Mechanisms and Control of Gene Transcription II Flashcards
1
Q
Polymerases in eukaryotes
A
- 3 types, distinguished by sensitivity to a-aminitin
- Pol I = rRNA
- Pol II = mRNA
- Pol III = tRNA + snRNA
2
Q
Promoter
A
- Core promoter = minimal portion of promoter needed for initiation at TSSS
- 34bp us of TSS = bs for POL
- Core promoter binds general TF specific for each type of polymerase
- Pol I promoter = UCE, core promoter, pppA/G
3
Q
Pol I TF
A
- UCR of Pol I binds UBF1 (removes nucleosome)
- Core promoter binds 4 proteins inc RRN3
- TBP interacts w/ TAF 63 + 110
- SL1 recognises promoter + recruits Pol1a
4
Q
Pol III promoter
A
- Unusual = promoter lies ds of TSS
- URS = us of TSS
- Core promoter = +55 and +80
- TFIIIC recognises core promoter w/ zinc fingers
- TFIIIB also binds
- RNA pol III recruited by TFIIIC/B
5
Q
Pol II promoter
A
- Has 6 more proteins: TFIIB,D,A,F,D + H
- Multiple different elements, x conserved, act in combination
- Tata box, BREu/d, Initiator elements, ds elements like DCE
- TBP binds minor groove + crates bend, associates w/ TAFII
- TFIIB binds DNA, TAF2 binds initiator, TAF9+6 interact w/ DPE
- TFIID recruitment = essential
6
Q
Conserved factors in initiation
A
- TATA binding protein
- Only acts at small no. of promoters transcribed by RNA Pol II
- Pol II = SL1, hRRN3 + UBP3
- Pol III = TFiiB, BRF, TFIIIC
- Pol I = TBP + TFP
7
Q
Eukaryotic RNA Pol III TF vs σ70
A
- TBP + σ bend DNA
- TFIIB controls start site selection vs σ
- TFIIE melts DNA vs σ
- Non-template strand captured by TFIIF vs σ domain 2
- Template strand interacts w/ TFIIB finger vs σ domain 3
- TFIIB competes w/ RNA of >10bp for saddle, structural Δ in sigma promotes release
8
Q
Types of promoter transcribed by Pol II
A
- Distinct types of core promoter for transcription initiation by RNA Pol II
- Yeast = 1 type, conserved TSS, also found in mammals, has TATA, Inr, MTE, DPE x CGI
- Type II = drives expression of house keeping genes, 70%, dispersed, have CGI, x TATA
- Type III = developmentally regulated promoter, mixed
- Vertebrates = mainly focused (either single TSS or distinct cluster of start sites) or dispersed (several start sites over 50-100nt, typically found in CPG islands)
- S cerevisiae = just focused, d melanogaster = focus + dispersed or mixed
9
Q
CPG islands
A
- Dinucleotide C followed by G = often methylated at 5 of Cys after DNA synthesis
- Similar to bacteria
- 20% of expected frequency as methylated lys is deam to T
- CpG islands = clusters of C/G dinucleotides that are not methylated, found at promoters
- Methylation prevent TF/promote TF binding
- Have ↑ frequency of bs for TFs just us of TSS
10
Q
Methods for studying promoter/enhancer
A
- Confirm promoter
- Either destroy by deletion of mutation (CRIPSR cas9) + see effect on gene expression
- Make a chimeric gene e.g. to see if HSE really HSE make chimeric gene w/ HSE added to gene w/o HSE
- Or could fuse promoter/enhancer to ‘reporter gene’
- Cor element alone x enough to give ↑ levels of expression of reporter gene, promoter/reporter gene hybrid is introduced into cells + activity of promoter assessed
11
Q
Promoter + enhancer for genes = modular organisation
A
- Human hsp70 gene promoter = modular structure
- Can mix and match regulatory element that binds TF w/ core element
- Enhancers have regulatory element, activates expression from promoter in response to signals
- Enhancers activate a promoter when placed up to 100- bp from promoter
12
Q
How do enhancers work?
A
- Using SV40 DNA
- Has enhancer, core promoter, x promoter regulatory elements
- CG box binds TF SPI, gives promoter w/ low level of expression
- Need enhancer, us of promoter
- C, B + part of A = 72bp repeat, 1 = enough, have 2
- A, B + C contain individual enhansons
- Experiment (fuse TATA box to reporter gene, plate w/ ↑ cells, transvect TATA + reporter in, 72bp repeat gives enhancer + cells survive, then use just C, some survive, take + grow w/ G418, see duplicated C, same w/ A + B
- Showed 2 protoenhancer needed
- Enhancers often composed of same sequence of elements found in promoter e.g. AP1 bs
- SV40 has multiple TF, bs mutually exclusive
13
Q
Superenhancer
A
- Control a gene cluster on chromatin loop anchored by CTCF DNA binding protein at each end
- Interact w/ Mediator
- E.g. in a or B globin loci, confer tissue + stage specific global mRNA expression
- Resembles an enhancer, 5/6 DNAse hypersensitive sites
- MFine tune gene expression
- Evidence (mRNA spiced + placed in human B global gene into mouse, took larger parts until cloned region of 60kB + found global genes expressed where should be)
14
Q
Transcription factors
A
- Binds to promoters + enhancers
- Proteins bind to DNA, especially in chromatin, creates DNAse I hypersensitive sites bends DNA
- ‘Footprint’ next to DNA hypersensitive site, protein binding means DNAse I x cleave
- Factors that influence ability of TF to bind = TF-TF interaction, TF-cofactor interaction, DNA modifications, DNA shape, genomic context
15
Q
AP1
A
- TFs often bind to promoter + enhancer as dimer
- Homodimer of C-Jun or heterodimer of C-Jun/c-Fos bind w/ different affinities
- AP1 recognition site has dyad symmetry
- AP1 TF bind AP1 have diff affinities (Fos:Jun hetemrodimer binds AP1 30x ↑ than Jun homodimer)
- Fos + Jun have 2 1/2 sites in NTD, CT leucine zipper
- Specificity of interactions btw Fos/Jun - residues of hydrophobic face (Leu zipper a+d) + residues surrounding hydrophobic face (e+g)
- Fos:Fos homodimers x form
16
Q
Detecting TF bound to DNA in vitro
A
- ChIP
- EMSA (radioactively labelled DNA incubated w/ nuclear protein, run on gel, compare to DNA w/o protein, complexes migrate slower than free DNA, Ab raised against protein complex, can Δ conditions
17
Q
Regulating processes via cooperativity
A
- E.g. reprogramming
- Most cells are differentiated
- Can add genes encoding TF Oct4, Klf4, could reprogram somatic cells back to pluripotent state
- If add different signals, can re-differentiate into different cell types e.g. fibroblast → neurone
- As reprogram, switch off somatic enhancers + switch on enhancers for pluripotency
18
Q
Modular TF experiment
A
- ‘Domain swap’ experiment
- GAL4 has NT DBD + TAD
- WT DBD + TAD binds enhancer us activator sequence → transcription
- Take away CTD + add different oligomerisation domain
- Replace activator w/ Lex operator
- Mix and match TAD + DBD
- Y2H (Gal4 DBD + bait, Gal4 TAD + prey)
19
Q
Consensus sequence vs responsiveness
A
- Based on steroid-thyroid hormone receptor family TF
- Respond to presence of hormones
- Bind to major groove on DNA
- Thyroid hormone bs = dyad symmetry, have GGT
- Also have DBD, TAD + hormone binding domain
- Experiment = oestrogen receptor + DBD of glucocorticoid receptor, aa substitution
20
Q
How do TF function
A
- us enhancer w/ ↑ bs + promoter w/ ↑ bs, work together
1. (like prokaryotes), A + B work to recruit RNAP + PIC to open DNA + get transcription, RLS = RNAP recruitment
2. Nucleosome depleted region → RNAP recruited → Pol stalls, waits for processing of transcript, RLS = release of promoter into early elongation + escape
21
Q
Enhancer
A
- Multiple transcription bs
- Enhancer activation coincides w/ DNAse I hypersensitivity
- Chromosomes around have specific PTMs
- Associated w/ divergent transcription
- 20x more enhancers than genes
- ‘Superenhancers’ - LCR that control major developmental switch
- Use NET-Seq
22
Q
How do enhancers interact with promoters
A
- Flies = enhancers show specificity for different types of promoter (housekeeping vs developmental)
- Housekeeping genes have proximal enhancers, developmentally regulated = distal enhancer
- Experiment (GALF4 fused to TF of interest)
- Coactivator = help enhancers talk to promoter, facilitate interaction btw TF bound to proximal/distal enhancer + PIC
- Co repressor = prevent enhancer talking to promoter
- Cohesin may stables the interactions
- TFIIH + mediator have CDks, act on RNAPII + help release from promoter
23
Q
Insulators + promoter interaction
A
- Not of insulator binding proteins e.g. CTCF (TF that interacts w/ DNA, needs to bind insulator)
- ICR = another insulator
- Methylation on females = different to paternal
- ICR separates promoter form enhancer
- When ICR = methylated, CTF x bind paternal but does bind maternal (no methylation), stops enhancer talking to promoter
24
Q
Chromatin + epigenetics
A
- DNA methylation + histone PTM influences DNA
- Imprint = different modification on female + male chromosomes at fertilisation
- Disregulation of chromatin = associated w/ ↑ disease
- Heritable epigenetic phenomena e.g. PEV + X chromosome inactivation
25
Q
Chromatin structure
A
- Chromatin neutralises -ve charge on backbone of DNA
- Compaction through chromatin itself + through loops within loops
- Has to be accessibly structure so can pack + unpack
- Histones in interphase nucleus, salt competes w/ histone
26
Q
Chromatin methylation
A
- CpG assoc w/ gene expression
- PTM associated w/ active/repressed genes
- DNMT = deposits methylation, TETS = remove methylation
- Readers of methylation = MeCBP
27
Q
PTM to histones
A
- Modify tails, amino-terminal regions
- Acetylation, methylation, ubiquitylation
- Writers = KAT, KMT, Ub ligases
- Erasers = KDCA, KDM, DUB
- Reader = bromodomain, chromodomain/ PHD
28
Q
Histone assembly
A
- Dimer of H3/4 or assemble tetramer (unclear)
- FACT/CAF put together + add H2A-H2B
- ↑ nucleosomes in a typical nucleus
- PTM to histones distinguish genes
- K4 = active, K9/K27 = active or repressed
29
Q
Chromatin remodelling ATP families
A
- Use ATP hydrolysis to pump DNA over surface of nucleosome
- 3 main families: SWI/SNF (bromo domain that reads Lys acetyl) ISWI family (SANT domain that reads unmodified tail), Mi-2 family (PHD finger, chromo domain reads methyl lysine)
- Experiment = add chaperones to nucleosomes, addd activator
- Nucleosomes = deposited in acetylated state, then modified by KAT or KDACs
- When DNA wrapped over nucleosome, DNA is bent + E like DNAse I access major groove of DNA in nucleosome
- 2 types of nucleosomal organisation at promoters: 1. micrococcal nuclease acts in linked, 2. nucleosomal contact at promoter
30
Q
Dynamic DNA methylation + histone modfiication
A
- Like E coli DNA = hemimethylated after replication
- DNA can be demethyl from 5 methyl cyst to 5hmc by TET
- DNA methyl transferases need SAM to methylate
- Methyl C = inactive promoter, de-methyl = active
- Insulators can also be non-methylated, can bind TF
31
Q
DNA methylation in development
A
- After fertilisation, sperm DNA demethyl, egg DNA slower demethyl
- Imprinted region remains methylated, cells that go from gonad demethyl slower than other gonad cells
- When produce gametes, sperm rapidly determines/re-establishes methylation in prenatal male germ cells, meth= in oocytes, slower
- TETs
32
Q
ChIP
A
- Allows access to where + what level histone modification are
- Cross-link DNA to protein, lyse cells, sonicate/ E digest, use Ab against different histone modifications, immunoprecipitate, analyse bound DNA
33
Q
Histone modification on genes
A
- Lys4 in H3 = active when methyl, next to TSS
- Lys36 methyl x deposited over body of gene
- Lys27 acetyl = repressive, assoc w/ promoter + enhancer
- Enhancer vs promoter, enhancer = ↑ rich in mono H34MeI than trimethyl
34
Q
Histone modification influenced by state of the cell
A
- Methylated
TET Es can also demethylate histones, a-ketoglutarate + O2 dependent, LSDs = demethylases, methyltransferases - Acetylation/deacetylation
Acetyl coA = substrate, butyrate inhibits HDAC
35
Q
Function of covalent modification to protein + histone
A
- Acetylation
Added w/ KAT, removed w/ HDAC, assoc w/ active chromatin, at promoters, DNA ↑ accessible - Reader proteins
‘Read’ epigenetic code on histones + DNA + recruit other factors to regions of chromatin, Kme = read by HP1/PHD1 - Methylation
- insulator binding protein = sensitive to binding at methylated site, MeCP2 binds methylated DNA, ↑ TF bind
36
Q
Reading histone code
A
- V specific
- Methyl-lysine binding domain can distinguish
- Different forms of methylation, Me1-3, tudor domain binds KMe2 x KMe3
- Different lys residues methylated
- Same methylated residues in different context
37
Q
Different H3K4Me binding protein
A
- CHD1 = chromatin remodelling ATPase
- PHD of YNG1 acetylates Lys314, leads to writing of chromatin mod at other sites, inhibits DNA methyltransferase
- BPTF PHD + bromodomain allow nucleosome movement only when other modifications are present
- Modifications can recruit PIC e.g. TFIID
- Spread K4Me
38
Q
Spreading of histone modification
A
- PHD on KMT MLL1 can methylate on L4 H3
- Once MLL1 deposited Lys 4, binds + modifies Lys4 on neighbouring nucleosome or other H3 tail
- Gen5 KAT deposits acetylation on K9H3, then bromodomain acts as KAT + put same mod
- Spreading continues until boundary element
39
Q
Additional factors recruited by readers
A
- MeCP2 binds methylated DNA + recruits Lys-deacetylase that de-acetylates neighbouring nucleosome
- Rhett syndrome
- Interaction site on MeCP2 acts as transcriptional repressor domain out of context
40
Q
Reader proteins + heterochromatin
A
- 2 types of chromatin in nucleus, heterochromatin + euchromatin (repressive)
- Heterochromatin = centromere, telomere, LINE/SINE
- Heterochromatin = either constitutive or facultative (inducible, assoc w/ genes, can be spread at a single nucleosome)
41
Q
2 types of spreading
A
- Suv39 KMT binds H3K9me3 to spread modification
- Suv39 can be recruited through reader
- HP1 reads H3K9Me3, can recruit Suv39 to allow ↑ Lys9 to be deposited
- Reader can also recruit locus-specific protein that changes region of nucleosome in different context
- HP1 recruits factors that control chromosome biology e.g. cohesin
- Lys 9 + HP1 = associated w/ heterochromatin
- Recruit transcriptional activators
42
Q
Position effect variegation
A
- Spreading of heterochromatin effects expression of nearby gene
- E.g. variegated eye colour in Drosphilia
- White gene = red eyes in euchromatin, always expressed, some flies undergo chromosome inversion so white gene closer to centromere, removes barrier for gene expression, get variegated response
- Experiment = flies fed mutagens, look for 2nd site mutations
- 2 types of mutation: 1. enhancer (↓ red ↑ white) 2. suppressor (↑ white, ↑ red)
- Su(var)2-5 mutation in HP1 + Su(Var)8-9 mutation in H3K9
43
Q
Specificity of modifications
A
- Centromere = constitutive heterochromatin
- H3K9Me3 = mark, HP1 = reader
- Experiment (screen for genes to silence gene inserted into heterochromatin, insert marker, if repressed x grown on Ura- medium, mutagenise + look for mutagens)
- Found mutations in TF or ATF + CREB family
- Underlying DNA recruits ATF/CREB TF which recruit HDAC + HMAT, allow conversion of active acetylated → repressed chromatin
44
Q
Lysine acetyl transferase
A
e. g. = CBP/p300 = domain for K acetylation, bromodomain (reader) so recruited to mark
- Nuclear hormone receptor recruit p300
- TF clockbnids directly to DNA w/ DBD, has KAT activity, recruits other factors
- Cyclin E, use E2F TF to mediate 3 states: 1. proliferating cell cycle, E2F recruits p300, acetylate + phosphorylate pRb so x interact w/ E2F, 2. Non-proliferating gene off, Rb x phosphorylated, competes w/ p300 for binding on E2F, HDAC deacetylated, 3. full off, Rb also has Suv39 methyl transferase, self-sustaining, HP1 recruits reader
45
Q
Steroid thyroid hormone superfamily
A
- Binds to nucleosomal DNA, dimer of receptors
- Then recruits factors to remodel nucleosome e.g. chromatin remodelling
- Chromatin remodelling = dynamic
- E.g. mammalian p52 promoter, binds oestrogen receptor at ERE, NucE/T x bind until nucleosome is unwrapped
- Experiment = reconstitute 2 nucleosomes that shield bs for promoter, measure GR bound over time
- As ER binds, it associates w/ KAT + recruits SWI/SNF
- As ER leaves promoter, its associated w/ KDAC
- Receptor cycling
- Receptor is targeted for degradation by binding proteosome
- If withdraw hormone need mechanism to switch gene rapidly, cycling of TBP
46
Q
How to get modification where required w/ ncRNA
A
- Genetic screen w/ S pome
- Showed mutation in genes encoding parts of RNAi silencing machinery
Cytoplasmic
- RNAi = Dicer, argonaute = Idr
- Dicer cuts ds RNA to 21-23bp RNA, cut into ss RNA, assoc w/ Argonaut, if homologous to mRNA ss binds, 5’OH = target for exosome, 5’P = substrates for Rat1
Nucleus
- In S phase, chromatin split into 2 strands, need to re-establish heterochromatin using RNA
- Use RITS to degrade nascent RNA → chromatin that stops transcription
- Dicer processes dsRNA → 21-23nt, spliced by Argo
- If homologous to ssRNA, Argo binds transcript
- RITS has chromatin remodelling, assoc w/ KDAC + KMT
- Removes acetylation + binds K9 methylation
47
Q
Reversing repression
A
- Heterochromatin = assoc w/ H1, makes stable nucleosome but transcription machinery x interact
- H1 = non-core histone, interacts w/ strands of DNA
- Linker DNA cleaved by MNase
- Certain TFs bind to nucleosomal arrays + displace H1 e.g. forkhead factors
- Act as molecular mimic of H1
- Bind enhancers + super enhancers as control major developmental switches e.g. distal enhancer
- Reprogram TF so when added together make IPAC state + get closed chromatin
48
Q
How are chromosomes organised in interphase nucleus
A
- Chromosomes occupy distinct territories in nucleus
- Equilibrium model for territories - chromatin in random conformation, DNA access hard
- Factal globule - best evidence
- Within territories, individual chromosomes move back + forth btw open + closed compartment
- Globules bring regions that are far in space together
- HiC - ligate RE ends to a linker w/ marker that aids in purification, cut to small sequences, de-cross link, amplify + deep sequence
- HiC = high interaction lie off diagonal, know DNA binding proteins,
49
Q
How does CTCF break boundaries
A
- CTCF binds boundaries of TADs
- Bind in head-to-head orientation
- CTCF recruits + interacts w/ ATPase cohesin + condensin that bring together 2 strands of DNA, pump DNA through ring, makes condensed structure
- Loop extrusion needs E
- HiC w/o CTCF → need CTCF
50
Q
Formation of heterochromatic compartments
A
- Chromatin is shaped by interplay of phase separation + TAD by active loop extrusion
51
Q
Is chromatin organisation assoc w/ gene expression
A
- Dogma = TAD boundaries restrict enhancer function
- In vertebrates CTCF binding is ↑ at boundaries of TADs
- CTCF acts as insulator in Drosphilia, act imprinted loci in mammals so thought TAD boundaries block enhancer
- Need to remove CTCF, ↓ Δ in gene expression
- Chromatin organisation x assoc w/ gene expression
52
Q
E.g. where CTCF binding does influence gene expression
A
- IgF2-H19
- CTCF only binds maternal chromosome when DNA x methylated
- Male chromosomes have interaction btw enhancer + H19 Igf2 promoter so enhancer enhances expression of Igf2
- Maternal chromosome, enhancer int. w/ H19 promoter, H19 expressed, Igf2 x
- Sub-TAD insulates Igf2 form enhancer, x formed in paternal
- CTCF also contains RNA binding domain that works w/ H19 hcRNA to create insulator to block enhancer-promoter interaction
53
Q
Regulation of human B-globin locus
A
- LCR has ↑ hypersensitivity site
- Genes expressed at different times of development
- LCR is an erythroid super-enhancer, controls expression of entire locus
- Direct region of global LCR control = chromatin opening, timing of DNA replication, expression of individual genes in temporal order
- LCR physically contacts promoters by forming loops in loops/domain
- Experiment = Ac-Lys Ab show Lys acetylation through a domain when genes have potential to be expressed
54
Q
X chromosome inactivation (Xi)
A
- In females, 1 of 2 X chromosomes is Barr body + expression of most genes repressed
- Active X chromosome + inactive has locus Xist
- Y has fewer genes than X, balances imbalance of X-linked genes btw sexes so level of expression is the same
- Flies + works ↑ gene expression in single X in males by acetyl lys16 on H4, opens structure
- Once X inactivation is initiated in female embryos, maintained in all somatic cells
55
Q
Order of events
A
- Embryonic stem cell in female has 2 X chromosome w/ Xist + Tsix RNA
- Choose what chromosome to be inactivate
- Xist coats inactivated chromosome
- Exclusion of Polii, loss of euchromatic marks
- Certain genes in Xi escape to periphery
- Using HiC, Xa + xi have distinct chromosome territories
- Xa = 100 distinct TADs, Xi - no TADs, has 2 large compartments
56
Q
Mechanism
A
- XIC produces mRNA transcripts from only inactive X chromosomes
- Xist made form gene in XIC
- Control of XIst expression itself = due to mRNA methylation
- Inactive X is maintained inactive through histone deacetylation + methyl
57
Q
Model for choosing Xi randomly
A
- XIC has ↑ mRNA
- Pre-Xi, Xist repressed by CTCF at promoter, Some X-X come together at centre involving CTCF + Six
- ncRNA jpx removes CTCF, Xist expressed
58
Q
How does Xist repress genes
A
- Binds SPEN that recruits Xist to Pol II + allows complexes that suppress transcription like NURD complex
- Active transcription facilitates repression
