control of transcription

transcription

DNA is copied into a single stranded RNA molecule, the transfer of genetic information from dsDNA to ssRNA

what is a holoenzyme

the holoenzymes is the core RNA polymerase and sigma70

process of transcription

-sigma70 allows the RNA polymerase to recognise and bind to promoter elements, areas that control txn upstream of the txn start sites

-initial interaction is known as a closed site

-RNA polymerase with sigma 70 pulls apart the strands over the txn start site so the polymerase has access to the template strand

-template moves through the polymerase, txn bubble moves along and is closed up behind

-after the mRNA is about 10 nucleotides long, the sigma factor is released and the rest of elongation is just carried out by the core polymerase

bacterial promoters

-within 60bp of the txn start site

-2 major sequence elements

Promoters = cis acting DNA regulatory element through which txn is initiated and controlled

-more efficient promoter = more RNA and more protein

-nucleotides upstream of txn start site have a minus designation and downstream are positive

-2 basic hexamers at -35 and -10

eukaryotic promoters

-divide into 2 basic regions -> core (basal) region which is located round the txn start site and the regulatory region which can be located over many kilobases

-first core promoter element was the TATA box: located around 30bp upstream, first 4 bases are often T A T A

-the initiator/inr is located over the txn start site but has a very loose sequence, pyrimidine rich sequence

-never find all of the elements together

CpG islands

Core promoter elements: CpG islands

-in mammals 60-70% of protein coding genes lack obvious TATA and inr

-txn initiation occurs at a lower rate and at several start sites

-associated with regions with a high frequency of CG sequences -> CpG islands

-in mammals most C residues followed by G a methylated

-generally C residues in CpG islands escape methylation

-important for promoter function

-methylation of CpG islands is associated with silencing aka txn is switched off

regulatory regions

-can be proximal (within a kilobase) or many kilobases away

-UAS (upstream activating sequence) & enhancer = activator binding sites

-URS (upstream repressing sequences) & silencer = repressor binding sites

tools to identify promoter elements

sequence comparison

reporter analysis

sequence comparison

-lining up promoters, and looking for sequences that are over-represented

-how the TATA box was found

-looking for common patterns

-may or may not have functional importance

reporter analysis

-reporter genes encode proteins whose levels can be easily measured

e.g. GFP, luciferase, LacZ

-amount of reporter protein provides a measure of gene expression

-reporters can be used to identify when a gene is expressed, where it is expressed, what signals it responds to and what factors and sequences control its expression

eukaryotic RNAP

RNAPI
RNAPII
RNAPIII

RNAPI

-transcribes ribosomal RNAs

-in the nucleolus

RNAPII

-transcribes mRNA, snRNAs, miRNAs

-in the nucleus

prokaryotic vs eukaryotic RNAPII

-overall looks similar, same crab claw shape in the bacterial polymerase

-eukaryotic has 12 subunits

-prokaryotic has 5 subunits, some subunits are analogous to eukaryotic

RNAPIII

-transcribes tRNAs, 5S RNAs, U6 RNA, 7S RNA

-in the nucleus

general transcription factors (GTFs)

-bacterial RNA polymerase requires σ-factor to recognise promoters

-they are RNA poll specific

-multi component factors

-form a complex over the TATA box

-recruit RNA pol II to the promoter

-direct initiation at the start-site

-in eukaryotes they have multiple transcription factors:

• TFIIA

• TFIIB

• TFIID

• TFIIE

• TFIIF

• TFIIH

TFIIA

-3 subunits

-stabilises TFIID binding

-anti repression function

TFIIB

-1 subunit

-recruits RNA pol II – TFIIF

-important for start site selection

TFIID

-13 subunits

-binds to the TATA box

-recruits TFIIB

-made up of TATA binding protein (TBP) (the central subunit) + TBP associated factors (TAFs)

-TBP alone can direct the assembly of the PIC on a TATA containing promoter

-cannot do this on a TATA-less promoter

-TBP can not support ‘activated’ transcription

-TAFs promote the interaction of TFIID with basal promoter elements and they interact with activators to promote transcription initiation

TFIIE

-2 subunits

-helps recruit TFIIH and modulates its activity

TFIIF

-2 subunits

-assists TFIIB recruiting RNA pol II

-stimulates RNA pol II elongation

TFIIH

-9 subunits

-promoter melting and clearance

-CTD kinase activity

-DNA repair coupling

-can be divided into two parts: CORE + CAK

-CAK module contains one of the kinases that phosphorylates the CTD of RNA pol II

-TFIIH contains an ATPase (called XPB) that is involved in promoter melting

pre-initiation complex assembly

-called an order of addition

-how people got them to assemble within a test tube

-useful as a framework to understand the process

-in vivo it won’t happen in this nice order

importance of the different GTFs

-key part is the binding of TFIID over the TATA box

-then add TFIIA and TFIIB, TFIIA helps stabilise TFIID

-TFIIB is critical, brings in RNA polymerase to the complex

-TFIIF and TFIIE then bind, TFIIE helps TFIIH to bind which is critical

-equivalent to the closed complex in bacteria

-promoter DNA is opened by TFIIH, helicase activity of the TFIIH separates the strands over the txn start site, requires ATP hydrolysis

-as pol II transcribes (called promoter clearance) it’s extensively phosphorylated on the C-terminal domain

-CTD is a series of repeats at the C-terminal end of the largest of pol II

-TFIID and TFIIA may stay behind

-TFIIB, TFIIE and TFIIH are released

-TFIIF moves down the template with pol II

function of UAS/enhancer elements

-basal txn = low/inactive

-activated txn = high

-only get high levels of transcription from a promoter if you have a core promoter, an initiator and some activator binding sites

classifications of UAS/enhancer elements

common sequence elements

response elements

common sequence elements

e.g. GC box, octamer, CAAT box

-often located close to the core promoter (promoter proximal)

-bind activators that are relatively abundant in the cell and constitutively (constantly) active

-job is to boost basal level of txn from a particular promoter

response elements

e.g.

- SRE: binds serum response factor, inducer is growth factors

- HSE: binds heat shock factor, inducer is heat shock

-bind factors whose activity is controlled in response to specific stimuli

what does the combination of elements determine

-the combination of elements in a promoter determines where and what level of genes are expressed, and what promoters are transcribed

enhancer location

-enhancer = will work irrespective of its location and orientation

-the enhancer can be downstream of the promoter

-DNA is flexible so activators associated with enhancers can be brought into close contact with general transcription factors at the core promoter many bases away

-activators associated with enhancers are brought into contact with GTF/RNA pol II at the core promoter by looping out of the intervening DNA

eukaryotic activators

-eukaryotic activators are modular

-the activation domain and the DNA binding domain are modular and work when separated

-nearly all have a single DNA binding domain but can have multiple activation domains

examples of DNA binding domains

leucine zipper, zinc finger, homeodomain, helix loop helix

activation domains

-often characterised according to their amino acid composition

- Acidic patch

-clusters of -vely charged residues

e.g. VP16

- Glutamine rich

-high gln content

e.g. SP1

- Proline rich

e.g. Jun

-lack of sequence conservation and structural information

-generally thought to be unstructured

-contain multiple short segments that work together in an additive fashion, will still work (just less well) if you truncate some away

-interact with other proteins in the transcriptional machinery (e.g. TAFs)

analysing activators in vitro

DNA footprinting

electrophoretic mobility shift assays

transcription assays

electrophoretic mobility shift assays

-also known as a gel shift

-measures the ability of a protein to bind a particular DNA sequence

-have a source of the activator protein which you mix with some radiolabelled probe DNA

-run on non-denaturing acrylamide gel

transcription assays

-measuring the function of the activator protein

-reconstitute the process of transcription in a test tube

-need RNA pol II, GTFs, DNA template, radiolabelled rNTPs

-requires the activator to have a functional DNA binding domain (DBD) and a functional activation domain (AD)

*may come up in exam

analysing activators in vivo

reporter assay

chromatin immunoprecipitation (ChIP)

reporter assay

-have a plasmid that will encode the activator

-second plasmid will have a binding site for that protein, controlling the expression of a reporter gene

-cotransfect the plasmid

-migrate into the nucleus and activate txn of the reporter gene

-reporter transcripts are made which you can then use to measure the ability of the protein to stimulate transcription

-can then investigate what sites and sequences are important

chromatin immunoprecipitation

-helped understand where proteins are bound in the cell

-can map all the binding sites

-take the cells and add a cross-linking agent, it cross-links all the proteins that are bound to the DNA

-glues the activator in place

-then isolate the chromatin and shear DNA

-use an antibody that is specific to protein, can then purify (precipitate) this

-can then reverse the cross-links and digest the protein, leaving the binding sites for that protein

-can either be analysed by PCR or being put through high-throughput sequencing

how do activators work

1. binding of one activator can promote the binding of another activator, referred to as cooperative binding

2. Major mechanism is that they stimulate assembly of the pre initiation complex

3. release stalled RNAP

4. modulation of chromatin

major mechanism is that they stimulate assembly of the pre initiation complex

-they provide additional contacts that stimulate the ability of RNAP to bind to the core promoter

Interact with:

-TFIID

-TFIIB: interactions between activation domain and TFIIB help bind to the promotor, TFIIB brings in RNAPII

-mediator

- Many activators cannot activate transcription in minimal in vitro transcription systems

- This suggested that activation must require additional factors

- Searches for such factors using yeast led to the discovery of the mediator complex

- Subsequently an analogous mediator complex was found in humans

- Mediator is a very large complex of approx. 22 polypeptides

- Can exist on its own or associated with RNAPII (through the c-terminal domain)

- EM studies have revealed that mediator is composed of three domains: head, middle and tail

- Many activators interact with specific mediator subunits

- Mediator provides a bridge between activators and RNAPII

- Mediator-activator interactions aid recruitment of RNAPII and therefore enhance PIC formation

release stalled RNAP (stimulate activity)

-can stall at or near to the promoter

-RNAP is assembled, open complex has occurred, first few nucleotides of RNA have been made and then RNAP gets stuck

-active activator proteins release stalled RNAPII

e.g. heat shock genes; in the absence of heat shock, RNAPII pauses after ~50nts; heat shock activates HSF transcription factor which interacts with RNAPII and releases it from the pause

modulation of chromatin

-re-modelling of chromatin allows complex formation

-eukaryotic DNA is not naked, its associated with histones which form the chromatin complex

-without it, chromatin tends to inhibit transcription hence the remodelling

packaging of eukaryotic DNA by chromatin

-all eukaryotic cells have to solve a major problem

-need to fit a lot of DNA in a small space

-the human genome is 2m in length and has to be packaged into a nucleus just a few microns in diameter

-achieved by assembling DNA in chromatin

-basic function of chromatin is to compact DNA

composition of DNA

-composed primarily of small basic proteins called histones

-2 major types of histones: core histones and linker histones

core histones

- core histones (H2A, H2B, H3, H4) are highly conserved; a globular domain made up of α-helices and loops

- they fold (called a histone fold) as the different globular domains binds forming loops

- 2 histone folds can then interact to form a ‘handshake’ interaction

- also have flexible N-terminal tails; the are highly basic and rich in lysine and arginine

- form repeating units called nucleosomes

- nucleosomes is composed of about 147bp of DNA wrapped twice around an octamer of histone proteins

- octamer is composed of a central H3-H4 tetramer and 2 flanking H2A-H2B dimers

- this octamer is not stable unless it has DNA wrapped around it

nucleosome organisation

- DNA passes directly from one nucleosome to the next

- Linker histones such as histone H1 bind to DNA between nucleosomes

- In vitro linker histones result in the formation of a thicker 30nm fibre

- Evidence suggests that a regularly folded 30nm fibre is unlikely to exist in vivo

evidence that chromatin inhibits transcription

in vitro reconstitution experiments

in vivo nucleosome positioning experiments

genetic studies in saccharomyces cervisiae (budding yeast)

in vitro reconstitution experiments

-adding a naked DNA template to RNAPII and transcription factors results in transcription

-adding a chromatin template to RNAPII and transcription factors results in no transcription

in vivo nucleosome positioning experiments

-numerous experiments have shown that nucleosomes are disrupted or lost during transcriptional activation

genetic studies in saccharomyces cervisiae (budding yeast)

-nucleosome number was experimentally controlled using a specifically engineered yeast strain

-chromosomal copies of H4 genes were deleted and a plasmid expressing H4 under the control of a ‘regulatable’ promoter is present

-promoter is the GAL4 promoter

-this is on when there is galactose in the medium but off when glucose is present

-therefore when glucose was added to the medium the expression of H4 was rapidly shut off

-this resulted in nucleosome depletion and the expression of many inducible genes

conflicting roles of the nucleosome in the nucleus

-has to compact DNA as well as forming a template for DNA transcription

-chromatin structure is dynamic

-cells have mechanisms to modulate chromatin structure

3 major mechanisms for the control of chromatin structure

histone variants

post-translational modification of histones

ATP dependent chromatin remodelling

histone variants

-histone variants are encoded by genes that differ from the highly conserved major types

-variants are expressed at very low levels compared to their conventional counterparts

-all the conventional histones, except H4 have variants

-histone variants confers novel structural and functional properties of the nucleosome which affect chromatin dynamics

post-translational modification of histones

-the N-terminal tails are the sites for extensive post-translational modification

-can be acetylation, methylation, ubiquitylation, phosphorylation etc

-key role is controlling gene expression

-histone modification state has been proposed to constitute a code that sets its transcriptional state

-could directly alter chromatin folding/structure

-could control the recruitment of non-histone proteins to chromatin (which in turn influence the recruitment/function of the transcriptional machinery)

• acetylation

• histone methylation

• lysine methylation and control of transcription

acetylation

-occurs on lysine residues

-mediated by histone acetyl transferases (HATs)

-the HATs will take acetyl-CoA and use that and add an acetyl group to the lysine

-acetylation can be readily reversed by HDACs (histone deactylases), therefore the acetylation state of histones is highly dynamic

-correlation between high levels of acetylation and transcription

-the first nuclear HAT was shown to be homologous to yeast GCN5, important because GCN5 was known to function as a transcriptional activator

-this confirmed that acetylation is a key component of transcriptional activation

-nuclear HATs are now known to function in large multisubunit complexes of two major types: GNAT family and MYST family

-activators recruit HATs to specific promoters

-many HAT complexes contain a specific subunit that interacts with activators e.g. Tra1, TRRAP

-some HATs are part of the general transcription machinery

how does acetylation mediate transcriptional activation

- Direct influence on chromatin structure

-due to acetylation, you lose the positive charge of the lysine

-this allows the chromatin fibre to compact

-not normally sufficient to have enough of an effect

- Directs the recruitment of bromodomain proteins

-acetyllysine directs this

-specific acetylated lysine residues are recognised by proteins with bromodomains

-bromodomain proteins often promote transcription

e.g. Bdf 1 (binds acetylated H4 and recruits TFIID)

e.g. TAF1 (TFIID subunit, also binds acetylated H4)

histone methylation

-can occur on lysine, and also arginine but less well understood

-lysines may be mono, di or tri methylated by HKMTs (often contain a SET domain)

-methylation is not readily reversible by demethylases do exist

-methylation does not affect charge so probably has only minor is any influence on chromatin structure

lysine methylation and control of transcription

-methylated lysines function as beacons/landing pads for particular proteins that have particular types of domains

-depending on the context methyl-lysine residues can function either as ‘activating’ or ‘repressing marks’

ATP-dependent chromatin remodelling

-cells have multiple remodelling complexes (most are multisubunit complexes)

-at least 22 in our cells

-all have a Snf2-related ATPase ‘helicase and NTP driven nucleic acid translocase’ superfamily 2 (SF2)

-at least 4 distinct families, which are characterized by additional domains and the architecture of the ATPase domain

-they use the energy from ATP hydrolysis to drive a variety of reactions with chromatin

-depending on the complex they can catalyse one or more of the reactions: sliding, unwrapping, eviction, spacing or histone variant exchange

how does the SWI/SNF remodel chromatin

-was the first complex to be isolated, came from yeast

-catalytic subunit is Snf2

-it hydrolyses 1000 ATP molecules/minute in the presence of DNA or nucleosomes

-Snf2 is related to DNA helicases

-DNA helicase use the energy from ATP hydrolysis to unwind nucleic acids

-Snf2 is thought to be a molecular motor that tracks along DNA and introduces torsion/stress which promotes movement of DNA relative to the nucleosome

-results in disruption of histone-DNA interactions and movement of the nucleosome

ATP-dependent and HAT complexes co-operate

-SWI/SNF and the GCN5 HAT regulate the same genes in yeast

-these HATs and ATP-dependent remodellers are commonly recruited to the same promoters

-bromodomains in Snf2 help tether it to acetylated nucleosomes

-HATs and ATP-dependent remodellers function co-operatively

yeast SWI/SNF

-general transcription: regulates the expression of 5% of the 6000 yeast genes

-cell cycle; regulates 25% of genes expressed at the end of mitosis

human: PBAF, cBAF, ncBAF

-3 related complexes with some subunits shared

-general transcription: needed for a number of txn factors; glucocorticoid and retinoid receptors, heat shock factor etc

-roles in cell cycle control via interaction with Rb and cyclin E

-roles in development, deletion in mice results in embryonic lethality

-role in tumour suppressor pathways; mutations are associated with a variety of tumour types

SWI/SNF and cancer

-at least 9 different genes encoding subunits of the SWI/SNF complexes are recurrently mutated in cancer, and these mutations are collectively present in nearly 25% of cancers

-mutations in specific SWI/SNF genes are enriched in particular cancer types

-different SWI/SNF gene mutations confer distinct cancer vulnerabilities in mouse models

-the tumour-suppressor activity of the SWI/SNF complexes most likely due roles in facilitating transcription factor function

-the identification of potential therapeutic vulnerabilities that arise from SWI/SNF gene mutations is leading to new areas of clinical investigation

-mutations in genes encoding SWI/SNF subunits, which include nonsense, frameshift and deletion mutations suggestive loss-of-function

repression of transcription

-cells commonly exploit chromatin structure to bring about transcriptional repression

-mediated by the recruitment of chromatin modifying factors e.g.

• histone deactylases (HDACs)

• ATP-dependent remodellers

• histone methylases (heterochromatin)

histone deacetylases

-whereas active regions of the genomes are hyperacetylated, repressed regions are hypoacetylated

-deacetylation is mediated by histone deacetylases (HDACs)

-just as HATs were shown to be transcriptional co-activators, HDACs function as co-repressors

-4 major groups: class I, II and IV (classical HDACs = zinc dependent), class III (Sir2 family, require NAD as a co-factor)

Co-repressor complexes:

- HDACs commonly function in the context of large multi-subunit complexes

- E.g. SIN3 co-repressor complexes, which are conserved from yeast to mammals

- Recruited to promoters by interaction with site-specific DNA binding proteins

ATP-dependent remodellers

-some commonly mediate transcriptional repression

-e.g. the NuRD complex which belongs to the Mi2/CHD family

-highly conserved in plants and animals, it is broadly expressed in most tissues and plays roles in normal differentiation and tumourogenesis

-multi-subunit complex that also contains HDACs

-closed chromatin = txn off, open chromatin = txn on

histone methylases

-2 basic types of chromatin = euchromatin: gene rich, potential to be transcribed; heterochromatin: gene poor, repetitive regions, transcriptional silencing e.g. centromeres and telomeres

-biochemical features of heterochromatin

- Hypoacetylation

- Specific histone H3 methylation

- Association of specific silencing factors

-assembly of heterochromatin

- Need some deacetylase to remove any acetyl marks on the lysine

- Acetyl group needs to be removed by histone deacetylase

- Lysine is now accessible to the histone transferase

- Suvar39 methylates histone H3 and lysine 9

- This methyl modification is then recognised by a specific protein called HP1

-heterochromatin protein 1 (HP1) is a chromodomain protein; chromodomains often recognise and bind to methylated lysine residues

-the chromodomain of HP1 is specific for H3 Lys9me2/3

-binding of HP1 is thought to compact nucleosomal arrays

-and act as a platform for the recruitment of further activities that prevent recruitment/ activity of RNAPII

analysis of heterochromatin using reporter silencing assays

-in fission yeast

-reporter gene reports on the activity

-when the ade6 gene is expressed you get the formation of white colonies on agar

-remove the ade6 gene from its normal locus and put it in the heterochromatic centromeric repeats

-the heterochromatin went over it and caused it to be transcriptionally silent

-this forms red colonies, they result from build up of a red pigment which is an adenine biosynthetic intermediate

heterochromatin: X-chromosome inactivation

-females have 2 X chromosomes, one of which is inactivated

-this equalises the number of X-linked genes expressed in males and females

-the inactivated X-chrm is seen in the nucleus as a condensed structure (Barr body) that is assembled into a specific form of heterochromatin

-formation of the Barr body is controlled by non coding RNAs Xist and Tsix

-very complicated process