and 35 µL of the refolded WT1 ZF (-

KTS) with or without DNA were mixed with 15 µL of 2X Native Sample Buffer ( 20 %

Glycerol, 0.004 % Bromophenol blue, 125 mM Tris pH 8.8). One native gel was run at

100 V for 55 minutes with the direction of migration towards the anode (+). The second

gel was run for 55 minutes at 100 V towards the cathode (-).

2.7. Gel Shift Assays

2.7.1. WT8 Preparation

WT8 was amplified from the pUC19 plasmid using the polymerase chain reaction

(PCR) and the following two primers; M13F 5’-CAC GAC GTT GTA AAA CGAC-3’

and M13R 5’-GGA TAA CAA TTT CAC ACA GG-3’ (Table 5). The 20 µL PCR

reaction contained the following reagents; 10 ng template (pUC19 with WT8), 6 pmoles

of M13F, 6 pmoles of M13R, 1 mM MgSO4, 2X Platinum Pfx amplification buffer

52

(Invitrogen), 0.5 mM dNTP’s, 0.5 µL of Platinum Pfx Polymerase (Invitrogen), and

dH2O up to 20 µL. The DNA was amplified in an Eppendorf Mastercycler epgradient

with an initial denaturation at 94 ºC for four minutes followed by 35 cycles of 30 seconds

at 94 ºC, 30 seconds at 45 ºC, and 60 seconds at 68 ºC. The PCR was concluded with a

four minute elongation step at 68 ºC. The WT8 product from this reaction was purified

using a QIAquick column (QIAGEN), following the PCR purification procedure outlined

in the QIAquick spin handbook (Qiagen,2002). The product was eluted in 60 µL of EB

Buffer (QIAGEN).

When labeling the DNA with the fluorescently labeled M13F probe (IRDyeTM

700 nm, LI-COR, USA) (Figure 9) the above reaction was used with the exception of the

labeled M13F primer. Labeled DNA was stored at 4 ºC in the dark.

2.7.2. Gel Shift with M13F Probe

All reactions were carried out in 10 µL volumes. Protein was diluted using 1.1X

Gel Shift Buffer (0.11 mg/mL poly dIC, 0.55 mM TCEP, 0.055 mg/mL BSA, 22 mM

Tris pH 7.5, 5.5 mM MgCl2, 110 mM KCl, 11 µM ZnCl2, 11 % v/v Glycerol). Once

protein was properly diluted, 10 ng of probe was added to each reaction. The reactions

were incubated at room temperature for 90 minutes in the dark. The entire reaction was

electrophoresed on a gel (0.5X TBE, 10 % polyacrylamide) for 70 minutes at 100 V and

4 ºC in ice water using 1X TBE running buffer. Gels were visualized on the LI-COR

Odyssey Imager (LI-COR, USA) at 700 nm.

53

2.7.3. Western of Gel Shift

The 0.5X TBE, 10 % polyacrylamide gel was transferred onto 0.45 µm

nitrocellulose membrane using an XBLOT cell (Invitrogen). The transfer buffer

contained 20 % methanol, 192 mM Glycine, 150 mM NaCl) and the transfer was

completed after one hour at 350 mA. The membrane was rinsed for five minutes in Tris

buffer solution (TBS: 20 mM Tris pH 7.6, 150 mM NaCl). Blocking was carried out

overnight at 15 °C in TBS with 0.5 % w/v non-fat dairy milk (NFDM). The membrane

was washed with TBST (TBS with 0.1 % v/v TWEEN 20) then incubated in the primary

antibody (either mouse anti histag, rabbit anti GST (Cell Signalling Technologies), or

rabbit anti WT1 ZF (Santa Cruz Biotechnologies Inc., USA) ) at a 1 : 5,000 dilution in

blocking overnight at 15 ºC with shaking (60 rpm). The membrane was washed in TBST

and subsequently incubated for one hour at 15 ºC with the secondary antibody (either

goat anti mouse HRP or goat α rabbit HRP) (Amersham) at a 1 : 5,000 dilution. After

rinsing with TBST a volume of 2 mL of Luminol reagent (Western Blotting Luminol

Reagent, Santa Cruz Biotechnology Inc., USA) was added, this was left to incubate in the

dark at room temperature for five minutes. The membrane was wrapped in plastic and

exposed to film, which was subsequently developed on a Kodak X-OMAT 2000A

Processor. To strip the membrane before probing with the second primary antibody, the

membrane was incubated for 30 minutes at 50 ºC in a stripping buffer (62.5 mM Tris pH

7.0, 100 mM β-mercaptoethanol, 2 % SDS). After stripping the membrane was

excessively washed with dH2O.

54

Table 5. Primers used in this study Primers used in this study in the PCR amplification of WT8 DNA with or without Probe as well as primers used in the amplification of SELEX libraries for Filter Binding Assays.

Primer Sequence Amplicon M13F 5’-CAC GAC GTT GTA AAA

CGAC-3’ M13R 5’-GGA TAA CAA TTT CAC

ACA GG-3’

WT8 in pUC19

M13Fprobe 5’**-CAC GAC GTT GTA AAA

CGAC-3’ M13R 5’-GGA TAA CAA TTT CAC

ACA GG-3’

WT8 with LICOR 700 probe

T7downstream primer 5’-GCG AAT TCT AAT ACG

ACT CAC TAT AGG GAG AGG ATA CAT CAC GTG-3’

T7upstream primer 5’-CGG AAG CTT CTG CTA

CAT GCA ATG G-3’

Amplification of SELEX PCR pools

** LICOR 700nm probe (See Figure 9)

55

C74H96N12Na4O27S6Si3 Mol. Wt.: 1954.22382

Figure 9. LICOR 700DX IRDye LICOR 700DX IRDye for fluorescently labeling commonly used in DNA sequencing reactions.

56

2.8. Filter Binding Assays

2.8.1. Preparation of RNA Selection library

The RNA selection libraries were prepared by Dr. Gary Zhai. They were

prepared according to the published method of Affinity Selection (Klug and

Famulok,1994).

2.8.2. Preparation of PCR pools from RNA Selection libraries

Pooled libraries were PCR’d using the original primers; T7downstream primer 5’-

GCG AAT TCT AAT ACG ACT CAC TAT AGG GAG AGG ATA CAT CAC GTG-3’

and the reverse T7upstream primer 5’-CGG AAG CTT CTG CTA CAT GCA ATG G-

3’(Table 5). PCRs were done by Dr. Paul Romaniuk.

2.8.3. RNA Preparation. In vitro RNA transcription and radiolabeling by run-off

transcription

Pel22 RNA and 5S RNA were the controls used in the filter binding assays

(FBA). They were transcribed with radiolabeling using the MAXIscript In Vitro

Transcription Kit (Ambion). The 5S RNA template DNA was contained in the pUC18

plasmid, the 5S gene was cloned downstream of the T7 promoter (Romaniuk et al.,1987)

and was linearized just downstream of the T7 promoter. The pUC18 plasmid containing

the 5S RNA DNA was linearized using DraI (pUC18 with pel22 required cleavage with

RsaI).

57

A full reaction of 20 µL was used, this reaction contained 1 µg of linearized

template (5S or pel22) DNA, 1X transcription buffer (Ambion), 0.5 mM of each

ribonucleotide (ATP, CTP, GTP), 0.025 mM UTP, 2 µL of α-32P UTP (800 Ci/mmol,

Amersham), and 2 µL of T7 enzyme mix (Ambion) containing the RNA polymerase.

For the pooled PCR RNA libraries, half reactions were done. The reactions were

incubated at 20 ºC for one hour. Following the incubation 20 µL of 2X gel loading buffer

(Ambion) was added to each reaction prior to gel loading. Each reaction was loaded onto

a Urea/polyacrylamide gel (8 % polyacrylamide, 8.3 M Urea, 1X TBE) that had been pre

run at 400 V and 20 ºC for 30 minutes in 1X TBE running buffer. Once samples were

loaded the gel was electrophoresed at 400 V for one hour at 20 ºC. The gel was exposed

to film (BIOMAX MR film, KODAK) for two to three minutes and developed on a

Kodak X-OMAT 2000A Processor (Kodak). Corresponding radiolabeled bands were cut

out and eluted overnight in 250 µL of RNA elution buffer (1 mM EDTA, 0.6 M

NH4OAc, 0.1 % SDS). The RNA was precipitated out of solution by the addition of 625

µL of ice cold 95 % Ethanol and 1.5 µg of glycogen. This was incubated at –70 ºC for

one hour. To pellet the RNA, samples were spun at Vmax (approximately 13,500 rpm) in

a microcentrifuge. Supernatant was disposed of in liquid radioactive waste, and the pellet

was dried using a Savant speed vacuum concentrator for two hours at 20 ºC. Once the

RNA was thoroughly dried, the full reactions were resuspended in 100 µL each of

nuclease free water (Gibco). Half reactions were resuspended in 50 µL each. The

radioactivity of the RNA preparations was determined using a 1214 Rackbeta liquid

scintillation counter in counts per minute. RNA was aliquoted into 500,000 cpm per tube

and stored at – 20 ºC (for no more than two weeks) until needed.

58

2.8.4. Nitrocellulose filter binding assay

All filter binding assays were done using 12-point dilution series ranging from 0

to 720 nM of protein in a final volume of 90 µL. The two proteins used in all the FBA’s

were WT1 ZF (-KTS) and WT1 ZF (+KTS). All protein dilutions were carried out in

1.1X TMK buffer (20 mM Tris, 100 mM KCl, 100 µg/mL BSA, 0.5 mM TCEP, 5 µM

ZnCl2, 5 mM MgCl2). Once the dilutions were prepared, 32P radiolabeled RNA with 0.01

mg/mL polydIC was added to each dilution at a concentration of approximately 5,000

cpm/reaction. To the 90 µL of protein dilution, 10 µL of RNA was added and each

reaction was allowed to equilibrate for 90 minutes at 20 ºC. After incubation, 80 µL of

each dilution reaction was vacuum filtered onto a pre-soaked 0.45 µm nitrocellulose

membrane (Whatman) using a dot-blot apparatus. The filter was dried at 50 ºC for ten

minutes, covered in plastic wrap and exposed overnight at room temperature to a

phosphor screen (Molecular Dynamics, USA). The phosphor screen was visualized and

assessed using the STORM 820 imaging system (Molecular Dynamics, USA).

59

Chapter 3. Results

3.1. Protein Purification

3.1.1. WT1 Zinc Finger Domain Peptides

In order to determine the optimum conditions for production of the large amounts

of protein required for crystallization, induction trials using 5 mL cultures of transformed

E. coli BL21 DE3* were conducted at 25, 30 and 37 °C. When the cell density reached an

O.D.600 between 0.67 to 0.85, protein expression from the pET16b constructs was

induced by the addition of 1 mM IPTG to the cultures. After a further two hour

incubation, cells were harvested, lysed, and separated into soluble and insoluble fractions.

Analysis of the fractions from each incubation temperature on a 15 % SDS-PAGE

demonstrated that the best yield of WT1-ZF protein was found in the insoluble fraction of

the 37 °C incubation (Figure 10).

Based upon these results, large scale protein production and purification was

carried out using 3-6 L cultures grown at 37 °C. Once the cell culture reached a density of

O.D.600 of 0.8, protein expression was induced by addition of 1 mM IPTG and the culture

was grown for a further four hours. After the cells were harvested, inclusion bodies were

prepared, solubilized in a buffer containing 6 M urea and batch purified using His-Select

resin (Sigma). The –KTS isoform of WT1-ZF eluted from the column with buffers

containing 5 M urea and a step gradient of 25 to 75 mM imidazole (Figure 11). The

+KTS isoform of WT1-ZF was purified in the same fashion and eluted from the column

with a step gradient of 15 to 200 mM imidazole (Figure 12).

Further purification was required. Fractions from the His-Select column were

pooled, concentrated and then applied to a size exclusion column (SEC). The column was

60

Figure 10. Expression Trial of WT1 ZF(-KTS). 15 % (4 % Stacking) SDS PAGE run at 200 V for 55 minutes. 20 µL of non-induced and induced pellet and supernatant samples were run at 10 OD600 / mL. Three temperatures were tested in the expression trials; 25, 30, and 37 °C. Arrow indicated WT1 ZF(-KTS) protein. Gel was stained with Coomassie Blue.

61

Figure 11. WT1 ZF(-KTS) purification on Ni-NTA resin. 15 % (4 % Stacking) SDS PAGE run at 200 V for 55 minutes. 20 µL of each imidazole fraction was loaded. Gel was stained with Coomassie Blue.

62

Figure 12. WT1 ZF(+KTS) purification on Ni-NTA resin. 15 % (4 % Stacking) SDS PAGE run at 200 V for 55 minutes. 20 µL of each imidazole fraction was heated then loaded onto gel. Gel was stained with Coomassie Blue.

63

eluted with a buffer containing 5 M urea and 200 mM NaCl. Fractions of 1.5 ml were

collected and those containing UV absorbing material were analyzed by 15 % SDS-

PAGE (Figures 13, 14). Fractions C4-C11 from the WT1-ZF (–KTS) purification were

pooled (Figure 13), as were fractions C5 to C9 of the WT1-ZF (+KTS) purification

(Figure 14).

Pooled protein fractions were supplemented with 1 mM DTT and stored at 4 °C.

The concentration of protein in each preparation was determined by UV spectroscopy in

elution buffer and was typically 1 mg/ml (50 µM). Final yields of purified protein ranged

from 3 to 9 mg/L of starting culture.

The apparent size of WT1-ZF(–KTS)/(+KTS) was 19 kDa when run on a 15 %

SDS-PAGE, while the actual predicted size based on its amino acid content was just over

20 kDa.

3.1.1.1. Refolding of WT1 Zinc Finger Domain Peptides

To refold WT1 ZF (-KTS) for crystallization trials the purified protein was slowly

dialyzed into Refolding Buffer A (10 mM Tris pH 6.0, 25 mM NaCl, 5 mM MgSO4, 2

mM KCl, 2 mM ZnCl2), which resulted in a negligible loss of protein. A lower pH of 6.0

was used to prevent the ZnCl2 from precipitating, as precipitation resulted in a significant

loss of protein. Before the crystallization trials commenced the protein was subsequently

dialyzed into Refolding Buffer B (Refolding Buffer A without ZnCl2) and then into either

dH2O or Refolding Buffer C (10 mM Tris pH 7.0, 25 mM NaCl, 5 mM MgSO4, 2 mM

KCl).

64

Figure 13. Gel filtration purification of WT1 ZF(-KTS).

b)

a)

a) SEC column was run at 0.5 mL/min with 1.5 mL fractions in 5 M Urea, 200 mM NaCl. b) Fractions showing UV absorbance were run on a 15 % (4 % Stacking) SDS PAGE at 200 V for 55 minutes. 20 µL of each fraction was heated then loaded onto gel. Gel was stained with Coomassie Blue.

65

b)

a)

Figure 14. Gel filtration purification of WT1 ZF (+KTS). a) SEC column was run at 0.5 mL/min with 1.5 mL fractions in 5 M Urea, 200 mM NaCl. b) Fractions showing UV absorbance were run on a 15 % (4 % Stacking) SDS PAGE at 200 V for 55 minutes. 20 µL of each fraction was heated then loaded onto gel. Gel was stained with Coomassie Blue

66

3.2. Forming a WTE-WT1 Zinc Finger Domain Peptide Complex

The addition of WTE to any concentration of refolded WT1 ZF (-KTS) resulted in

complete precipitation. Based on these findings WTE was added to the unfolded purified

protein and the two were refolded together in the following buffer; 5 mM DTT, 500 mM

NaCl, 0.005 % v/v TWEEN 20, 10 % v/v Glycerol, 5 mM MgCl2, 0.5 mM ZnCl2, and 50

mM Tris pH 7.5. When complexed in this manner no loss of WT1 was noted.

To ensure that a complex was in fact formed two native gels were run using

polyacylamide gel electrophoresis. One gel was run cathode (-) to anode (+), while the

second gel was run in the reverse direction. As can be seen from the two native gels

(Figure 15), the uncomplexed WT1 ZF (-KTS) contains an overall positive intrinsic

charge and migrates towards the cathode (-), whereas the complexed WT1 ZF (-KTS)

/WTE has an overall negative charge and thus migrates towards the anode (+).

3.3. Crystallization of WTE - WT1 ZF Complex

Numerous attempts to crystallize the WTE/WT1 ZF (-KTS) complex were

unsuccessful. The crystal screens utilized in this study were as follows: Crystal Screen I

(Hamptons Research), Crystal Screen II (Hamptons Research), and CSSI with Bis-Tris

pH 7.2. A comparison of freshly purified protein vs. old protein, both at concentrations

of 8 mg/mL protein with a 1:1 stoichiometry of protein:DNA, was also investigated. No

promising precipitate or possible crystals were seen in any of the screens. All crystal

trays were set up with 1 µL: 1 µL drops of protein/DNA complex : well solution using

both the sitting drop and hanging drop method.

67

Figure 15. Migration of WT1 ZF(-KTS) refolded with and without WTE DNA. 12.5 % Native polyacrylamide gels run at 100 V for 50 minutes. Stained using Coomassie blue. Right: Native gel run from Cathode to Anode. Left: Native gel run from Anode to Cathode.

68

3.4. Protein Purification of Ciao-1

3.4.1. Purification of GST-Ciao-1

Large scale production of GST-Ciao-1 proved difficult. High quantities of Ciao-1

appeared to have an affinity for GST and there was substantial degradation of the GST-

Ciao-1 into its parts. Unfortunately due to this inherent affinity between the two proteins

it was not possible to achieve the desired separation. Based on these results a construct

of Ciao-1 in pET30a with a C-terminal His tag was used to produce recombinant protein.

3.4.2. Purification of pET30a Histag-Ciao-1

In order to determine the optimum conditions for production of the large amounts

of protein required for crystallization, induction trials using 5 mL cultures of transformed

E. coli BL21 DE3* were conducted using the same growth conditions as the GST-Ciao-1

construct. The cells were grown at 37 °C until the cell density reached an O.D.600 of 0.8.

Protein expression from the pET30a construct was induced by the addition of 1 mM

IPTG to the cultures. Upon induction, the temperature was dropped to 25 °C and the

cells were incubated 8 hours. Cells were harvested, lysed, and separated into soluble and

insoluble fractions. Analysis of the fractions from a 15 % SDS-PAGE demonstrated that

there was good yield of Ciao-1 protein in the soluble fraction.

Large scale protein production and purification was carried out using 3 L cultures

grown at 37 °C. After the cells were harvested and lysed, the lysate was purified using

His-Select resin (Sigma). The protein was eluted from the column with buffers

containing a step gradient of 40 to 75 mM imidazole (Figure 16).

69

Figure 16. Ni-NTA column purification of Ciao-1 with c-terminal his tag. 10 % (4 % Stacking) SDS PAGE run at 200 V for 55 minutes. 20 µL of each imidazole fraction was heated then loaded onto gel. Gel was stained with Coomassie Blue.

70

As can be seen from the SDS-PAGE of column fractions from the purification,

the Ciao-1 protein is not homogeneous and further purification was required. Fractions

from the His-Select column were pooled, concentrated to 2 mL and applied to a size

exclusion column (SEC). The column was eluted with a buffer containing 20 mM Tris

pH 8. Fractions of 1.5 mL were collected and those containing UV absorbing material

were analyzed by 10 % SDS-PAGE (Figure 17a). Fractions C9 to D9 were pooled

(Figure 17b).

Pooled protein fractions were stored at 4 °C. The concentration of protein in each

preparation was determined by UV spectroscopy in elution buffer and was typically 2.5

mg/mL. Yields of purified protein were typically 8 mg/L of starting culture.

The apparent size of Ciao-1 was 44 kDa when run of a 10 % SDS-PAGE, while

the actual predicted size based on its amino acid content was just over 39 kDa. Although

it is usually desired to cleave off any affinity tags prior to crystallization the Ciao-1

protein proved unstable and quickly decayed without the tag (Figure 18).

3.5. Crystallization of Ciao-1

All the crystallization trials were set up with an 8 mg/mL concentration of Ciao-1,

as there was severe precipitation above this point. Drops were set up with 1 µL of

protein and 1 µL of well solution in sitting drop trays. The following crystal screens

were used; Crystal Screen I (Hampton Research), Crystal Screen II (Hampton Research),

71

b)

a)

Figure 17. Gel filtration purification of Ciao-1 with c-terminal his tag. a) SEC column run at 0.5 mL/min in 20 mM Tris pH 8.0, fractions were eluted at 1.5 mL. b) SEC fractions were then run on a 10 % (4 % Stacking) SDS PAGE at 200 V for 55 minutes. 20 µL of each fraction was heated then loaded onto gel. Gel stained with Coomassie Blue.

72

Ciao-1 cleaved

Ciao-1 at R.T.

Ciao-1 at 4 °C

MW

kDa

175

83

62

47.5

32.5

Figure 18. Ciao-1 his tag cleavage. 10 % SDS PAGE gel of the cleavage of Ciao-1 with C-terminal his tag. Cleaved at room temperature overnight with Enterokinase (Sigma). The two controls included the Ciao-1 protein at room temperature overnight in the cleavage buffer as well as the Ciao-1 stored at 4°. Gel was stained with Coomassie Blue.

73

PEG/ION screen (Sigma reagents). The following well conditions gave possible crystals;

30 % PEG 1,500 as well as 10 % PEG 1,000 with 10 % PEG 8,000. These conditions

were then optimized in the following fashion. Both sitting and hanging drop trays were

set up with varying percentages (10 – 40%) of PEG 1,000, PEG 2,000, PEG 3,350, PEG

1,000/8,000, PEG 5,000, PEG 6,000, PEG 8,000, and PEG 10,000. The best

crystallization conditions (20 % PEG 3,350 and 30% PEG 8,000) were further optimized

using an additive screen (see Table iv in Appendix). After optimization the crystals were

found to reside only in the protein skin and were not sturdy enough to remove without

damaging. Due to these findings no further examinations were done.

3.6. Co-Crystallization of Ciao-1 and WT1 ZF(-KTS) or (+KTS)

Many efforts were made to combine these two proteins, which had been

previously shown to partner in vivo (Johnstone et al.,1998; Johnstone et al.,1999). Both

proteins were dialyzed into the same buffer (20 mM Tris pH 8.0 or Buffer C) and titrated

together at various concentrations ranging from 1 to 50 μM. We were never able to

achieve a homogenous mixture, the proteins always precipitated upon combination.

Placing the two together in 5 M urea solution was also attempted, but each protein

required different pH units to refold, so they could not be refolded together.

3.7. Filter Binding Assay

The fundamental theory behind filter binding assays is the fact that nitrocellulose

membrane retains protein while nucleic acids (RNA/DNA) will pass through unless

bound by protein. Hence when using radiolabeled RNA or DNA, the radiation seen on

74

the membrane can only be attributed to that which is bound by its protein binding partner.

Binding affinities for the different RNA aptamers and selection pools was determined by

fitting the FBA data to the following simple bimolecular equilibrium equation.

Kd = [Protein]Free [RNA]Free

[Protein· RNA]complex

In this case, [Protein]Free = [Protein]Total as the amount bound by the RNA is negligible

when compared to free protein concentration.

Two assumptions must be made when performing FBAs. 1) There is always

incomplete retention of RNA on nitrocellulose, to normalize all data we must then

assume that the fraction of RNA bound at the plateau of the binding curve is 1.0 for each

binding experiment. 2) All the protein used in the reaction is assumed 100% active.

Equilibrium binding curves were created using Kaleidagraph software (Synergy

Software, Reading, PA) by plotting the fraction of RNA bound on the y-axis vs the total

protein concentration on the x-axis. For binding curves with more than one trial standard

deviations were plotted.

Fraction of RNA bound = [Protein]Total / (Kd + [Protein]Total).

Hence where 50 % of the RNA is bound on the generated curve Kd is equal to the

protein concentration.

75

3.7.1. Quantitative filter binding of WT1 ZF (-KTS) and (+KTS) with the RNA aptamer Pel22 and 5S rRNA

The equilibrium binding of the zinc finger domains of the two WT1 isoforms

(WT1 ZF (-KTS),WT1 ZF (+KTS)) to the RNA aptamer Pel22 as well as to 5S rRNA

were measured using a nitrocellulose binding assay (Romaniuk, 1985; Romaniuk, 1990).

WT1 ZF(-KTS) and (+KTS) both bind to a consensus binding region within the

Pel22 sequence (Figure 19). Published results (Zhai et al.,2001) indicate that the Kd

values for WT1 ZF (-KTS) and WT1 ZF(+KTS) with Pel22 are 13.8 nM and 22.8 nM,

respectively. The RNA aptamer Pel22 was thus used as the positive control for the

subsequent filter binding assays.

5S rRNA was the negative control used for these FBAs as neither isoform has

been shown to demonstrate any significant affinity (WT1 ZF (-KTS) Kd = >1000 nM,

WT1 ZF (+KTS) Kd = >1000 nM) (Zhai et al.,2001).

The binding curves from three or more independent experiments for Pel22 and 5S

rRNA with (-KTS) and (+KTS) are shown in Figure 19. These results are in agreement

with the published results stated above in that the Kd values for WT1 ZF (-KTS) and

WT1 ZF(+KTS) with Pel22 are 10.0 nM and 20.3 nM respectively. The results shown

for the 5S rRNA in Figure 19 were also in agreement with published results as the Kd

values were determined to be >1000 nM for both the (-KTS) and the (+KTS) WT1 ZF

isoform.

76

[WT1(-KTS)], M [WT1(+KTS)], M

76

Figure 19. Filter binding assay controls. Top left: Sequence of pel22 RNA (Zhai et al.,2001) used in Filter binding assay. Consensus sequence: GAUAUGGUG. Top right: Secondary structure of pel22 as deduced by mfold version 3.2 (Mathews et al.,1999; Zuker,2003). Bottom: Binding curves of both WT1 ZF(-KTS) [Right] and WT1 ZF(+KTS) [Left] with controls were generated by Kaleidagraph. Blue: Pel22, Red: 5S RNA. Each point denotes the mean of three independent experiments; error bars represent standard deviation of the mean calculation.

77

Both the pel22 and 5S RNA were examined alongside each subsequent binding

assay to ensure that the conditions were ideal for complex formation, however to make

the figures less confusing they were not shown.

3.7.2. Quantitative filter binding of WT1 ZF (-KTS) and (+KTS) with the SELEX pooled PCR libraries

The binding curves from three or more independent experiments for four of the

generated SELEX libraries with WT1 ZF (-KTS) and WT1 ZF (+KTS) are shown in

Figure 20. The Kd values of WT1 ZF (-KTS) for the SELEX amplification rounds were

69(±21) nM (round 15), 0.95(±0.20) nM (round 17), 0.75(±0.020) nM (round 19), and

360(±73) nM (round 20). The Kd values of WT1 ZF(+KTS) for the SELEX

amplification rounds were 88(±27) nM (round 15), 0.80(±0.30) nM (round 17),

0.67(±0.080) nM (round 19), and 160(±41) nM (round 20) (Figure 20, Table 6). These

results are as expected in that there is a trend seen where the latter libraries have higher

affinities with the exception of the 20th round of selection. The selection that we were

expecting to see was not statistically significant in rounds 15 to 19. When taking the

standard deviation into account the Kd values for the two isoforms overlapped in these

three rounds of selection. The SELEX libraries were created in the hopes of introducing

a preference for the WT1(+KTS) isoform over the (-KTS) isoform. The 20th round of

selection did show a 1.4X affinity for the (+KTS) isoform that was significant, in that

there was no overlap of standard deviation. To further investigate this lack of expected

selection six aptamers were tested from the 20th round of amplification.

78

3.7.3. Quantitative filter binding of WT1 ZF (-KTS) and (+KTS) with select aptamers from the 20th round of selection The binding curves from three independent experiments (unless otherwise stated)

for the RNA aptamers 2, 26, 29, 48, 58, and 61 with WT1 ZF (-KTS) and (+KTS) are

shown in Figures 21 to 26. The Kd values for WT1 ZF(-KTS) with RNA aptamers 3, 26,

29, 48, 58, and 61 were determined to be 510 nM, 2.3 (±0.50) nM, >1000 nM, 96(±26)

nM, 180 nM, and 580 nM respectively (Figure 21 to 26, Table 6). For the WT1 ZF

(+KTS) isoform with the RNA aptamers 3, 26, 29, 48, 58, and 61 the Kd values were

shown to be 200 nM, 18(±0.92) nM, 360 nM, 580(±180) nM, 67 nM, and 250 nM,

respectively (Figure 21 to 26, Table 6). In general all the aptamers tested (with the

exception of 26, and 48) showed a slightly greater affinity for the +KTS variant, but the

difference was not large and could not be statistically analyzed as these four aptamers (3,

29, 58, 61) were only tested in one independent experiment. The two aptamers that were

tested in three or more independent experiments showed a significant preference for the

(-KTS) isoform. The Kd values determined from the mean of the independent

experiments, for aptamers 26 and 48, showed no overlap when taking standard deviation

into account.

79

Figure 19. Binding assay comparison of the SELEX libraries 15, 17, 19 and 20. Top left: Binding curve of SELEX round 15 plotted using Kaleidagraph. Top right: Binding curve of SELEX round 17 plotted using Kaleidagraph. Bottom left: Binding curve of SELEX round 19 plotted using Kaleidagraph. Bottom Right: Binding curve of SELEX round 20 plotted using Kaleidagraph. Blue: WT1 ZF(-KTS), Red: WT1 ZF(+KTS). Each point denotes the mean of three independent experiments; error bars represent standard deviation of the mean calculation. Table 6. WT1 ZF isomer comparison of RNA aptamer binding affinities. Kd values for the various RNA aptamers tested from SELEX round 20 as well as the pooled previous SELEX rounds. Values were determined using nitrocellulose filter binding assays and data was analyzed using Kaleidagraph. Standard deviation is shown for those binding studies whose mean was determined from three or more independent Tri Figure 20. Binding assay comparison of the SELEX libraries 15, 17, 19 and 20.

[Protein], M [Protein], M

[Protein], M [Protein], M

Top left: Binding curve of SELEX round 15 plotted using Kaleidagraph. Top right: Binding curve of SELEX round 17 plotted using Kaleidagraph. Bottom left: Binding curve of SELEX round 19 plotted using Kaleidagraph. Bottom Right: Binding curve of SELEX round 20 plotted using Kaleidagraph. Blue: WT1 ZF(-KTS), Red: WT1 ZF(+KTS). Each point denotes the mean of three independent experiments; error bars represent standard deviation of the mean calculation.

80

Table 6. WT1 ZF isomer comparison of RNA aptamer binding affinities. Kd values for the various RNA aptamers tested from SELEX round 20 as well as the pooled previous SELEX rounds. Values were determined using nitrocellulose filter binding assays and data was analyzed using Kaleidagraph. Standard deviation is shown for those binding studies whose mean was determined from three or more independent trials.

RNA WT1 ZF (+KTS) (M) WT1 ZF (-KTS) (M) 5S >10-6 >10-6 pel22 2.0 (±0.36) x10-8 1.0 (±0.08) x10-8 SELEX Round 15 8.8 (±2.7) x10-8 6.9 (±2.1) x10-8 SELEX Round 17 8.0 (±3.0) x10-10 9.5 (±2.0) x10-10 SELEX Round 19 6.7 (±0.80) x10-10 7.5 (±0.20) x10-10 SELEX Round 20 1.6 (±0.41) x10-7 3.6 (±0.73) x10-7 3 2.0 x10-7 5.1 x10-7

26 1.7 (±0.92) x10-8 2.3 (±0.50) x10-9 29 3.6 x10-7 >10-6 48 5.8 (±1.8) x10-7 9.6 (±2.6) x10-8 58 6.6 x10-8 1.8 x10-7

61 2.5 x10-7

5.8 x10-7

81

[Protein], M

Figure 21. Binding assay for RNA aptamer 3 from the 20th SELEX library. Top left: Sequence of RNA used in Filter binding assay. Top right: Secondary structure of aptamer 3 as deduced by mfold version 3.2 (Mathews et al.,1999; Zuker,2003). Bottom left: Binding curve generated by Kaleidagraph. Blue: WT1 ZF(-KTS), Red: WT1 ZF(+KTS). Each point denotes one independent experiment; no standard deviation was calculation.

82

[Protein], M Figure 22. Binding assay for RNA aptamer 26 from the 20th SELEX library. Top left: Sequence of RNA used in Filter binding assay. Top right: Secondary structure of aptamer 26 as deduced by mfold version 3.2 (Mathews et al.,1999; Zuker,2003). Bottom left: Binding curve generated by Kaleidagraph Blue: WT1 ZF(-KTS), Red: WT1 ZF(+KTS). Each point denotes the mean of three independent experiments; error bars represent standard deviation of the mean calculation.

83

Figure 23. Binding assay for RNA aptamer 29 from the 20th SELEX library.

[Protein], M

Top left: Sequence of RNA used in Filter binding assay. Top right: Secondary structure of aptamer 29 as deduced by mfold version 3.2 (Mathews et al.,1999; Zuker,2003). Bottom left: Binding curve generated by Kaleidagraph Blue: WT1 ZF(-KTS), Red: WT1 ZF(+-KTS). Each point denotes one independent experiment; no standard deviation was calculation.

84

[Protein], M

Figure 24. Binding assay for RNA aptamer 48 from the 20th SELEX library. Top left: Sequence of RNA used in Filter binding assay. Top right: Secondary structure of aptamer 48 as deduced by mfold version 3.2 (Mathews et al.,1999; Zuker,2003). Bottom left: Binding curve generated by Kaleidagraph Blue: WT1 ZF(-KTS), Red: WT1 ZF(+KTS). Each point denotes the mean of three independent experiments; error bars represent standard deviation of the mean calculation.

85

Figure 25. Binding assay for RNA Aptamer 58 from the 20th SELEX library.

[Protein], M

Top left: Sequence of RNA used in Filter binding assay. Top right: Secondary structure of aptamer 58 as deduced by mfold version 3.2 (Mathews et al.,1999; Zuker,2003). Bottom left: Binding curve generated by Kaleidagraph Blue: WT1 ZF(+KTS), Red: WT1 ZF(-KTS). Each point denotes one independent experiment; no standard deviation was calculation.

86

[Protein], M

Figure 26. Binding assay for RNA aptamer 61 from the 20th SELEX library. Top left: Sequence of RNA used in Filter binding assay. Top right: Secondary structure of aptamer 61 as deduced by mfold version 3.2 (Mathews et al.,1999; Zuker,2003). Bottom left: Binding curve generated by Kaleidagraph Blue: WT1 ZF(-KTS), Red: WT1 ZF(+KTS). Only one trial was run so no standard deviation was calculated.

87

Chapter 4. Discussion

4.1. WT1

4.1.1. Purification of WT1 ZF isoforms

In this study we were able develop a protocol for a simple large scale purification

of WT1 ZF(-KTS) and (+KTS) which may prove useful for future structural studies.

There was some concern over the large amounts of degradation seen when

purified WT1 ZF was run on SDS PAGE gels (Figure 11, 12). It was postulated that

perhaps the protein was simply unstable, however when the samples were not heated

before loading onto a denaturing SDS gel no deterioration was seen (data not shown).

This would seem to indicate that the degradation seen in the SDS PAGE gel was a

byproduct of heating the protein. The purified protein also proved to be quite stable

when kept at 4 °C in 5 M urea and 200 mM NaCl. Protein stored in this fashion was just

as effective in the FBAs, if not slightly more so, than that of a frozen aliquot of the same

protein.

4.1.2. Crystallization of WT1

It has proven quite complex in the past to crystallize other zinc finger proteins as

they have a tendency to oxidize. As a result, an inert atmosphere would be preferred.

Most zinc finger studies are done using NMR, as this method has the advantage of

keeping the protein in argon saturated solutions. One other reason that the apo-WT1 ZF

might not have crystallized is the flexibility of the protein in solution. There is a flexible

linker region between each of the four zinc fingers, and in solution without substrate or

88

binding partner, these four fingers are free to move. This freedom of movement may not

be conducive to symmetric crystal formation. Using the full-length protein may lend

apo-WT1 some structural stability in future crystallization trials. To date, producing

large amounts of full-length WT1 has proved elusive, as the latest published article on

such showed disappointing yields (Geng and Carstens,2006).

The protein used in this study was prepared from inclusion bodies, and as such

there is a chance that a small percentage of the protein was folded improperly. While a

small amount of unfolded WT1 would not affect the overall activity of the sample, to any

noticeable amount, in a FBA, it could result in unstable crystallization conditions.

4.1.2.1. Co-Crystallization of WT1 ZF with Ciao-1

For the co-crystallization of WT1 ZF with Ciao-1, the main obstacle proved to be

combining the two proteins in solution. DTT was used to maintain a reducing

environment throughout this study. DTT is known to chelate metals and may be

interfering with the coordination of zinc by the histidines and cysteines in the zinc

fingers. If this is the case, argon saturated buffers would allow the removal of DTT,

which may provide a more stable ZF protein to interact with Ciao-1.

4.1.2.2. Co-Crystallization of WT1 ZF with WTE

In the case of the WTE DNA and WT1 ZF protein we were able to combine the

two in an unfolded state. By using small enough dialysis tubing (1,000 MW) to ensure

that the DNA oligos would not permeate, the 1:1 solution was slowly dialyzed into the

WT1 ZF refolding solution with 10 % glycerol. The complex was verified using native

89

gel electrophoresis and can be seen in Figure 15, however this method was entirely

qualitative. No crystals were obtained of the WT1 ZF (-/-) / WTE complex. As

previously mentioned the oxidation of the zinc fingers was the most likely explanation.

DTT was used to counter this result, but argon saturation would likely have proved

superior as DTT may be interacting too extensively with the zinc ions. It was also not

known what percentage of the complex was with double stranded DNA. If there was

only a small amount of complex in the solution then achieving crystals would be

unlikely.

4.1.3. Filter Binding Assays

The data generated in this study (Table 6) showed no evidence to support the

results found previously by Zhai (unpublished data) that showed aptamers 26 and 48

displayed a binding preference for the (+KTS) isoform. The two aptamers that were

tested in three or more independent trials (26, and 48), were shown to have a statistically

significant selectivity for the (-KTS) isoform. This data was statistically significant in

that the Kd values did not overlap when taking standard deviation into account. The

other four aptamers (3, 29, 58, 61) were tested with one trial and as such, it cannot be said

with any certainty that these findings are correct as more independent trials must be run

to determine the statistical relevance.

The computer generated secondary structures of the RNA aptamers tested

(Figures 21 to 26), showed no clear correlation between structure and isoform binding.

The structures shown were only a sample of many secondary structure predictions

generated using the mfold program. Those chosen demonstrated the lowest free energy.

90

We cannot assume that the RNA molecules actually resemble these structures in vivo, as

there are many different parameters known to affect secondary RNA structure, such as

temperature, salt and Mg++ concentration (Bukhman and Draper,1997; Shiman and

Draper,2000; Zhai et al.,2001). The latest version of mfold is set up so that the

temperature of structure formation is always 37 °C. The FBAs of this study were all

carried out at 20°C.

4.1.4. Supershift Assay

A good protocol was obtained in this study for a fluorescently labeled DNA for

supershift assay, although there does seem to be some downfalls when looking at the

binding with WT1 (-KTS). The binding seen was approxiamately 100 fold less than

expected. This may have been caused by the LI-COR tag itself, which is quite large

(Figure 9). The tag may be impeding the binding of the WT1 ZF protein to the DNA

strand and thus causing a apparent decrease in affinity. Once a large enough

concentration of the WT1 ZF protein is present, binding can be seen. To overcome this

problem a longer DNA molecule could be used. This would ensure that the binding site

is far removed from the LICOR tag, and thus should abrogate any interference (at least

directly) with the DNA/ ZF protein binding site. As a qualitative analysis this protocol is

quite feasible, and has the added advantage of not having to use radioactivity. The

method however, has yet to be optimized to give accurate quantitative results.

91

4.2. Ciao-1

4.2.1. Protein Purification

Purification of the Ciao-1 WD40 protein proved simple enough when using a his

tagged construct. The protocol determined in this study produced large quantities of pure

his tagged protein. It was noted that this protein was more unstable at higher

concentrations.

Purification of the GST tagged construct proved unfeasible for large-scale, as

there was degradation of the protein resulting in the GST tag being cleaved off. While

this would normally prove beneficial, there appeared to be some affinity between the

GST and the Ciao-1 protein. Unfortunately, the separation of the two proved quite

difficult.

Other protein binding partners for Ciao-1 have yet to be elucidated. As Ciao-1 is

known to be a WD40 protein, it has the ability to bind on three separate surfaces. More

detailed study of the binding between Ciao-1 and WT1 are required, as it is not yet

known which of the three surfaces of Ciao-1 bind to WT1. To date there are only two

papers published on this WD40 protein (Johnstone et al.,1998; Johnstone et al.,1999),

both were in regards to the relationship with WT1.

4.2.2. Crystallization of Ciao-1

As in the case of WT1, the crystallization of Ciao-1, was not successful. Unlike

the WT1 ZF protein, Ciao-1 was a full-length protein produced in soluble form. This

alone, would suggest that it is a better candidate for further crystallization trials. The

crystallization attemps in this study were done with high concentrations of the Ciao-1

92

protein. As it is now known that the Ciao-1 his tagged protein is unstable in these

conditions, one could try the crystallization trials with lower concentrations. A recent

publication showed the crystallographic structure of the yeast homolog of Ciao-1

(Srinivasan et al.,2007).

The purification of Cia1 was almost identical to our purification of Ciao-1, in fact

the only difference was in the type of SEC (Sephacryl S300) column used. They

crystallized the his-tagged Cia1 using the following conditions. Purified protein was

concentrated to 12 mg/mL and crystallized using the hanging drop method against a

reservoir containing 22 % PEG 4000, 20 mM Tris-HCl (pH 8.5), and 100 mM CaCl2.

While the authors do not mention the solution that their protein was stored in, they do

indicate that 10% glycerol was used in their storage buffer and as such they did not

require any other cryoprotectant for x-ray diffraction. Given the similarities to our

method of purification, the addition of glycerol to the storage buffer of Ciao-1 might

allow us to replicate their crystal growth conditions. The Cia1 structure would be a great

model for molecular replacement if Ciao-1 crystals were obtained.

93

5.0. Conclusion

The SELEX library that was created prior to this study did not exhibit any

specificity for the (+KTS) isoform over the (-KTS).

The attempted crystallization of WT1 ZF with DNA was recently solved by

another group but structural studies can still be carried out with other binding partners, of

particular interest will be the protein binding partner.

The Ciao-1 crystal structure also proved to be elusive, however, given what we

now know about the crystallization of the yeast homologue Cia1 this is still a decent

candidate for further crystallization trials. Perhaps all that is needed is some glycerol in

the storage buffer.

All encompassing binding proteins, such as WT1, are very important in the

human body. They play a role in cellular differentiation, development and prolonged

stability. It is their ability to bind to such diverse proteins/nucleic acids that allows them

to play so dominant a role in the living organism. The more information that is obtained

about WT1, the more we come to understand the complexity and fragility of the human

body. It has taken two decades to come to the meager understanding we have now of the

roles and functions of this amazing protein, perhaps within the next two decades we will

finally be able to visualize the whole story, if indeed we ever do.

94

Literature Cited

Ambion "MAXIScript In Vitro Transcription Kit. ." MAXIScript Manual: 5.

Armstrong, J. F., K. Pritchard-Jones, W. A. Bickmore, N. D. Hastie and J. B. Bard (1993). "The expression of the Wilms' tumour gene, WT1, in the developing mammalian embryo." Mech Dev 40(1-2): 85-97.

Bard, J. B., J. F. Armstrong and W. A. Bickmore (1993). "WT1, a Wilms' tumour gene." Exp Nephrol 1(4): 218-23.

Bardeesy, N. and J. Pelletier (1998). "Overlapping RNA and DNA binding domains of the wt1 tumor suppressor gene product." Nucleic Acids Res 26(7): 1784-92.

Beckwith, J. B. (1969). "Macroglossia, omphalocele, adrenal cytomegaly, gigantism and hyperplastic visceromegaly." Birth Defects

188-196.

Bergmann, L., C. Miething, U. Maurer, J. Brieger, T. Karakas, E. Weidmann and D. Hoelzer (1997). "High levels of Wilms' tumor gene (wt1) mRNA in acute myeloid leukemias are associated with a worse long-term outcome." Blood 90(3): 1217-25.

Bickmore, W. A., K. Oghene, M. H. Little, A. Seawright, V. van Heyningen and N. D. Hastie (1992). "Modulation of DNA binding specificity by alternative splicing of the Wilms tumor wt1 gene transcript." Science 257(5067): 235-7.

Bor, Y. C., J. Swartz, A. Morrison, D. Rekosh, M. Ladomery and M. L. Hammarskjold (2006). "The Wilms' tumor 1 (WT1) gene (+KTS isoform) functions with a CTE to enhance translation from an unspliced RNA with a retained intron." Genes Dev 20(12): 1597-608.

Borel, F., K. C. Barilla, T. B. Hamilton, M. Iskandar and P. J. Romaniuk (1996). "Effects of Denys-Drash syndrome point mutations on the DNA binding activity of the Wilms' tumor suppressor protein WT1." Biochemistry 35(37): 12070-6.

Bouhouche, N., M. Syvanen and C. I. Kado (2000). "The origin of prokaryotic C2H2 zinc finger regulators." Trends Microbiol 8(2): 77-81.

Bruening, W., P. Moffett, S. Chia, G. Heinrich and J. Pelletier (1996). "Identification of nuclear localization signals within the zinc fingers of the WT1 tumor suppressor gene product." FEBS Lett 393(1): 41-7.

Bruening, W. and J. Pelletier (1996). "A non-AUG translational initiation event generates novel WT1 isoforms." J Biol Chem 271(15): 8646-54.

Bukhman, Y. V. and D. E. Draper (1997). "Affinities and selectivities of divalent cation binding sites within an RNA tertiary structure." J Mol Biol 273(5): 1020-31.

95

Call, K. M., T. Glaser, C. Y. Ito, A. J. Buckler, J. Pelletier, D. A. Haber, E. A. Rose, A. Kral, H. Yeger, W. H. Lewis and et al. (1990). "Isolation and characterization of a zinc finger polypeptide gene at the human chromosome 11 Wilms' tumor locus." Cell 60(3): 509-20.

Caricasole, A., A. Duarte, S. H. Larsson, N. D. Hastie, M. Little, G. Holmes, I. Todorov and A. Ward (1996). "RNA binding by the Wilms tumor suppressor zinc finger proteins." Proc Natl Acad Sci U S A 93(15): 7562-6.

Coyle, P., E. Tichelman, R. Pauw, J. Philcox and A. Rofe (2003). "Zinc inhibition of hepatic fructose metabolism in rats." Biol Trace Elem Res 92(1): 41-54.

Dallosso, A. R., A. L. Hancock, K. W. Brown, A. C. Williams, S. Jackson and K. Malik (2004). "Genomic imprinting at the WT1 gene involves a novel coding transcript (AWT1) that shows deregulation in Wilms' tumours." Hum Mol Genet 13(4): 405-15.

Dauter, Z., V. S. Lamzin and K. S. Wilson (1997). "The benefits of atomic resolution." Curr Opin Struct Biol 7(5): 681-8.

Davies, J. A., M. Ladomery, P. Hohenstein, L. Michael, A. Shafe, L. Spraggon and N. Hastie (2004). "Development of an siRNA-based method for repressing specific genes in renal organ culture and its use to show that the Wt1 tumour suppressor is required for nephron differentiation." Hum Mol Genet 13(2): 235-46.

Davies, R. C., C. Calvio, E. Bratt, S. H. Larsson, A. I. Lamond and N. D. Hastie (1998). "WT1 interacts with the splicing factor U2AF65 in an isoform-dependent manner and can be incorporated into spliceosomes." Genes Dev 12(20): 3217-25.

Dey, B. R., V. P. Sukhatme, A. B. Roberts, M. B. Sporn, F. J. Rauscher, 3rd and S. J. Kim (1994). "Repression of the transforming growth factor-beta 1 gene by the Wilms' tumor suppressor WT1 gene product." Mol Endocrinol 8(5): 595-602.

Dong, J., R. Lai, K. Nielsen, C. A. Fekete, H. Qiu and A. G. Hinnebusch (2004). "The essential ATP-binding cassette protein RLI1 functions in translation by promoting preinitiation complex assembly." J Biol Chem 279(40): 42157-68.

Drummond, I. A., H. D. Rupprecht, P. Rohwer-Nutter, J. M. Lopez-Guisa, S. L. Madden, F. J. Rauscher, 3rd and V. P. Sukhatme (1994). "DNA recognition by splicing variants of the Wilms' tumor suppressor, WT1." Mol Cell Biol 14(6): 3800-9.

Elrod-Erickson, M., T. E. Benson and C. O. Pabo (1998). "High-resolution structures of variant Zif268-DNA complexes: implications for understanding zinc finger-DNA recognition." Structure 6(4): 451-64.

Elrod-Erickson, M., M. A. Rould, L. Nekludova and C. O. Pabo (1996). "Zif268 protein-DNA complex refined at 1.6 A: a model system for understanding zinc finger-DNA interactions." Structure 4(10): 1171-80.

96

Fairall, L., J. W. Schwabe, L. Chapman, J. T. Finch and D. Rhodes (1993). "The crystal structure of a two zinc-finger peptide reveals an extension to the rules for zinc-finger/DNA recognition." Nature 366(6454): 483-7.

Fischbach, B. V., K. L. Trout, J. Lewis, C. A. Luis and M. Sika (2005). "WAGR syndrome: a clinical review of 54 cases." Pediatrics 116(4): 984-8.

Fong, H. K., J. B. Hurley, R. S. Hopkins, R. Miake-Lye, M. S. Johnson, R. F. Doolittle and M. I. Simon (1986). "Repetitive segmental structure of the transducin beta subunit: homology with the CDC4 gene and identification of related mRNAs." Proc Natl Acad Sci U S A 83(7): 2162-6.

Foster, J. (2006). "Contribution of Individual Zinc Fingers of WT1 in RNA aptamer binding." Masters Thesis, University of Victoria, Victoria, BC, Canada.

Geng, J. and R. P. Carstens (2006). "Two methods for improved purification of full-length mammalian proteins that have poor expression and/or solubility using standard Escherichia coli procedures." Protein Expr Purif 48(1): 142-50.

Hamilton, T. B., K. C. Barilla and P. J. Romaniuk (1995). "High affinity binding sites for the Wilms' tumour suppressor protein WT1." Nucleic Acids Res 23(2): 277-84.

Hamilton, T. B., F. Borel and P. J. Romaniuk (1998). "Comparison of the DNA binding characteristics of the related zinc finger proteins WT1 and EGR1." Biochemistry 37(7): 2051-8.

Hammarskjold, M. L. (2001). "Constitutive transport element-mediated nuclear export." Curr Top Microbiol Immunol 259: 77-93.

Hammes, A., J. K. Guo, G. Lutsch, J. R. Leheste, D. Landrock, U. Ziegler, M. C. Gubler and A. Schedl (2001). "Two splice variants of the Wilms' tumor 1 gene have distinct functions during sex determination and nephron formation." Cell 106(3): 319-29.

Hastie, N. D. (1994). "The genetics of Wilms' tumor--a case of disrupted development." Annu Rev Genet 28: 523-58.

Heckman, C., E. Mochon, M. Arcinas and L. M. Boxer (1997). "The WT1 protein is a negative regulator of the normal bcl-2 allele in t(14;18) lymphomas." J Biol Chem 272(31): 19609-14.

Henning, K. A., L. Li, N. Iyer, L. D. McDaniel, M. S. Reagan, R. Legerski, R. A. Schultz, M. Stefanini, A. R. Lehmann, L. V. Mayne and E. C. Friedberg (1995). "The Cockayne syndrome group A gene encodes a WD repeat protein that interacts with CSB protein and a subunit of RNA polymerase II TFIIH." Cell 82(4): 555-64.

97

Hewitt, S. M., G. C. Fraizer, Y. J. Wu, F. J. Rauscher, 3rd and G. F. Saunders (1996). "Differential function of Wilms' tumor gene WT1 splice isoforms in transcriptional regulation." J Biol Chem 271(15): 8588-92.

Hewitt, S. M., S. Hamada, T. J. McDonnell, F. J. Rauscher, 3rd and G. F. Saunders (1995). "Regulation of the proto-oncogenes bcl-2 and c-myc by the Wilms' tumor suppressor gene WT1." Cancer Res 55(22): 5386-9.

Inoue, K., H. Sugiyama, H. Ogawa, M. Nakagawa, T. Yamagami, H. Miwa, K. Kita, A. Hiraoka, T. Masaoka, K. Nasu and et al. (1994). "WT1 as a new prognostic factor and a new marker for the detection of minimal residual disease in acute leukemia." Blood 84(9): 3071-9.

Jin, L., B. W. Guzik, Y. C. Bor, D. Rekosh and M. L. Hammarskjold (2003). "Tap and NXT promote translation of unspliced mRNA." Genes Dev 17(24): 3075-86.

Johnstone, R. W., R. H. See, S. F. Sells, J. Wang, S. Muthukkumar, C. Englert, D. A. Haber, J. D. Licht, S. P. Sugrue, T. Roberts, V. M. Rangnekar and Y. Shi (1996). "A novel repressor, par-4, modulates transcription and growth suppression functions of the Wilms' tumor suppressor WT1." Mol Cell Biol 16(12): 6945-56.

Johnstone, R. W., N. Tommerup, C. Hansen, H. Vissing and Y. Shi (1999). "Structural organization, tissue expression, and chromosomal localization of Ciao 1, a functional modulator of the Wilms' tumor suppressor, WT1." Immunogenetics 49(10): 900-5.

Johnstone, R. W., J. Wang, N. Tommerup, H. Vissing, T. Roberts and Y. Shi (1998). "Ciao 1 is a novel WD40 protein that interacts with the tumor suppressor protein WT1." J Biol Chem 273(18): 10880-7.

Kendrew, J. C., G. Bodo, H. M. Dintzis, R. G. Parrish, H. Wyckoff and D. C. Phillips (1958). "A three-dimensional model of the myoglobin molecule obtained by x-ray analysis." Nature 181(4610): 662-6.

Kim, J., J. M. Lee, P. E. Branton and J. Pelletier (1999). "Modification of EWS/WT1 functional properties by phosphorylation." Proc Natl Acad Sci U S A 96(25): 14300-5.

Kispal, G., K. Sipos, H. Lange, Z. Fekete, T. Bedekovics, T. Janaky, J. Bassler, D. J. Aguilar Netz, J. Balk, C. Rotte and R. Lill (2005). "Biogenesis of cytosolic ribosomes requires the essential iron-sulphur protein Rli1p and mitochondria." EMBO J 24(3): 589-98.

Klug, S. J. and M. Famulok (1994). "All you wanted to know about SELEX." Mol Biol Rep 20(2): 97-107.

98

Kreidberg, J. A., H. Sariola, J. M. Loring, M. Maeda, J. Pelletier, D. Housman and R. Jaenisch (1993). "WT-1 is required for early kidney development." Cell 74(4): 679-91.

Kriwacki, R. W., S. C. Schultz, T. A. Steitz and J. P. Caradonna (1992). "Sequence-specific recognition of DNA by zinc-finger peptides derived from the transcription factor Sp1." Proc Natl Acad Sci U S A 89(20): 9759-63.

Laity, J. H., H. J. Dyson and P. E. Wright (2000). "Molecular basis for modulation of biological function by alternate splicing of the Wilms' tumor suppressor protein." Proc Natl Acad Sci U S A 97(22): 11932-5.

Laity, J. H., B. M. Lee and P. E. Wright (2001). "Zinc finger proteins: new insights into structural and functional diversity." Curr Opin Struct Biol 11(1): 39-46.

Lander, E. S., L. M. Linton, B. Birren, C. Nusbaum, M. C. Zody, J. Baldwin, K. Devon, K. Dewar, M. Doyle, W. FitzHugh, R. Funke, D. Gage, K. Harris, A. Heaford, J. Howland, L. Kann, J. Lehoczky, R. LeVine, P. McEwan, K. McKernan, J. Meldrim, J. P. Mesirov, C. Miranda, W. Morris, J. Naylor, C. Raymond, M. Rosetti, R. Santos, A. Sheridan, C. Sougnez, N. Stange-Thomann, N. Stojanovic, A. Subramanian, D. Wyman, J. Rogers, J. Sulston, R. Ainscough, S. Beck, D. Bentley, J. Burton, C. Clee, N. Carter, A. Coulson, R. Deadman, P. Deloukas, A. Dunham, I. Dunham, R. Durbin, L. French, D. Grafham, S. Gregory, T. Hubbard, S. Humphray, A. Hunt, M. Jones, C. Lloyd, A. McMurray, L. Matthews, S. Mercer, S. Milne, J. C. Mullikin, A. Mungall, R. Plumb, M. Ross, R. Shownkeen, S. Sims, R. H. Waterston, R. K. Wilson, L. W. Hillier, J. D. McPherson, M. A. Marra, E. R. Mardis, L. A. Fulton, A. T. Chinwalla, K. H. Pepin, W. R. Gish, S. L. Chissoe, M. C. Wendl, K. D. Delehaunty, T. L. Miner, A. Delehaunty, J. B. Kramer, L. L. Cook, R. S. Fulton, D. L. Johnson, P. J. Minx, S. W. Clifton, T. Hawkins, E. Branscomb, P. Predki, P. Richardson, S. Wenning, T. Slezak, N. Doggett, J. F. Cheng, A. Olsen, S. Lucas, C. Elkin, E. Uberbacher, M. Frazier, R. A. Gibbs, D. M. Muzny, S. E. Scherer, J. B. Bouck, E. J. Sodergren, K. C. Worley, C. M. Rives, J. H. Gorrell, M. L. Metzker, S. L. Naylor, R. S. Kucherlapati, D. L. Nelson, G. M. Weinstock, Y. Sakaki, A. Fujiyama, M. Hattori, T. Yada, A. Toyoda, T. Itoh, C. Kawagoe, H. Watanabe, Y. Totoki, T. Taylor, J. Weissenbach, R. Heilig, W. Saurin, F. Artiguenave, P. Brottier, T. Bruls, E. Pelletier, C. Robert, P. Wincker, D. R. Smith, L. Doucette-Stamm, M. Rubenfield, K. Weinstock, H. M. Lee, J. Dubois, A. Rosenthal, M. Platzer, G. Nyakatura, S. Taudien, A. Rump, H. Yang, J. Yu, J. Wang, G. Huang, J. Gu, L. Hood, L. Rowen, A. Madan, S. Qin, R. W. Davis, N. A. Federspiel, A. P. Abola, M. J. Proctor, R. M. Myers, J. Schmutz, M. Dickson, J. Grimwood, D. R. Cox, M. V. Olson, R. Kaul, N. Shimizu, K. Kawasaki, S. Minoshima, G. A. Evans, M. Athanasiou, R. Schultz, B. A. Roe, F. Chen, H. Pan, J. Ramser, H. Lehrach, R. Reinhardt, W. R. McCombie, M. de la Bastide, N. Dedhia, H. Blocker, K. Hornischer, G. Nordsiek, R. Agarwala, L. Aravind, J. A. Bailey, A. Bateman, S. Batzoglou, E. Birney, P. Bork, D. G. Brown, C. B. Burge, L. Cerutti, H. C. Chen, D. Church, M. Clamp, R. R. Copley, T. Doerks, S. R. Eddy, E. E. Eichler, T. S.

99

Furey, J. Galagan, J. G. Gilbert, C. Harmon, Y. Hayashizaki, D. Haussler, H. Hermjakob, K. Hokamp, W. Jang, L. S. Johnson, T. A. Jones, S. Kasif, A. Kaspryzk, S. Kennedy, W. J. Kent, P. Kitts, E. V. Koonin, I. Korf, D. Kulp, D. Lancet, T. M. Lowe, A. McLysaght, T. Mikkelsen, J. V. Moran, N. Mulder, V. J. Pollara, C. P. Ponting, G. Schuler, J. Schultz, G. Slater, A. F. Smit, E. Stupka, J. Szustakowski, D. Thierry-Mieg, J. Thierry-Mieg, L. Wagner, J. Wallis, R. Wheeler, A. Williams, Y. I. Wolf, K. H. Wolfe, S. P. Yang, R. F. Yeh, F. Collins, M. S. Guyer, J. Peterson, A. Felsenfeld, K. A. Wetterstrand, A. Patrinos, M. J. Morgan, P. de Jong, J. J. Catanese, K. Osoegawa, H. Shizuya, S. Choi and Y. J. Chen (2001). "Initial sequencing and analysis of the human genome." Nature 409(6822): 860-921.

Larsson, S. H., J. P. Charlieu, K. Miyagawa, D. Engelkamp, M. Rassoulzadegan, A. Ross, F. Cuzin, V. van Heyningen and N. D. Hastie (1995). "Subnuclear localization of WT1 in splicing or transcription factor domains is regulated by alternative splicing." Cell 81(3): 391-401.

Lee, S. B. and D. A. Haber (2001). "Wilms tumor and the WT1 gene." Exp Cell Res 264(1): 74-99.

Lee, S. B., K. Huang, R. Palmer, V. B. Truong, D. Herzlinger, K. A. Kolquist, J. Wong, C. Paulding, S. K. Yoon, W. Gerald, J. D. Oliner and D. A. Haber (1999). "The Wilms tumor suppressor WT1 encodes a transcriptional activator of amphiregulin." Cell 98(5): 663-73.

Li, D. and R. Roberts (2001). "WD-repeat proteins: structure characteristics, biological function, and their involvement in human diseases." Cell Mol Life Sci 58(14): 2085-97.

Little, N. A., N. D. Hastie and R. C. Davies (2000). "Identification of WTAP, a novel Wilms' tumour 1-associating protein." Hum Mol Genet 9(15): 2231-9.

Lu, D., M. A. Searles and A. Klug (2003). "Crystal structure of a zinc-finger-RNA complex reveals two modes of molecular recognition." Nature 426(6962): 96-100.

Machin, G. A. and W. T. McCaughey (1984). "A new precursor lesion of Wilms' tumour (nephroblastoma): intralobar multifocal nephroblastomatosis." Histopathology 8(1): 35-53.

Madden, S. L., D. M. Cook, J. F. Morris, A. Gashler, V. P. Sukhatme and F. J. Rauscher, III (1991). "Transcriptional repression mediated by the WT1 Wilms tumor gene product." Science 253(5027): 1550-3.

Maheswaran, S., C. Englert, P. Bennett, G. Heinrich and D. A. Haber (1995). "The WT1 gene product stabilizes p53 and inhibits p53-mediated apoptosis." Genes Dev 9(17): 2143-56.

100

Maheswaran, S., S. Park, A. Bernard, J. F. Morris, F. J. Rauscher, 3rd, D. E. Hill and D. A. Haber (1993). "Physical and functional interaction between WT1 and p53 proteins." Proc Natl Acad Sci U S A 90(11): 5100-4.

Malik, K. T., V. Poirier, S. M. Ivins and K. W. Brown (1994). "Autoregulation of the human WT1 gene promoter." FEBS Lett 349(1): 75-8.

Mathews, D. H., J. Sabina, M. Zuker and D. H. Turner (1999). "Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure." J Mol Biol 288(5): 911-40.

McTaggart, S. J., E. Algar, C. W. Chow, H. R. Powell and C. L. Jones (2001). "Clinical spectrum of Denys-Drash and Frasier syndrome." Pediatr Nephrol 16(4): 335-9.

Metzger, M. L. and J. S. Dome (2005). "Current therapy for Wilms' tumor." Oncologist 10(10): 815-26.

Miller, J., A. D. McLachlan and A. Klug (1985). "Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes." EMBO J 4(6): 1609-14.

Miller, R. W., J. F. Fraumeni, Jr. and M. D. Manning (1964). "Association of Wilms's Tumor with Aniridia, Hemihypertrophy and Other Congenital Malformations." N Engl J Med 270: 922-7.

Murzin, A. G. (1992). "Structural principles for the propeller assembly of beta-sheets: the preference for seven-fold symmetry." Proteins 14(2): 191-201.

Muto, R., S. Yamamori, H. Ohashi and M. Osawa (2002). "Prediction by FISH analysis of the occurrence of Wilms tumor in aniridia patients." Am J Med Genet 108(4): 285-9.

Nakagama, H., G. Heinrich, J. Pelletier and D. E. Housman (1995). "Sequence and structural requirements for high-affinity DNA binding by the WT1 gene product." Mol Cell Biol 15(3): 1489-98.

Nolte, R. T., R. M. Conlin, S. C. Harrison and R. S. Brown (1998). "Differing roles for zinc fingers in DNA recognition: structure of a six-finger transcription factor IIIA complex." Proc Natl Acad Sci U S A 95(6): 2938-43.

Oka, Y., A. Tsuboi, T. Taguchi, T. Osaki, T. Kyo, H. Nakajima, O. A. Elisseeva, Y. Oji, M. Kawakami, K. Ikegame, N. Hosen, S. Yoshihara, F. Wu, F. Fujiki, M. Murakami, T. Masuda, S. Nishida, T. Shirakata, S. Nakatsuka, A. Sasaki, K. Udaka, H. Dohy, K. Aozasa, S. Noguchi, I. Kawase and H. Sugiyama (2004). "Induction of WT1 (Wilms' tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression." Proc Natl Acad Sci U S A 101(38): 13885-90.

101

Omichinski, J. G., P. V. Pedone, G. Felsenfeld, A. M. Gronenborn and G. M. Clore (1997). "The solution structure of a specific GAGA factor-DNA complex reveals a modular binding mode." Nat Struct Biol 4(2): 122-32.

Park, S., M. Schalling, A. Bernard, S. Maheswaran, G. C. Shipley, D. Roberts, J. Fletcher, R. Shipman, J. Rheinwald, G. Demetri and et al. (1993). "The Wilms tumour gene WT1 is expressed in murine mesoderm-derived tissues and mutated in a human mesothelioma." Nat Genet 4(4): 415-20.

Pavletich, N. P. and C. O. Pabo (1991). "Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 A." Science 252(5007): 809-17.

Pavletich, N. P. and C. O. Pabo (1993). "Crystal structure of a five-finger GLI-DNA complex: new perspectives on zinc fingers." Science 261(5129): 1701-7.

Qiagen (2002). ""QIAprep miniprep handbook."." Qiagen: 22-23.

Qiagen (2002). ""QIAquick spin handbook."." Qiagen: 18.

Rauscher, F. J., 3rd, J. F. Morris, O. E. Tournay, D. M. Cook and T. Curran (1990). "Binding of the Wilms' tumor locus zinc finger protein to the EGR-1 consensus sequence." Science 250(4985): 1259-62.

Reizner, N., S. Maor, R. Sarfstein, S. Abramovitch, W. V. Welshons, E. M. Curran, A. V. Lee and H. Werner (2005). "The WT1 Wilms' tumor suppressor gene product interacts with estrogen receptor-alpha and regulates IGF-I receptor gene transcription in breast cancer cells." J Mol Endocrinol 35(1): 135-44.

Richard, D. J., V. Schumacher, B. Royer-Pokora and S. G. Roberts (2001). "Par4 is a coactivator for a splice isoform-specific transcriptional activation domain in WT1." Genes Dev 15(3): 328-39.

Romaniuk, P. J., I. L. de Stevenson and H. H. Wong (1987). "Defining the binding site of Xenopus transcription factor IIIA on 5S RNA using truncated and chimeric 5S RNA molecules." Nucleic Acids Res 15(6): 2737-55.

Rupprecht, H. D., I. A. Drummond, S. L. Madden, F. J. Rauscher, 3rd and V. P. Sukhatme (1994). "The Wilms' tumor suppressor gene WT1 is negatively autoregulated." J Biol Chem 269(8): 6198-206.

Sakamoto, Y., M. Yoshida, K. Semba and T. Hunter (1997). "Inhibition of the DNA-binding and transcriptional repression activity of the Wilms' tumor gene product, WT1, by cAMP-dependent protein kinase-mediated phosphorylation of Ser-365 and Ser-393 in the zinc finger domain." Oncogene 15(17): 2001-12.

Saxen, L. and H. Sariola (1987). "Early organogenesis of the kidney." Pediatr Nephrol 1(3): 385-92.

102

Schuh, R., W. Aicher, U. Gaul, S. Cote, A. Preiss, D. Maier, E. Seifert, U. Nauber, C. Schroder, R. Kemler and et al. (1986). "A conserved family of nuclear proteins containing structural elements of the finger protein encoded by Kruppel, a Drosophila segmentation gene." Cell 47(6): 1025-32.

Segal, D. J., J. W. Crotty, M. S. Bhakta, C. F. Barbas, 3rd and N. C. Horton (2006). "Structure of Aart, a designed six-finger zinc finger peptide, bound to DNA." J Mol Biol 363(2): 405-21.

Shiman, R. and D. E. Draper (2000). "Stabilization of RNA tertiary structure by monovalent cations." J Mol Biol 302(1): 79-91.

Smith, T. F., C. Gaitatzes, K. Saxena and E. J. Neer (1999). "The WD repeat: a common architecture for diverse functions." Trends Biochem Sci 24(5): 181-5.

Srinivasan, V., D. J. Netz, H. Webert, J. Mascarenhas, A. J. Pierik, H. Michel and R. Lill (2007). "Structure of the yeast WD40 domain protein Cia1, a component acting late in iron-sulfur protein biogenesis." Structure 15(10): 1246-57.

Stoll, R., B. M. Lee, E. W. Debler, J. H. Laity, I. A. Wilson, H. J. Dyson and P. E. Wright (2007). "Structure of the Wilms tumor suppressor protein zinc finger domain bound to DNA." J Mol Biol 372(5): 1227-45.

Venter, J. C., M. D. Adams, E. W. Myers, P. W. Li, R. J. Mural, G. G. Sutton, H. O. Smith, M. Yandell, C. A. Evans, R. A. Holt, J. D. Gocayne, P. Amanatides, R. M. Ballew, D. H. Huson, J. R. Wortman, Q. Zhang, C. D. Kodira, X. H. Zheng, L. Chen, M. Skupski, G. Subramanian, P. D. Thomas, J. Zhang, G. L. Gabor Miklos, C. Nelson, S. Broder, A. G. Clark, J. Nadeau, V. A. McKusick, N. Zinder, A. J. Levine, R. J. Roberts, M. Simon, C. Slayman, M. Hunkapiller, R. Bolanos, A. Delcher, I. Dew, D. Fasulo, M. Flanigan, L. Florea, A. Halpern, S. Hannenhalli, S. Kravitz, S. Levy, C. Mobarry, K. Reinert, K. Remington, J. Abu-Threideh, E. Beasley, K. Biddick, V. Bonazzi, R. Brandon, M. Cargill, I. Chandramouliswaran, R. Charlab, K. Chaturvedi, Z. Deng, V. Di Francesco, P. Dunn, K. Eilbeck, C. Evangelista, A. E. Gabrielian, W. Gan, W. Ge, F. Gong, Z. Gu, P. Guan, T. J. Heiman, M. E. Higgins, R. R. Ji, Z. Ke, K. A. Ketchum, Z. Lai, Y. Lei, Z. Li, J. Li, Y. Liang, X. Lin, F. Lu, G. V. Merkulov, N. Milshina, H. M. Moore, A. K. Naik, V. A. Narayan, B. Neelam, D. Nusskern, D. B. Rusch, S. Salzberg, W. Shao, B. Shue, J. Sun, Z. Wang, A. Wang, X. Wang, J. Wang, M. Wei, R. Wides, C. Xiao, C. Yan, A. Yao, J. Ye, M. Zhan, W. Zhang, H. Zhang, Q. Zhao, L. Zheng, F. Zhong, W. Zhong, S. Zhu, S. Zhao, D. Gilbert, S. Baumhueter, G. Spier, C. Carter, A. Cravchik, T. Woodage, F. Ali, H. An, A. Awe, D. Baldwin, H. Baden, M. Barnstead, I. Barrow, K. Beeson, D. Busam, A. Carver, A. Center, M. L. Cheng, L. Curry, S. Danaher, L. Davenport, R. Desilets, S. Dietz, K. Dodson, L. Doup, S. Ferriera, N. Garg, A. Gluecksmann, B. Hart, J. Haynes, C. Haynes, C. Heiner, S. Hladun, D. Hostin, J. Houck, T. Howland, C. Ibegwam, J. Johnson, F. Kalush, L. Kline, S. Koduru, A. Love, F. Mann, D. May, S. McCawley, T. McIntosh, I. McMullen, M. Moy, L. Moy, B. Murphy, K. Nelson,

103

C. Pfannkoch, E. Pratts, V. Puri, H. Qureshi, M. Reardon, R. Rodriguez, Y. H. Rogers, D. Romblad, B. Ruhfel, R. Scott, C. Sitter, M. Smallwood, E. Stewart, R. Strong, E. Suh, R. Thomas, N. N. Tint, S. Tse, C. Vech, G. Wang, J. Wetter, S. Williams, M. Williams, S. Windsor, E. Winn-Deen, K. Wolfe, J. Zaveri, K. Zaveri, J. F. Abril, R. Guigo, M. J. Campbell, K. V. Sjolander, B. Karlak, A. Kejariwal, H. Mi, B. Lazareva, T. Hatton, A. Narechania, K. Diemer, A. Muruganujan, N. Guo, S. Sato, V. Bafna, S. Istrail, R. Lippert, R. Schwartz, B. Walenz, S. Yooseph, D. Allen, A. Basu, J. Baxendale, L. Blick, M. Caminha, J. Carnes-Stine, P. Caulk, Y. H. Chiang, M. Coyne, C. Dahlke, A. Mays, M. Dombroski, M. Donnelly, D. Ely, S. Esparham, C. Fosler, H. Gire, S. Glanowski, K. Glasser, A. Glodek, M. Gorokhov, K. Graham, B. Gropman, M. Harris, J. Heil, S. Henderson, J. Hoover, D. Jennings, C. Jordan, J. Jordan, J. Kasha, L. Kagan, C. Kraft, A. Levitsky, M. Lewis, X. Liu, J. Lopez, D. Ma, W. Majoros, J. McDaniel, S. Murphy, M. Newman, T. Nguyen, N. Nguyen, M. Nodell, S. Pan, J. Peck, M. Peterson, W. Rowe, R. Sanders, J. Scott, M. Simpson, T. Smith, A. Sprague, T. Stockwell, R. Turner, E. Venter, M. Wang, M. Wen, D. Wu, M. Wu, A. Xia, A. Zandieh and X. Zhu (2001). "The sequence of the human genome." Science 291(5507): 1304-51.

Wang, W., S. B. Lee, R. Palmer, L. W. Ellisen and D. A. Haber (2001). "A functional interaction with CBP contributes to transcriptional activation by the Wilms tumor suppressor WT1." J Biol Chem 276(20): 16810-6.

Wang, Z. Y., Q. Q. Qiu, K. T. Enger and T. F. Deuel (1993). "A second transcriptionally active DNA-binding site for the Wilms tumor gene product, WT1." Proc Natl Acad Sci U S A 90(19): 8896-900.

Wiedemann, H. R. (1964). "[Familial Malformation Complex with Umbilical Hernia and Macroglossia--a "New Syndrome"?]." J Genet Hum 13: 223-32.

Wolfe, S. A., L. Nekludova and C. O. Pabo (2000). "DNA recognition by Cys2His2 zinc finger proteins." Annu Rev Biophys Biomol Struct 29: 183-212.

Yamagami, T., H. Sugiyama, K. Inoue, H. Ogawa, T. Tatekawa, M. Hirata, T. Kudoh, T. Akiyama, A. Murakami and T. Maekawa (1996). "Growth inhibition of human leukemic cells by WT1 (Wilms tumor gene) antisense oligodeoxynucleotides: implications for the involvement of WT1 in leukemogenesis." Blood 87(7): 2878-84.

Yang, L., Y. Han, F. Suarez Saiz and M. D. Minden (2007). "A tumor suppressor and oncogene: the WT1 story." Leukemia 21(5): 868-76.

Yarunin, A., V. G. Panse, E. Petfalski, C. Dez, D. Tollervey and E. C. Hurt (2005). "Functional link between ribosome formation and biogenesis of iron-sulfur proteins." EMBO J 24(3): 580-8.

104

Ye, Y., B. Raychaudhuri, A. Gurney, C. E. Campbell and B. R. Williams (1996). "Regulation of WT1 by phosphorylation: inhibition of DNA binding, alteration of transcriptional activity and cellular translocation." EMBO J 15(20): 5606-15.

Zhai, G., M. Iskandar, K. Barilla and P. J. Romaniuk (2001). "Characterization of RNA aptamer binding by the Wilms' tumor suppressor protein WT1." Biochemistry 40(7): 2032-40.

Zuker, M. (2003). "Mfold web server for nucleic acid folding and hybridization prediction." Nucleic Acids Res 31(13): 3406-15.

105

Appendix A) Protein Parameters and sequence of WT1 ZF (-/-) in pET16b

10 20 30 40 50 60 MGHHHHHHHH HHSSGHIEGR HMRRVPGVAP TLVRSASETS EKRPFMCAYP GCNKRYFKLS 70 80 90 100 110 120 HLQMHSRKHT GEKPYQCDFK DCERRFFRSD QLKRHQRRHT GVKPFQCKTC QRKFSRSDHL 130 140 150 160 KTHTRTHTGE KPFSCRWPSC QKKFARSDEL VRHHNMHQRN MTKLQLAL

Number of amino acids: 168 Molecular weight: 20165.0 Theoretical pI: 10.50 Formula: C868H1354N302O231S14 Total number of atoms: 2769 Extinction coefficients: Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water. Ext. coefficient 10470 Abs 0.1% (=1 g/l) 0.519, assuming ALL Cys residues appear as half cystines Ext. coefficient 9970 Abs 0.1% (=1 g/l) 0.494, assuming NO Cys residues appear as half cystines

106

B) Protein Parameters and sequence of WT1 ZF (-/+) in pET16b

10 20 30 40 50 60 MGHHHHHHHH HHSSGHIEGR HMRRVPGVAP TLVRSASETS EKRPFMCAYP GCNKRYFKLS 70 80 90 100 110 120 HLQMHSRKHT GEKPYQCDFK DCERRFFRSD QLKRHQRRHT GVKPFQCKTC QRKFSRSDHL 130 140 150 160 170 KTHTRTHTGK TSEKPFSCRW PSCQKKFARS DELVRHHNMH QRNMTKLQLA L

Number of amino acids: 171 Molecular weight: 20481.3 Theoretical pI: 10.52 Formula: C881H1378N306O236S14 Total number of atoms: 2815 Extinction coefficients: Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water. Ext. coefficient 10470 Abs 0.1% (=1 g/l) 0.511, assuming ALL Cys residues appear as half cystines Ext. coefficient 9970 Abs 0.1% (=1 g/l) 0.487, assuming NO Cys residues appear as half cystines

107

C) Protein Parameters and Sequence of Ciao-1 in pET30

10 20 30 40 50 60 METKDSLVLL GRVPAHPDSR CWFLAWNPAG TLLASCGGDR RIRIWGTEGD SWICKSVLSE 70 80 90 100 110 120 GHQRTVRKVA WSPCGNYLAS ASFDATTCIW KKNQDDFECV TTLEGHENEV KSVAWAPSGN 130 140 150 160 170 180 LLATCSRDKS VWVWEVDEED EYECVSVLNS HTQDVKHVVW HPSQELLASA SYDDTVKLYR 190 200 210 220 230 240 EEEDDWVCCA TLEGHESTVW SLAFDPSGQR LASCSDDRTV RIWRQYLPGN EQGVACSGSD 250 260 270 280 290 300 PSWKCICTLS GFHSRTIYDI AWCQLTGALA TACGDDAIRV FQEDPNSDPQ QPTFSLTAHL 310 320 330 340 HQAHSQDVNC VAWNPKEPGL LASCSDDGEV AFWKYQRPEG LVEHHHHHH Number of amino acids: 349 Molecular weight: 39121.3 Theoretical pI: 5.02 Formula: C1712H2574N484O536S19 Total number of atoms: 5325 Extinction coefficients: Extinction coefficients are in units of M-1 cm-1, at 280 nm. Ext. coefficient 105055 Abs 0.1% (=1 g/l) 2.685, assuming ALL Cys residues appear as half cystines Ext. coefficient 103930 Abs 0.1% (=1 g/l) 2.657, assuming NO Cys residues appear as half cystines

108

Table i. Hamptons Crystal Screen 1 Formulation of all 50 well solutions from Crystal Screen 1(Hamptons Research).

109

Table ii. Hamptons Crystal Screen 2 Formulation of all 48 well solutions from Crystal Screen 2 (Hamptons Research).

110

Table iii. Hamptons PEG/ION Screen Formulation of all 48 well solutions from PEG/ION Screen (Hamptons Research).

111

Table iv: Additives used in Ciao-1 Optimization Formulation of all 24 additives. Additives were diluted 1/10 in well solution.

Tube # Additive Stock concentration 1 DMSO 30 % 2 iProH 30 % 3 EtOH 30 % 4 Glycerol 50 % 5 MeOH 30 % 6 MgCl2 0.1 M 7 Ethylene Glycol 30 % 8 MnCl2 0.1 M 9 Dextran 2.5 % 10 Urea 0.1 M 11 Na2EDTA 0.1 M 12 LiCl 1 M 13 Glycine 1 M 14 NaCl 2 M 15 CaCl2 0.1 M 16 NaI 1 M 17 D-Glucose 30 % (w/v) 18 Glycerol 30 % 19 D-Sucrose 30 % (w/v) 20 Guanidine HCl 1 M 21 Sodium Citrate 1.6 M 22 CsCl 1 M 23 MPD 50 % 24 Benzamidine HCl 20 % (w/v)