Three Functional Genomic Approaches to Biochemical

and Screen-Based Analyses of Topics in Cellular Biology

Jovana J. Grbić

Schultz Laboratory

April 09, 2007

Talk Outline—Part I

• Genomic Profiling of Runx3 Downstream Target Genes in a Gastric Cancer Model System

• Generation and Use of a Novel shDNA Library Targeting the Mouse Kinome in the Discovery of Osteogenesis Regulators

• Elucidating the Biological Role of the Protein Interaction Between Bmi1 and Pontin52

Runx3

• Member of the highly conserved Runt domain family of transcription factors• Thought to be the most ancient of the three genes, both due to its length and regulation of neurogenesis of the

monosynaptic reflex arc• 128-amino acid Runt domain regulates binding of Runx proteins to a consensus DNA sequence and mediates

interaction with core-binding factor-β• Cellular Roles:

– Development and survival of dorsal root ganglia neurons (axonal projection)– CD4+/CD8+ T cell development– Myeloid expression/Immune regulation– Chondrocyte differentiation– Gastric epithelia differentiation and growth

Runx3 Cellular Mechanism

• Part of TGFβ supersignalig network--directs activation/repression of genes through DNA binding on transcriptional domain

• Downstream signaling targets/mechanisms largely unknown

???

???

Gastric Cancer• Most frequent gastrointestinal malignancy • Second most-common cause of cancer-related

death in the world• Some gene alterations have been associated with

gastric cancers (E-cadhedrin, p53, TGFβ receptor)• Many chromosomal loci are lost in gastric cancers

(including 1p, 5q, 7q, 12q, 17p, 18q)• Underlying mechanisms of oncogenesis and tumor

progression are still very poorly understood

Causal Link to Gastric Cancer

• Runx3 loci selectively ablated in GC cell lines (FISH)

• Hemizygous hypermethylation of CpG island

• Runx3 expression able to reverse tumor growth in culture and in vivo

Establishing a Working Cell Line

M AGSSNU-1SN

U-1

6

AZATSA

--

-- -

+ -

+ +

+ -- -

+ -

+ +

+

Runx3

5’-aza-2’-deoxycytidine

Demethylation:

MS-PCR:

runx3CpG island

(~890 bp)

F

R

CGATTGGCTGTGCGACGCGTCGCTCCGCCAGCCCCGCCCCGCGGGCCCCGGGGGTACTAA CGATTGGTTGTGCGACGCGTCGTTTCGTTAGTTCCGTTCCGCGGGTTTCGGGGGTATTAA CGATTGGTTCTGCGACGCGTCGTTTCGTCAGTTTCGTTTCGCGGGTTTCGGGGGTATTAA CCCCGCGCGGGCGGCCGCGGCCCCGCCACTTGATTCTGGAGGATTTGTTCTGGGGCTGCG TCCCGCGCGGGCGGTCGCGGTTTCGTTATTTGATTTTGGAGGATTTGTTTTGGGGTTGCG CTTCGCGCGGGCGGTCGCGGTTTCGTTATTTGATTTTGGAGGATTTGTTCTGGGGTTGCG GCCGCGGAGTCGGGGCGGCCGCGGGCGAGCTTCGGGGCGGGAGGCGGCGGCAGCGGCACA GCCGCGGAGTCGGGGCGGTCGCGGGCGAGTTTCGGGGCGGGAGGCGGCGGTAGCGGTATA GTCGCGGAGTCGGGGCGGTCGCGGGCGAGTTTCGGGGCGGGAGGCGGCGGTAGCGGTACA GCCCCGCGCGGGCCCCGCCGCGGCCCAGGCAGCCGGGACAGCCACGAGGGGCGGCCGCAC GTTTCGCGCGGGTTCCGTCGCGGTTTAGGTAGTCGGGATAGCTACGAGGGGCGGTCGTAC GTTTCGCGCGGGTCTCGTCGCGGTCTAGGTAGTCGGGATAGTTACGAGGGGCGGTCGTAC GCGGGGCCGCGCGCCGAGGATGCGGGACTAGCCGGGCAGGCTGCGGGCGGCCGTCGGGCC GCGGGGTCGCGCGTCGAGGATGCGGGACTAGTCGGGTAGGCTGCGGGCGGTCGTCGGGCT GCGGGGTCGCGCGTCGAGGATGTGGGATTAGTCGGGTAGGTTGCGGGCGGTCGTCGGGCT AGCGAGGCCTCGCAGCGGGCGGGCCCTGGCGAGTAGTGGCCGGGCGCCGCCCCCTGCGCC AGCGAGGTTTCGCAGCGGGCGGGTTTTGGCGAGTAGTGGTCGGGCGTCGCTTTTTGCGTT AGCGAGGTTTCGTAGCGGGCGGGTTTTGGCGAGTAGTGGTCGGGTGTCGTTTTTTGCGTT CTGAGGCCCGGGCCCCGCCGCTTCTGCTTTCCCGCTTCTCGCGGCAGCGGCGGCCGAGGA TTGAGGTTCGGGTTCCGCCGTTTTTGTTTTTTCGTTTCTCGCGGTAGTGGCGGTCGAGGA TTGAGGTTCGGGTTCCGTCGTTTTTGTTTTTTCGTTTTTCGCGGTAGTGGCGGTCGAGGA GGCGCCCGCGCCGGCCGCCCCCGGGGGAAGCCGCGCCGTCTCCGCCTGCCCGGCGCCCTG GGCGTTCGCGTCGGCCGCTTCCGGGGGAAGTCG--------------------------- GGCGTTCGCGCCGGTCGTCTTCNGGGGAAGTCGCGTCGTTTTCGTTTGTCTGNCGTTTTG

WT Runx3 sequenceAGS (after sodium bisulfite)SNU-1 (after sodium bisulfite)

RT-PCR

Runx3

1 2 3 4M

Runx3 Profiling Strategy

M - + + AG

S W

TA

ZA

-tre

ated

Ove

rexp

ress

ion

AZAtreated

Over-expression

VectorRunx3

β-actin

Generate ComprehensiveExpression Profile

Overlap signatures andAnalyze convergent dataExtract mRNAs in

duplicate

HybridizeOnto UA133Affy Chip

Dr. John Walker

Runx3 Upregulated Genes

AGS+AZA

AGS+AZA

Runx3 stable

Runx3 stable

35 17 23

3359 76

Upregulated Genes:

Downregulated Genes:Gene ID AGS+AZA AGS_runx

3

Sterile alpha motif domain containing 4 (SAMD4) 4.92 4.49

hydroxyprostaglandin dehydrogenase 15-(NAD) 4.07 4.54

solute carrier family 2, member 3 3.38 3.37

solute carrier family 1, member 3 3.32 3.54

Molecule interacting with Rab13 2.89 3.25

A kinase (PRKA) anchor protein 2 2.62 2.82

lipase protein 2.49 2.15

tumor necrosis factor receptor superfamily, member 6 2.49 2.52

neuropilin 1 2.33 2.24

parvulin hPar14 2.33 2.10

beta tubulin, polypeptide 2.30 2.51

transmembrane 4 superfamily member 1 2.00 2.02

Anti-Proliferative Capacity of Upregulated Genes

• Several candidate genes display proliferative inhibition in GC cell line

• AKAP and hP14 both shown to have cell cycle regulatory roles

• None of the upregulated genes could inhibit cell growth beyond 30-50%

• Possible combinatorial effect in tumor suppression

Runx3

AKAP2

hPar14

β-actin

SAMD4

A) B)

C)

Empty Vector

Runx3 AKAP2 SAMD4 hPar14

0

100000

200000

300000

400000

500000

600000

700000

0 1 2 3 4 5 6

Day

Flu

ores

cenc

e In

tens

ity

Col

ony

#

Gene Transfected

Empty Vector

Runx3

SAMD4

AKAP2

Figure 2. (A) Northern blot reconfirmations of gene expression, (B) fluorescence-based cell proliferation assays and (C) colony formation assays with crystal violet staining and quantitation shown below

1 2 3

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

E V R u n x3 S A M D 4 A K A P h P a r 1 4

Runx3 Downregulated Genes

Gene ID Accession # AGS+AZA AGS_runx3

phosphoenolpyruvate carboxykinase 2

NM_004563 -5.3 -5.41

regulator of G-protein signalling 5 NM_003617 -3.6 -3.55

C/EBPβ NM_005194 -3.0 -3.44

HRMT1L2 NM_001536 -3.1 -3.11

Evi-1 X54989 -2.9 -2.92

STK38L (NDR2) NM_015000 -2.1 -2.64

galactosylceramidase NM_000153 -2.3 -2.37

cysteinyl-tRNA synthetase NM_139273 -2.1 -2.34

REG1α NM_002909 -2.4 -2.28

G protein-coupled receptor 49 NM_003667 -2.6 -2.23

Kruppel-like factor 5 (intestinal) NM_001730 -2.2 -2.11

AGS+AZA

AGS+AZA

Runx3 stable

Runx3 stable

35 17 23

3359 76

Upregulated Genes:

Downregulated Genes:

AGS+AZA Runx3 stable

•Tumorigenesis and Cancer Progression•Selectively Overexpressed in Cancer•Other Disease Regulatory Roles

Genomic Analysis• Four-gene central

network: IL-6, C/EBPβ, TNF, NFE2L2

• All involved with some aspect of cancer progression or tumor viability

• Secondary interactions of downregulated genes: cell proliferation, tumorigenesis, apoptosis, metastasis

Conclusions

• Runx3 is a master tumor suppressor—regulates combination of genes as an extended network

• More emphasis on downregulation of oncogenes than upregulation of other suppressors

• Data consistent with the established strong causal link between Runx3 silencing and cancer advancement

Talk Outline—Part II

• Genomic Profiling of Runx3 Downstream Target Genes in a Gastric Cancer Model System

• Generation and Use of a Novel shDNA Library Targeting the Mouse Kinome in the Discovery of Osteogenesis Regulators

• Elucidating the Biological Role of the Protein Interaction Between Bmi1 and Pontin52

RNAi: Function and Potential• RNA shown to interfere

with certain native functions of endogenous genes/biological functions

• Can also be introduced exogenously to force gene silencing

• Wide array of current methods for cellular siRNA delivery

• Advent of vector-based hairpin incorporation methods hold promise for medicinal and high-throughput applications

5’-CUUACGCUGAGUACUUCGAdTdTdTdTGAAUGCGACUCAUGAAGCU-5’

AGGTGGACATAACTTACGCTGAGTACTTCGATTTGTCCGTTCGG 5’ 3’

CDS

0 1 2 3 4

GC5

0 1 2 3 4

GC3

0 4 8 9 12 16 19 GC of the oligo

AA 5TA 4AT 2TT 2NA 1NN 0

TT 5TA 4AT 2AA 2TN 1NN 0

F = W5·F5+ W3·F3 + WGC ·FGC + WGC5 ·FGC5+ WGC3 ·FGC3

F5F3

Algorithmic Sequence Design

Dr. Serge Batalov

(Favorability)

Final Sequence Generation

5 unique sequences:Specificity, Fidelity, Ideal Parameters

1) Parameter input2) Additional algorithm values3) Putative sequence candidates generated4) Smith-Waterman similarity search5) Unique sequences vetted for shDNA cloning

High Throughput Library Construction

Dr. Anthony Orth, Dr. Sheng Ding, Alicia Linford, Myleen Medina

High-throughputmini-preps, plating into 384-well format

Primer PCRs

Transfection into E.Coli

Ligation into pDONR vector

Total library consists of 5 siDNA targets per gene, targeting approximately500 total murine kinases(Approximately 85-90% sequence fidelity)

Kinases as Targets for Control of Lineage-Specific

Differentiation• Approximately 518

kinases (1.7% of human genes); mouse orthologs for 510—good model system

• Mediate most signal transduction in cells—involved in a large number of biological processes

• Mesenchymal stem cell differentiation: bone regeneration vs. other lineages (fat, muscle, cartilage)

Osteogenesis Screening

Plate 4

0

500

1000

1500

2000

2500

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Well

Flu

ore

sc

en

ce

Alkaline Phosphatase Fluorescence Assay

Cbfa1 re-confirmation

0

100

200

300

400

500

600

2099

-G4

2103

-H3

2106

-C8

2106

-F10

2095

-G2

2103

-G5

2100

-F5

2102

-G2

2105

-G4

2100

-G2

2074

-C10

2078

-B3

2074

-A7

2078

-C2

2073

-B2

2078

-G3

2076

-A2

2080

-G10

2080

-E2

2084

-C3

2084

-B3

2080

-C11

cont

rol

RL

U

Cbfa1 Reporter Assay

2 rounds of ALP screening and Cbfa1 confirmation:

87 primary hits validated by both methods

Dr. Xu Wu

MSC

Osteoblasts

Adipocytes

SkeletalMyocytes

Chondrocytes shDNA libraryscreen

10T½

shDNAtransfection

Day 1

Day 3ReplaceMedia

Day 6Stain with

ALP enzyme

Day 3LuciferaseReadout

Hit Characterization

Cbfa1 Reporter Assay

0.0200.0400.0600.0800.0

1000.01200.01400.01600.01800.02000.0

BMP-4treated

EV Chkb CDK9 Srpkl

Lum

ines

cenc

e (R

LU)

B)

B-actin

ALP

OPN

OC

Collagen I

BMP4

WT

CD

K9

Chkl

Srpk

1C)

Prognostication of chemoresponsiveness of germ cell tumors

80%NM_016795Srpk1

Rostrocaudal muscular dystrophy70%NM_007692Chkb

Adipogenesis, cardiomyocyte enlargement, nuclear localization

85%NM_130860CDK9

Biological FunctionKnockdown Efficiency (25 ng/rxn)

Accession #Hit

Prognostication of chemoresponsiveness of germ cell tumors

80%NM_016795Srpk1

Rostrocaudal muscular dystrophy70%NM_007692Chkb

Adipogenesis, cardiomyocyte enlargement, nuclear localization

85%NM_130860CDK9

Biological FunctionKnockdown Efficiency (25 ng/rxn)

Accession #Hit

ALP Osteopontin

BMP4-treated

EmptyVector

CDK9

ALP Osteopontin

Srpk1

Chkb

A)

Conclusion

• Successful construction of a vector-encoded shDNA library targeting the murine kinome

• Initial screening efforts have yielded several candidate kinases putatively involved in osteogenesis

• Follow up (in progress) will include other shDNA sequences and genomic characterization of hits

Talk Outline—Part III

• Genomic Profiling of Runx3 Downstream Target Genes in a Gastric Cancer Model System

• Generation and Use of a Novel shDNA Library Targeting the Mouse Kinome in the Discovery of Osteogenesis Regulators

• Elucidating the Biological Role of the Protein Interaction Between Bmi1 and Pontin52

Hematopoiesis

• HCSs give rise to the collective immune system

• Stem cell niche provides essential signaling pathways/factors via MSCs for HCS self-renewal

• Delicate balance between self-renewal and differentiation

Bmi1: Regulation of HSCs

• Intrinsic factors also contribute to HSC self-renewal

• Polycomb group repressive complex 1 member Bmi1 indispensable to HSC maintenance: forced overexpression and knockout studies

• Direct repression of p14/p16 locus

• Putative links to Wnt, SHH pathways

• Cooperative oncogenic capacity with c-Myc

Bmi1, Stem Cells and Cancer

•Important role for Bmi1 in self-renewal capacity of hematopoietic and leukemic stem cells•Prognostic ability for patient survival (prostate cancer)•Involved in human medulloblastomas

Identify regulators of BMI-1 (cDNA, siRNA screens; pull-down)

FLAGActin FLAGBMI-1

WT FLA

GActin

FLAGBM

I1

64

82

48

Anti-FLAG Ab

FLAG-A

ctin

FLAG-B

MI-1

293T

MALDI-TOF

Hit picks, etc.

IPMS Hit Peptide Abundancy KIF-23 3 peptide fragments HSP70 protein 3 peptide fragments Skb1 homolog (PRMT5) 4 peptide fragments MEL-18* 8 peptide fragments HSP60 protein 4 peptide fragments CAP-1 2 peptide fragments RPA-1 3 peptide fragments UTP18 4 peptide fragments Beta-5 tubulin (TUBB) 5 peptide fragments Ring finger protein 1* 11 peptide fragments Bmi-1† 20 peptide fragments Pontin-52 (RUVBL1) 6 peptide fragments RBBP7 5 peptide fragments Bystin (BYSL) 4 peptide fragments Ring finger protein 2* 6 peptide fragments WDR77 3 peptide fragments *known Bmi-1 interactor

†bait protein

IP-MS Design and Execution

Pontin52• Pontin52 is a AAA+type ATPase• Essential cofactor for oncogenic

transformation by c-Myc• Regulates beta-catenin-mediated

neoplastic transformation and T-cell factor target gene induction via effects on chromatin remodeling

• E2F-dependent histone acetylation and recruitment of the Tip60 acetyltransferase complex to chromatin in late G1

• Pontin and Reptin regulate cell proliferation in early Xenopus embryos in collaboration with c-Myc and Miz-1

• Enzyme-dependent activation/regulation (rarity of AAA+ ATPase distribution) lends credibility to drugability/SM targeting

Myc/Pontin52-inducedColonies in primary REFs(ablated by null mutant)

SymAtlas Expression Correlation

BMI-1 Pontin52 c-Myc

HSC Progenitors

T and B Cells

Almost fingerprint-like degree of expression homology, specifically along blood-related cell lineages

FLAG bead IP;Anti-Pontin52 Antibody

Lane

1 (F

LAGActi

n)

Lane

2 (b

lank)

Lane

3 (

FLAGBM

I1)

85

60

50

BMI-1 interacts with Pontin-52 under native conditions

Interaction also verified with co-IP (FLAG BMI and HA Pontin)

Po

ntin

52M

EL

-18

FLAG IP (anti-HA ab)

FLAG IP (anti-HA ab)

O/E (anti-HA ab)

O/E (anti-HA ab)

1 2 3 4 5

*

*positive control

293T cells:

B=Bmi1

A=Actin

P=Pontin52

B A P B+A B+P

Silencing ConfersCancer Cell Death

scrshDNA Pontin 52BMI-1

DAOY

PC-3

A549

(a)

(b)

(c)

(d)

•Loss of Bmi1 established as incurring apoptosis in cancer cells

•Parallel effects with Pontin52???

Knockdown Efficiency

Link to Bmi1 p16 Pathway?

• WI38 fibroblasts serve as ideal model for senescence (intact p16 expression)

• Bmi1 silencing shown to inversely activate p16 levels

• Similar effect for Pontin52

• No off-target effects observed

Conclusion

• Bmi1 complexes with Pontin52 under low-stringency conditions

• Possibly linked via Myc/p16 signaling pathways

• Future efforts towards inhibition and in vivo models of stem cell/tumor regulation

Shh

PTCH SMOH Wnt ?

Gli1 Gli2 β - catenin

Pontin52

N - Myc

?

Bmi1

Cyclin D1/D2

E2F1

E4F1

p19 arf

p53

p16

Acknowledgements

Schultz Group (TSRI):

• Dr. Qihong Huang

• Dr. Sheng Ding

• Dr. Xu Wu

• Dr. Aaron Willingham

• Functional Genomics Subgroup

• Dr. Lubica Supekova

• Cookie Santamaria, Tanya Gresham, Toni Martin, Emily Remba, Michelle Davis

Dr. Peter G. Schultz

GNF:• Dr. John Walker (Profiling)• Dr. Eric C. Peters (Mass Spec)• Dr. Markus Warmuth (and Warmuth Group)• Dr. Serge Batalov• Dr. Anthony Orth (siDNA library)

– Alicia Linford, Myleen Medina, Brendan Smith, Abel Gutierrez

Committee:

• Dr. Benjamin Cravatt

• Dr. Peter Vogt

• Dr. Floyd Romesberg

Graduate Office:

• Marilyn Rinaldi, Stacy Evans, Diane Kreger

Family and Friends