BioSci 145A lecture 13 1 © copyright Bruce Blumberg 2000. All rights reserved BioSci 145A Lecture...

BioSci 145A lecture 13 1 ©copyright Bruce Blumberg 2000. All rights reserved

BioSci 145A Lecture 13 - 2/19/2002Transcription factors II

• Topics we will cover today– Hybrid tetracycline regulated system– implications of transgenic technology– Principles of gene regulation– Identification of regulatory elements– Identification of regulatory element binding proteins– Functional analysis– Transcription factors - introduction– Modulation of transcription factor activity

• transcription factor resources– http://transfac.gbf-braunschweig.de/TRANSFAC/– http://bioinformatics.weizmann.ac.il/transfac/

• detailed transcription factor database– http://copan.bioz.unibas.ch/homeo.html

• collected information about homeobox genes– http://biochem1.basic-ci.georgetown.edu/nrr/nrr.html

• nuclear receptor resource

• references– nuclear transport Nakielny and Dreyfuss (1999) Cell

99, 677-690.– Nuclear pore structure Daneholt (1997) Cell 88, 585-

588.


Regulated expression - hybrid tetracycline systems

• Background– references

• Kringstein et al (1998) PNAS 95, 13670-75• Baron et al (1999) PNAS 96, 1013-1018• Blau and Rossi (1999) PNAS 96, 797-799

– utilizes highly engineered tet and reverse tet proteins to get specific effects

• properties of the system– what happens when one puts proteins into the cell that

respond differently to the same effector compound?• If they can dimerize with each other• can not dimerize with each other• or if they do not dimerize and bind to different

and non-overlapping operator sequences– different function, can heterodimerize

• in this case, a fair number will make unproductive heterodimers and interfere with desired effect

• this will also be problematic even if the two dimers have been engineered to bind different targets

• so for maximum effect, we must prevent heterodimerization between effectors that

– can bind to different sequences– have different functions (activator vs

repressor


Regulated expression - hybrid tetracycline (contd)

– Opposite function - same DNA target• use pure tetR and VP16-rtetR• at low dox, the repressor will dominate• as dox increases, the repressor will dissociate and

VP16-rtetR will activate• increases dynamic range of activation ~105 fold

– increases sensitivity as well

Heterodimers possible

same DNA targets

different DNA targets

Heterodimers not possible

functional discrimination works



– Opposite function - different DNA target

• use std tet with one type of tetO to regulate gene A

• and rev tet with another type of tetO to regulate gene B

• in the absence of dox, gene A will be activated while B will be silent

• in the presence of high dox, gene A will be inactivated and gene B will be activated



• Applications

– activator and repressor

• very sensitive regulation of responsive gene expression

• tightly regulated expression over 5 logs

• can readily measure effects of subtle changes in gene expression

– how much change in expression is required to get effects?

– Do effects differ at different levels of expression?

– Activator/repressor two targets

• can create “conditional mutants” that have one activity at low levels of effector substance and another at high levels

• can regulate two different genes or two alleles of a single gene

– mutually exclusive expression

– or expression of neither

• can repeatedly switch between two states and observe effects at high resolution

• possible to perturb intracellular equilibria in small increments and follow the effects on phenotype



• Caveats and pitfalls– best done with stable cell lines

• advantages– much more versatile than ecdysone– possible to fine tune expression of a single gene, or

two genes with unprecendented resolution– can make conditional mutants without genetics

• eg in model systems not amenable to genetics such as Xenopus or chicken

– can make conditional replacements in vivo• knock the repressible tetO into an endogenous

gene• introduce a transgene under the control of the

activatable tetO• breed these mice with a line expressing the dual

tet repressors• increasing dox will inactivate the endogenous

gene and activate the transgene• disadvantages

– technically demanding– multiple steps required– may not work as well as predicted due to complexity– virus-based systems may not work in ES cells– Clontech’s TRE-effector plasmids have high

background - need to be remade


Regulated gene expression - summary

• what system do you need to use?

– Transgenic animals?

– Gene therapy?

– Cell culture?

• How much tolerance is there for modulation of non target genes?

– Eg glucocorticoids, estrogens, progestins and thyroid hormones are very important physiologically and cannot be modulated without collateral effects in adults

• however, these are very good in the early embryo of model organisms

• How stringent must the regulation be?

– The more stringent the requirements for control the greater the likelihood that complex techniques will be required

• eg hybrid tetracycline

• Is there a need for regulating multiple genes?

– Hybrid tet is the only way to go

• What are the commercial implications?

– Licenses may be required for various technologies

• tet is controlled by BASF

• ecdysone by the Salk Institute

– depending on the license agreement, one technique might be preferred (eg ecdysone)


Gene transfer technology - implications

• Genetics and reverse genetics

– gene transfer and selection technology speeds up genetic analysis by orders of magnitude

– virtually all conceivable experiments are now possible

• all questions are askable

– much more straightforward to understand gene function using knockouts and transgenics

• gene sequences are coming at an unprecedented rate from the genome projects

• Knockouts and transgenics remain very expensive to practice

– other yet undiscovered technologies will be required to understand gene function.

• Clinical genetics

– Molecular diagnostics are becoming very widespread as genes are matched with diseases

• huge growth area for the future

• big pharma is dumping billions into diagnostics

– room for great benefit and widespread abuse

• diagnostics will enable early identification and treatment of diseases

• but insurance companies will want access to these data to maximize profits


Gene transfer technology - implications (contd)

• gene therapy– new viral vector technology is making this a reality

• efficient transfer and reasonable regulation possible– long lag time from laboratory to clinic, still working with

old technology in many cases– The Biotech Death of Jesse Gelsinger. Sheryl Gay

Stolberg, NY Times, Sunday Magazine, 28 Nov 99• http://www.frenchanderson.org/history/biotech.pdf

• protein engineering– not as widely appreciated as more glamorous techniques

such as gene therapy and transgenic crops– better drugs, eg more stable insulin, TPA for heart attacks

and strokes, etc.– more efficient enzymes (e.g. subtilisin in detergents)– safe and effective vaccines

• just produce antigenic proteins rather than using inactivated or attenuated organisms to reduce undesirable side effects

• metabolite engineering– enhanced microbial synthesis of valuable products

• eg indigo (jeans)• vitamin C

– generation of entirely new small molecules• transfer of antibiotic producing genes to related

species yields new antibiotics (badly needed)– reduction of undesirable side reactions

• faster more efficient production of beer


Gene transfer technology - implications (contd)

• transgenic food

– gene transfer techniques have allowed the creation of desirable mutations into animals and crops of commercial value

• disease resistance (various viruses)

• pest resistance (Bt cotton)

• pesticide resistance

• herbicide and fungicide resistance

• growth hormone and milk production

– effective but necessary?

– negative implications

• pesticide and herbicide resistance lead to much higher use of toxic compounds

• results are not predictable due to small datasets

• at least one herbicide (bromoxynil) for which resistance was engineered has since been banned

• plants as producers of specialty chemicals

– still very underutilized since plant technology yet lags behind techniques in animals

– great interest in using plants as factories to produce materials more cheaply and efficiently

• especially replacements for petrochemicals

– plants and herbs are the original source of many pharmaceutical products hence it remains possible to engineer them to overproduce desirable substances


Principles of gene regulation

• Why does gene expression need to be controlled anyway?

– Primary purpose in multicellular organisms is to execute precise developmental decisions so that

• proper genes are expressed at

– appropriate time

– correct place

– at the required levels

• so that development, growth and differentiation proceed correctly

– maintenance of homeostasis

• produce required substances in appropriate amounts

– nutrients, cofactors, etc.

• degrade undesired substances from

– diet

– metabolism

– injury

• inter and intracellular signaling processes

• Where are the control points?

– Activation of gene structure

– initiation of transcription

– processing of the transcript to mRNA

– transport of mRNA to cytoplasm

– translation of mRNA

– processing and stability of protein


Principles of gene regulation (contd)

• Activation of gene structure

– genes are active only in cells where they are expressed

– structure of gene determines whether it is can be transcribed or not

– activation of an active structure may be one of the first steps in gene regulation

• modification of DNA

– methylation of DNA inactivates genes

– active genes are hypomethylated

• modification of histones

– methylation and acetylation of histones activates gene expression

» acetylase activates

• active genes are in an open, hypomethylated coformation. associated histones are hyperracetylated

– one of the primary responsibilities of cell-type specific transcription factors is to facilitate the formation of an active chromatin conformation

• majority of alleged co-activator and co-repressor proteins are relatively non-specific modifiers of chromatin conformation that interact with specific factors targeting chromatin remodeling



• Initiation of transcription

– Once the DNA template is accessible, the next requirement is to form the initiation complex

• although other forms of regulation are important, the majority of regulatory events occur at the initiation of transcription

– genes under common control share response elements (aka cis-cting elements, enhancers)

• these sequences are presumed to be recognized by specific protein(s)

• the protein(s) functions as a transcription factor needed for RNA polymerase to initiate

• the active protein is only available when the gene is to be expressed

– response elements are often cell-type or tissue-specific

• because binding proteins are cell-type specific

• but this is a tautology

– each gene has multiple response elements

• each regulatory event depends on the binding of a protein to a particular response element

• any one of these can independently activate the gene

• combinatorial regulation by multiple elements and proteins is a central mechanism by which levels of gene expression are modulated



– cis-acting control elements can be located many kilobases away from the transcriptional start site

• in intergenic regions

• in introns

• some elements may be quite close to TATA box or other intitiator elements

– cis-acting elements are responsible for allowing the recruitment of TBP and assembly of the initiation complex.


Transcription factors and the preinitiation complex

• Model for cooperative assembly of an activated transcription initiation complex at the TTR promoter in hepatocytes.

– Four activators enriched in hepatocytes plus ubiquitous AP-1 factors bind to sites in the hepatocyte-specific enhancer and promoter-proximal region of the TTR gene

– Activation domains of the bound activators interact extensively with co-activators, TAF subunits of TFIID,Srb/mediator proteins and general transcription factors. This causes looping of DNA and formation of stable initiation complex

– Highly cooperative nature of complex assembly prevents initiation complex from forming in other cells that lack all four of the hepatocyte-enriched transcription factors.



• processing of the transcript to mRNA

– RNA is synthesized as an exact copy of DNA

• heterogeneous nuclear RNA (hnRNA)

– hnRNA gets capped and polyadenylated

– introns are spliced out by the spliceosome, a large complex of RNA and proteins.

• exons can also be spliced out as well. Alternative splicing may produce proteins with new functions.

– Molecular mechanisms underlying alternative splicing are still only poorly understood

– regulation of alternative splicing is important in the CNS and for sex determination

– splice junctions are read in pairs

• spliceosome binds to a 5’ splice donor and scans for a lariat sequence followed by a 3’ splice acceptor

• mutations in either site can lead to exon skipping

– principle underlying gene trapping

– mRNA is now ready for transport to cytoplasm

– some organisms perform trans splicing between mRNAs

• another way to generate mRNA diversity



• transport of mRNA to cytoplasm

– capping, polyadenylation and splicing of mRNA are prerequisites to transport

– macromolecules are specifically transported bidirectionally though nuclear pores

• direction controlled by nuclear import and export signals in macromolecules

– fully processed mRNAs are packaged into ribonucleoprotein particles, mRNPs

• hnRNP proteins contain nuclear export sequences

– These are transported through the pore complex, unwinding as they do so

– On the cytoplasmic side of the pore, the mRNA is stripped from the RNP by binding to ribosomes

– those with signal sequences are paused and subsequently associate with ER

– those without are translated directly



• translation of mRNA

– by default, mRNAs are all translated

– efficiency of translation is important for protein levels.

• regulatory genes tend to be poorly translated

– two primary mediators of efficiency

• consensus around the ATG

– optimum is ACCACCATGG

– most important factor is a G following ATG (A gives about 40% of protein

– underlined sequence will give very high levels of translation - NcoI site

• stability of mRNA in the cytoplasm varies

– many short lived mRNAs have multiple copies of the sequence AUUUA in 3’ UTR



• stability of mRNA (contd)

– others mRNAs are specifically degraded, e.g. transferrin

– in the absence of iron, a specific protein (IRE-BP) binds to a region of the transferring mRNA containing AUUUA sequences

– this protects the mRNA from degradation, transferrin is synthesized and iron accumulates

– iron binds to IRE-BP and dissociates it from mRNA

» AUUUA mediates degradation


Identification of regulatory elements

• Given a gene of interest, how does one go about studying its regulation?

– First step is to isolate cDNA and genomic clones.

– Map cDNA to genomic sequence

• identify introns, exons

• locate approximate transcriptional start

– recognizing elements, e.g. TATA box

– 5’ primer extension or nuclease mapping

• get as much 5’ and 3’ flanking sequence as is possible

– fuse largest chunk of putative promoter you can get to a suitable reporter gene.

– Test whether this sequence is necessary and sufficient for correct regulation

• how much sequence is required for correct regulation?

– what is correct regulation?

» In cultured cells

» in animals?

– typical result is the more you look, the more you find.

• questions are usually asked specifically. That is, what part of the putative promoter is required for activity in cultured liver cells?

– doesn’t always hold in vivo.


Identification of regulatory elements (contd)

• Promoter mapping

– nuclease footprinting of promoter to identify regions that bind proteins

– make various deletion constructs

• Previously made by ExoIII deletions or insertion of linkers (linker scanning)

• typical method today is to PCR parts of the promoter and clone into a promoterless reporter

– map activity of promoter related to deletions

• incremental changes in activity indicate regions important for activity

– test elements for activity


Identification of binding proteins

• How to identify what factors bind to putative elements?

– examine the sequence

• does it contain known binding sites?

• if yes, do such proteins bind to the isolated element in gel-shift experiments?

– do the elements bind proteins from nuclear extracts?

• gel shift (EMSA) experiments

– clone the elements into reporters with minimal promoters.

• do these constructs recapitulate activity?

• Biochemical purification of binding proteins

– tedious, considerable biochemical skill required

– two basic approaches

• fractionate nuclear extracts chromatographically and test fractions for ability to bind the element in EMSA

• DNA-affinity chromatography

– multimerize the element and bind to a resin

– pass nuclear extracts across column and purify specific binding proteins

– protein microsequencing

– predict DNA sequence from amino acid sequence

• look in the database

• prepare oligonucleotides and screen library


Identification of binding proteins (contd)

• Biochemical purification of binding proteins (contd)

– advantages

• gold standard

• if you can purify proteins, this will always work

– disadvantages

• slow, tedious

• need good protein sequencing facility

• biochemical expertise required

• expense of preparing preparative quantities of nuclear extracts

• Molecular biological approaches

– oligonucleotide screening of expression libraries (Singh screening)

• multimerize oligonucleotide and label with 32P

• screen expression library to identify binding proteins

• advantages

– straightforward

– much less biochemical expertise required

– relatively fast

• disadvantages

– can’t detect binding if multiple partners are required

– fair amount of “touch” required



• Molecular biological approaches (contd)

– yeast one-hybrid assay

• clone element of interest into a reporter construct (e.g. -gal) and make stable yeast strain

• transfect in aliquots of cDNA expression libraries that have fragments of DNA fused to yeast activator

• if the fusion protein binds to your element then the reporter gene will be activated

• advantages

– somewhat more of a functional approach

– eukaryotic milieu allows some protein modification

• disadvantages

– slow, tedious purification of positives

– can’t detect dimeric proteins

– sensitivity is not so great

AD

Bait elements Reporter(s)His lacZ



• Molecular biological approaches (contd)

– expression cloning (sib screening)

• clone element of interest (or promoter) into a suitable reporter construct (e.g. luciferase)

• transfect (or inject, or infect, etc) pools (~10,000 cDNAs each) of cDNA expression libraries and assay for reporter gene

• retest positive pools in smaller aliquots (~1000)

• repeat until a pure cDNA is found

– advantages

– functional approach

– presumably using the appropriate cell type so modifications occur

– possibility to detect dimers with endogenous proteins

– disadvantages

• VERY TEDIOUS

• very slow, much duplication in pools, extensive rescreening is required

• could be expensive



– in vitro expression cloning (IVEC)

• transcribe and translate cDNA libraries in vitro into small pools of proteins (~100)

• EMSA to test protein pools for element binding

• unpool cDNAs and retest

• advantages

– functional approach

– smaller pools increase sensitivity

• disadvantages

– can’t detect dimers

– very expensive (TNT lysate)

– considerable rescreening still required

– tedious, countless DNA minipreps required



– hybrid screening system 1

• begin with cDNA libraries in 384-well plates, 1 cDNA per well

• pool cDNAs using robotic workstation

• prepare DNA with robotic workstation

• transcribe and translate protein in vitro

• test for ability to bind DNA element using sensitive, high-throughput assay

– fluorescence

– radioactive assay

• retest components of positive pools

• advantages

– very fast, only two steps required, ~ 2 weeks

– little work required

• disadvantages

– expense of robotics

– won’t detect dimers (unless 1 partner known)

– expense of reagents (TNT, radionuclides, fluorescent labels



– hybrid screening system 2

• prepare reporter cell line with element or promoter driving reporter gene (e.g. luciferase)

• prepare cDNA pools as in system 1

• use robotic workstation to transfect cDNA libraries into reporter cells

• assay for reporter gene

• advantages

– very fast

– truly functional approach

– use of cells allows modifications

– can detect dimers if one partner is already present in cell

• disadvantages

– expense of equipment

• OK, you have your element and binding protein, now what?

– functional analysis depends on type of protein you are dealing with

– goal will be to prove that this protein is necessary and sufficient to confer regulation onto the promoter, in vivo

• many just stop at works on the element


Transcription factors bind to regulatory elements

• The response element binding proteins you have carefully identified are transcription factors.

– There are many types. The primary mode of classification is via the type of DNA-binding domains and intermolecular interactions (next time)

• Features of transcription factors

– typically these proteins have multiple functional domains

• can frequently be rearranged or transferred

– DNA-binding domains

• these domains take many forms that will be discussed next time

• see also the list in TRANSFAC

http://transfac.gbf-braunschweig.de/TRANSFAC/

– Activation domains

• these are polypeptide sequences that activate transcription when fused to a DNA-binding domain

• these are diverse in sequence, 1% of random sequences fused to GAL4 can activate

• many activation domains are rich in acidic residues and assume an amphipathic -helix conformation when associated with coactivator proteins

• interact with histone acetylases that destabilize nucleosomes and open chromatin


Transcription factors bind to regulatory elements (contd)

• Features of transcription factors (contd)

– repression domains

• functional converse of activation domains

• short and diverse in amino acid sequence

– some are rich in hydrophobic aa

– others are rich in basic aa

• some interact with proteins having histone deacetylase activity, stabilizes nucleosomes and condenses chromatin

• others compete with activators for the same sequence and contacts with the transcription machinery

– protein:protein interaction domains

• these are diverse in sequence but do contain structural motifs

• leucine zipper

• helix-loop-helix

BioSci 145A lecture 13 1 © copyright Bruce Blumberg 2000. All rights reserved BioSci 145A Lecture...

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