Epigenetics

Question 1

describe the 2 main components of the epigenetic code

Answer 1

DNA methylation:
methyl marks added to DNA bases –> repress gene activity

Histone modifications:
different molecules can be added to ‘tails’ of histones –> alter activity of DNA wrapped round them

Question 2

why has the definition for epigentics changed from ‘modifications of DNA not affecting primary structure’

Answer 2

‘reconfiguring of DNA activity without altering primary structure’ – new

change from seeing epigentics as ‘chemical add ons’ of DNA –> seeing it as active reprogramming of gene expression and chromatin behaviour

Question 3

why is gene expression important in cell fate

Answer 3

balance between proliferation and differentiation in development

phenotype product of expressed genes –> genes that are active and silenced

Question 4

what 3 ways is gene expression controlled

Answer 4

Trans-acting factors –> short-term/responsive regulatyion via transcription factors

DNA methylation

Histone modifications

Question 5

describe the gene regulation via ‘Trans-acting factors’

Answer 5

short-term, responsive via transcription factors

factors bind regulatory sequences –> promoter/enhancer

= can recruit co-factors that ‘fine-tune’ activity –> activate or repressor

Question 6

describe the gene regulation via DNA methylation

Answer 6

chemical modificaytion of DNA –> CH3 added to cytosines

silences genes blocking TFs binding or by attracting proteins (MBDs) that compact chromatin via Histone modifications

= covalent modification –> permanent unless removed –> can be passed down to daughter cells = heritable

= long term, stable,heritable repression

Question 7

describe gene regulation via histone modifications

Answer 7

histone proteins have tails that can be modified (H3/4 especially)

= affect how tight or loose DNA is packed –> accesibility

acetyls and methyls (methyls can activate or repress depending where placed)

= ‘volume knobs’, not as permanent as DNA methylation, easily reversible , but more stable than TF control

Question 8

name 1 key role of epigentics in normal cells

Answer 8

In all cells DNA sequence is identical yet different cells need to behave differently

eg:
X-chromosome inactivation

females silence 1 X-chromosome to balance gene dosage between XX females and XY males

Xist is a lncRNA –> expressed on X-chromosome to be silenced –> coat X-chromosome ‘in cis’ –> scaffold to recuit chromati-modifuing complexes = silenced

Question 9

where are methyl groups added in DNA methylation

Answer 9

5th carbon of cytosine ring

= 5-methykcytosine (5mC)

catalysed by DNA methyltransferases –> DNMTs

= uses S-adenosylmethionine (SAM) as methyl donor

Question 10

name the methyl donro that DNMTs use to tag DNA in epigentics

Answer 10

S-adenosylmethionine (SAM)

Question 11

describe the difference in the roles in methylation of DNMT1 vs DNMT3A + DNMT3B

Answer 11

DNMT1 = ‘keeper’

= in cancer DNMT1 maintains the new marks in replication/daughter cells added by DNMT3a/b

DNMT3A/B = ‘Writers’ (create new marks)

= in cancer can hypermethylation TSG promoters/enhancers

Question 12

what percentage of CpG dinucletides are methylated in mammals

Answer 12

70%

CpGs = sites where a cytosine is followed by a guanine on the same strand

= many genes have unmethylated CpG-regions near promoters called CpG isalnds –> very long and unmethylated to allow trancription to occor

Question 13

name the main enzyme involved in DNA Demethylation

Answer 13

TET

= Ten-Eleven-Translocation proteins

hydoxylation followed by further oxidation

= causes base-excision and replacement with unmethylated cytosine

Question 14

what binds to 5mC following action of DNMT on DNA and acts as ‘readers’

Answer 14

MBD proteins

Methyl-CpG-binding domain proteins

= read the methylation and trigger downstream effect

recruits HAT/HDACs

Question 15

describe a simple pathway to remodel chromatin with methylation of DNA

Answer 15

DNMT adds CH3

MBD binds

HDAC recruited in HDAC-containg repressor complex (Sin3)

chromatin compaction due to more positive charge on histones

= MBD-HDAC links DNA marking to histone modifications

Creates a silencing loop: methylated DNA –> attract HDACs

deacetylate histone –> compact chromatin

= more methylation possible –> DNMTs clustered together and able to spread to nearby CpGs

Question 16

describe how the ‘histone code’ is important in influencing DNA methylation using H3K9 as an example

Answer 16

histone modifications attract or block methylation machinery - DNMTs

1. H3K9 methylationis a repressive histone mark

Recruits Hp1 (Heterochromatin-protein-1)

HP1 is the bridge between H3K9me3 and DNMT

= K3K9me3 promotes nearby CpG methylation/repression

creates reinforcing loop:
H3K9me3 → HP1 → DNMT1 → DNA methylation → maintenance of heterochromatin/repressed state

2. H3K9 acetylation –> prevents Hp1 binding

= weaker attraction reduces attraction and prevents DNMT machinery binding

Question 17

where are acetyl groups added to on histone tails

Answer 17

lysine residues - Ks

= neutralise positive charge on histones –> loosen binding

= prevent Hp1/adaptor protein recruiting methylation machinery

Question 18

how are histione modifications controlled

Answer 18

Readers , Writers and Erasers

Question 19

describe structure of nucleosomes

Answer 19

DNA wrapped around histone octamers –> 2 copies of H2A,H2B,H3 and H4

= form ‘beads on a string’

each histone has N-terminal tails that stick out and can be chemically modified

= especially H3/4

Question 20

describe how DNA methylation affects HISTONE methylation for STRONG repression

Answer 20

DNA currently has no methylation on it and chromatin is open/active

DNMTs are recruited by Transcription factors or methylation marks on histones

= DNA is methylated

MBDs bind methylated DNA via ‘chromodomains’

= recruit co-repressors and HDACs
= remove acetyl mark on histones ALLOWING methyl marks to be added

HMTs recruited via MBDs add H3K9me3

HP1 binds K3K9me3

= via Chromodomains

HP1 creates tight packed inactive heterochromatin via a self-reinforcing loop

Question 21

describe how HISTONE methylation affects DNA methylation for STRONG repression

Answer 21

histone methyl marks like H3K9me3 act as signal and recruit HP1

HP1 attract DNMT1 –> methylate CpG DNA

methyl DNA recruit MBD proteins –> more HDACs –> further compact

= self-reinforcing loop
= H3K9me3 is STRUCTURAL repression and very strong

Question 22

describe gene imprinting using Igf2 (insulin-like growth factor 2 receptor) as as an example

Answer 22

we inherit 2 alleles 1 from each parent and both are expressed –> imprinted genes break rule

= 1x alelle is expressed and 1x is silenced

Paternal Igf2r expressed and maternal silenced

Igf2 and H19 located close together and share enhancers –> only 1 is expressed depedning which parents chromosome its on

Maternal:
H19+ Igf2-
imprinting control region (ICR) between the 2 is unmethylated
CTCF insulator binds to ICR
CTCF block enhancer activating Igf2 –> only H19

Paternal:
Igf2+ H19-
ICR methylated
CTCF cannot bind –> enhancer free to activate Igf2
H19 silenced as promoter is methylated

Question 23

how were Polycomb group proteins discovered in Drosophilia

Answer 23

random mutagenisis experimenys in drosophila embryos

= homeotic transfromations –> structures that develop in one body seg,ent appeared in another

‘sex combs’ found on front legs of male flies appeard on other legs –> loss of repression of Homeotic (Hox) genes

= ability to maintain silence of Hox genes –> affected genes found to encode Polycomb group proteins (PcG)

Question 24

describe and explain the functions of the 2 main Polycomb complexes in epigentic silencing

Answer 24

PRC2 –> contains EZH1/2

= H3K27 histone methyltransferase
= trimethylates H3K27me3

PRC1 –> contains CBX (chromodomain) complex, SAM and RING

= CBX binds H3K27me3 mark
= RING domain ubiquitinates H2A
= SAM forms ‘daisy chain’ of nucleosomes to compact chromatin via polymerisation

Question 25

describe the mechanism of Polycomb-mediated gene silencing

Answer 25

PRC2 (EZH2 containing) trimethylates H3K27 --> H3K27me3 PRC1 reads mark --> CBX proteins bind mark and cluster PRC1s together = weak multivalent interactions SAM domains of PHC protein polymerises forming 'daisy chain' of nucleosomes and compacting chromatin RING ubiquitinates H2A on lysine 221 = blocks Transcription Factor access and RNA polymerase-2 loading = also recruits PRC2 in positive feedback loop for more silencing nearby compaction is the key mechansim of silencing REINFORCED by H2A ubiquitination

Question 26

what kindve domains do Embryonic stem cells have in their DNA

Answer 26

bivalent domains - repressive and active = H3K27me3 (repressive) H3K4me3 (activator) allow ESCs to stay pluripotent while allowing key developmental genes 'primed' for activation or repression = 'poised' transcriptional state = RNA polymerase and base Transcription Factors are present AND loaded but transcription is very low

Question 27

what happens to bivalent domains of embryonic stem cells in differentiation and name the enzyme involved

Answer 27

bivalent domain resolved into one of two 'monovalent' states - active/represseed UTX demethylases remove H3K27me3 mark on genes that need to be activated for differentiation via TRANSCRIPTION FACTORS = PCR1/2 complexes reinforce H3K27me3 on genes that remain silent chosen chromatin state is stably inherited through cell divisions = lineage specific gene expression

Question 28

describe 2 ways of epigentic deregulation of promoters in cancers

Answer 28

1. Turning off TSG/growth inhibitors = hypermethylation of promoter regions = repressive histone mark - H3K9/27me3 2. Turning on oncogenic/growth-promoting genes = hyPOmethylation (removal of repressive histone methyls and DNA methyls (TET) = activating histone marks (H3K4me3)

Question 29

what state are CpG islands near promoters usaully found in normal cells and why

Answer 29

unmethylated = allow transcription of normal regulatory genes In cancer cells: Mutations/changes in upstream pathways cause: 1. local hypermethylation of TSGs promoters 2. global HYPOmethyaltion in non-promoter regions can activate oncogenes

Question 30

name 2 histone marks that are repressive

Answer 30

H3K27me3 H3K9me3 = me3 means trimethylated

Question 31

name one way that we can use epigentics in diagnostics and name a type of treatment that targets the effects of epigentics

Answer 31

DNA methylation patterns can be used as biomarkers to detect cancer = hypermethylation of TSG promoters used to detect cancer i 5-azacytidine (AZA) is DNMT inhibitor = reverse the abnormal methylation of cancer and reactivate silenced genes

Question 32

what does the microRNA - miR-127 repress normally but silenced in cancer

Answer 32

miR-127 normally silences the oncogene Blc6 = BLC6 is a proto-oncogene for proliferation and survival In cancer miR-127 is silenced via promoter hypermethylation and histone deacetylation = allows Bcl6 to drive proliferation Combined effect of AZA (DNMT inhibitor) and PBA (HDAC inhibitor) reactivates miR-127 = Bcl6 is silenced again halting cancer progression

Question 33

describe what a genome-wide promoter methylation analysis in wilms tumour can include via MeDIP-chip (methylated DNA ImmunoPrecipitaton-chip)

Answer 33

Fragment DNA from Wilms tumour and normal kidney tissue and label with an antibody that binds to 5-mC = 5-methyl-Cytosine = areas which are methylated are labelled seperate and wash away unemtheylated DNA via pull-down assay = you know have samples for methylated regions of DNA in Wilms tumour and normal = MeDIP -> Methylated DNA immunoprecipitation Add different coloured dyes to the samples hybridise the different coloured samples to a promoter tiling tray -> 'Chip' = tray contains oligiomers of more than 24,000 gene promoters Each DNA fragment binds to its complimntary probe on the 'chip'/tray after washing compare colour of spots to identify WHICH promoters are more methylated in Wilms tumour than normal

Question 34

what did scientists discover doing a MeDIP-chip on Wilms tumour and what genes were specifically found here

Answer 34

large 800 kilobase region on chromosome 5 that was abnormally methylated = more than 50 genes --> 6% of all genes on chromosome 5 Protocadherin (PCDH) genes --> cell adhesion and communication

Question 35

in Wilms tumour an analysis found abnormal methylation of what genes on chromosome 5 and how might this relate to what we know about wilms tumour

Answer 35

Protocadherin (PCDH) genes = cell adhesion and signalling --> letting cells know what 'type' they are Wilms tumour cells look and behave like fetal kidney cells = hypermethylation of PCDH locks cells in immature state preventing normal kidney differentiation --> behave like fetal kindey cells = E-cadherin repressed and = MET gene needed for biurifiaction to form uretic bud in kidney formation

Question 36

what is inside the destruction complex that targets B-catenin in Wnt pathway

Answer 36

APC,AXIN and GSK3β = binds β-catenin --> tag for destruction active β-catenin replaces co-repressors for co-activators on TCF/LEF Transcription factors on Wnt promoters

Question 37

how can epigentic silencing affect the Wnt pathway via SFRPs (secreted Frizzled-Related Proteins)

Answer 37

SFRPs are Wnt antagonists that resemble Frizzled receptors = bind Wnt ligands in extracellular space Inhibits activation of Frizzled receptor to induce B-catenin signalling Hypermethylation of SFRP genes in cancer allows promotes uncontrolled Wnt signalling = more Wnt availabelt ot bind receptor = increase in β-catenin proliferativ/survival signalling

Question 38

what does the enzyme MGMT do and why is it an important consideration when treating glioblastoma

Answer 38

MGMT = O6-methylguanine-DNA methyltransferase DNA repair enzyme = removes alkyl/methyl groups from guanine --> fix damage from alkylating agents MGMT repairs damage caused by chemotherapy drugs = chemotherapy relies on apoptosis due to damaged DNA so repair inhibits this methylating MGMT promoter --> silence MGMT --> DNA damage unrepaired --> cancer cell dies

Question 39

what is EZH2 and its canionical vs non-canionical roles

Answer 39

Enhancer of Zeste Homolog 2 Histone methyltransferase (HMT) = catalytic subunit of Polycomb Repressive Complex 2 (PCR2) Main function (canionical) = H3K27me3 trimethylation on histone tails for DEVELOPMENTAL silencing = H3K27me3 recruits PRC1 -> chromatin compaction -> transcription OFF Non-canionical (PRC2 independant) - Methylate non-histone protein targets = Methylate transcription factors -> STAT3 -> enhance TF activity EZH2 can act as a transcriptional repressor or activator depending on context

Question 40

name the microRNA that suppresses EZH2 mRNA that can be muated in cnacer and the result of this

Answer 40

miR-101 suppresses EZH2 mRNA = mutated --> no suppression --> increase EZH2 production = more H3K27me3 --> gene silencing and tumour growth globally/genome wide

Question 41

what happens in heterozygous mutation in EZH2 at Y641(catalyytic domain) that promotes lymphomas and myeloid cancers

Answer 41

normal EZH2 --> good at first methylation = H3K27 --> H3K27me1 --> not so good at later steps to produce me3 mutant EZH2 --> bad at me1 step BUT very good at me2/3 step = mutantion changes substrate preference = heterozygyous mutation allows mutant and wild-tyoe enzymes to cooperate to create MUCH MORE H3K27me3 (strongest repressive mark) than either alone could promotes lyphoma growth --> Y641 mutation --> silence germinal center exit genes --> only B-cells in GC lymphomas rely on this program --> lymphoma specific

Question 42

how would a mutation at K27 to Methionine in H3.3 (histone) cause epigentic effects globally and locally leading to glioblastoma (brain)

Answer 42

Lysine 27 -> Methionine in the histone H3.3 variant = K27M binds and inhibits EZH2 in PCR2 complex = cant make H3K27me3 repressive mark global genome silencing 'unlocked' --> gene expression increase H3.3 is not everywhere = found at high concentration at regulatory regions/enhancers = These sites experience extremely LOW H3K37me3 due to PCR2/EZH2 getting stuck = Intense local chromatin disturbancesq/overactivation (remeber H3K27me3 is a repressive mark) Combination of global loss of represssion turning on too many genes AND local enhancer regions being overactive of lineage/growth specific genes = 'stuck-precursor' stem-like cell blocking neural differentiation = glioblastoma

Question 43

describe the H3.3-K27M mutation causing glioblastoma and who does it primarily affect

Answer 43

inhibits EZH2 = catalytic subunit of PRC2 = global loss of of H3K27me3 repressive mark AND intense overactivation at local H3.3 sites cells stuck in precursor/stem-like state found in 19% pediatric children glioblastomas and 0% adults glioblastoma

Question 44

describe the effects on methylation when IDH1 is mutated - (enzyme in TCA)

Answer 44

IDH1 mutation changes substrate specificity of enzyme = normally convert Isocitrate -> a-KG = Mutant converts a-KG -> 2-HG 2-HG is an oncometabolite that resembles a-KG = competetively inhibits a-KG dioxygenases = DNA and histone de-methylases = TET and HDMs Global hypermethylation and differentiation genes stuck inactive = failure to activate lineage programs for differentiation -> stem-like/precursor state = same effect as H3.3 mutation but this causes HYPER not HYPO methylation

Question 45

describe what is caused by the IDH1 mutation and who it mainly affects (epigentics)

Answer 45

Hpermethylation due to inhibition of a-KG dioxygenases via 2-HG --] TET/HDMs = IDH1 substrate specificity change very common in adult gliomas - 77%

Question 46

what is OLIG2 and why do we care about it in K27M tumours

Answer 46

OLIG2 is a oncogenic transcription factor that maintains neural progenitor/stem-like state = repress p53 activity Normally PRC2 via EZH2 adds H3K27me3 to OLIG2 locus = repressive mark allowing differentiation Lysine -> methionine at position 27 in H3.3 causes local and global HYPOmethylation = EZH2 binds and is sequestered by methionine on H3.3 OLIG2 is particularly affected due to high concentration of H3.3 at its enhancer regions = OLIG2 stuck 'on' = p53 suppressed = cell cycle 'brake' is lost and apoptosis inhibited = stuck in neural progenitor/stem-like state

Question 47

how can Oncogenic Ras cause EZH2 redistribution and how does this lead to cancer. /glioblastoma

Answer 47

Ras signalling activates Erk via MAPK Erk phosphorylates EZH2 lowering its affinity for H3 on HOX genes = phospho-EZH2 cannot efficiently bind or trimethylate H3K27 on normal targets/HOX genes Phosphorylated EZH2 shifts binding affinity to transcription factors instead of histones = binds EMX2 ascociated TFs and recruited to EMX2 promoter via protein-protein interactions with TF Places repressive mark on genes that should be active = HOX genes activated and EMX2 repressed = loss of normal forebrain development = glioblastoma formation

Question 48

what do HOX and EMX2 genes code for (link to glioblastoma formation via PCR2 reprogramming in oncogenic Ras signalling)

Answer 48

HOX --> code for 'where different body regions should form' in embryogenesis EMX2 --> codes for transcription factor for normal forebrain differentiation = PCR2 in Ras signalling moved from HOX to EMX2 --> Erk changes EZH2s binding prefernec to EMX2 transcription factors = brain does not devlop properly = abnormal forebrain development/glioblastoma

Question 49

what is HOTAIR and its role in cancer cells and where are its genes found

Answer 49

Long non-coding RNA = acts as a scaffold that 'drags' PCR2 to promoters of metastasis SUPPRESSOR genes PCR2 adds H3K27me3 allowing cells to be invasivee/metastsatic = HOTAIR coded by HOX genes

Question 50

where is HOTAIR lncRNA transcribed from and its role

Answer 50

HOX genes --> then moves to different HOX gene = produced at chromosome 12 and moved to chromosome 2 = HOX-c ------> HOX-d HOTAIR acts as scaffold recruiting PRC2 to metastasis suppressor genes = cells are more invasive and metastatic due to lack of suppressors produced

Question 51

what can EZH2 loss of function mutations cause using leukemia/blood cells as example

Answer 51

PCR2 loss = global loss of H3K27me3 repressive mark blood cell development requires strict 'stepwise' differntiation = each stage requires silencing of stemness/progenitor genes and activation of differentiation JAK/STAT and other cytokine 'immature' pathways remain ON = cells NOT able to turn them off/silence Differentiation stuck as stemness programs stay ON and dominate = haematopoitic stem cells remain undifferentiated and proliferate = leukemia

Question 52

why might EZH2 inhibitors not work in neuroblastoma

Answer 52

EZH2 can drive cancer via its canionical CATALYTIC function by dysregulation of H3K27me3 marks = catalytic EZH2 inhibitors block this function HOWEVER EZH2 can bind MYC-N directly Non-canionically = Transcriptional coactivator of growth-promoting genes WITHOUT CATALYTIC methyltransferase activity MYC-N is neuroblastoma for neural lineage MYC-N–EZH2 interaction does not require methyltransferase activity = catalytic inhibitor has no affect = inhibiting H3K27me3 formation --> EZH2 still helps MYCN drive oncogenic transcription big affect in MYC-N amplified neuroblastomas

Question 53

EZH2 catalytic inhibitors may not work in neuroblastomas due to Non-canionical function of EZH2 with MYCN - what can we do instead

Answer 53

PROTACs = bind EZH2 and E3 Ubiquitin ligase and bring them together proteasomal degredation = Needed as neuroblastomal cells use EZH2s protein interactions NOT its methyltransferase catalytic activity blocks canionical and non-canionical EZH2 functions

Question 54

what does the enzyme IDH1 normally do and what changes in some mutations

Answer 54

enzyme in the TCA cycle = Convert Isocitrate to a-KG and NADPH mutant IDH1 changes substrate specificity = a-KG --> 2-HG (oncometabolite) 2-HG resembles a-KG --> competetively inhibit a-KG dioxygenases = TET and KDMs (demethylases) --> global DNA hypermethyltion = also PHD enzymes which hydroxylaye Hif1a

Question 55

what does the oncometabolite 2-HG produced by the mutant form of IDH1/2 cause epigenetically

Answer 55

2-HG inhibits KDM and TET while a-KG activates them = KDM is a lysine demythlase for histones 2-HG inhibits DNA and histone methyltransferases = DNA and histone hypermethylation Affects on TSGs and differentiation genes = proliferative and stem-like cell

Question 56

what is BRD4 and what does it do

Answer 56

BRD4 is a BET = 'Bromodomain and extra-terminal' family protein BETs bind acetylated histones via Bromodomains = recruits transcription machinery boosting transcription of growth promoting/survival genes = MYC-N High 'global amplifier' increase cancer progression

Question 57

role of BET inhibitors in MYC-N suppression

Answer 57

BETs normally recruit co-activators to acetylated histones = boost transcription of growth/survival genes BET inhibitors block BRD4 from binding acetylated histones = prevent MYC-N transcription inhibit production of proliferative/surival genes = works well for MYC driven cancers = Neuroblastoma

Question 58

explain why methylation can be activation or repression marks

Answer 58

methylation does not change the charge of histones UNLIKE acetylation = effect depends on WHERE it methylates and WHICH 'reader' proteins bind to that site 1. H3K9me3 = recruits HP1 = silences STRUCTURAL genes 2. H3K27me3 = 'written' by EZH2 on PRC2 = TSG and differentiation gene repression/silencing - DEVELOPMENTAL 3. H3K4me3 = recruits transcription machinery and chromatin modifiers = activation mark at promoters

Question 59

explain the differnt proteins that bind to: - DNA-methyls - Histone-methyls - Histone-acetyls

Answer 59

Acetyl - Bromodomain containing proteins (BET such as BRD4): = recruit transcription machinery and chromatin modifiers for activation Histone-methyls - Chromodomain family proteins : = CBX-H3K27 = HP1-H3K9 = recruit chromatin modifiers depending on site of methyl mark on histone DNA-methyls - MBDs = also contain chromodomains

Question 60

role of CBX, RING and SAM domains in PRC1 complex

Answer 60

CBX - Chromdomain containing = binds H3K27me3 repressive mark and pulls nucleosomes together via weak multivalent interactions between PRC1s RING - E3 Ubiquitin ligase domain = Ubiquitinates H2A on lysine 119-> NOT that important for compaction of chromatin = Blocks RNA polymerase-II loading and RECRUITS PCR2 in positive-feedback loop SAM - Sterile alpha motif domains = domains polymerise connecting multiple PRC1 complexes = 'daisy chains' nucleosomes into compact cluster

Question 61

life cycle of Polycomb protein repression and relief of silencing

Answer 61

Question 62

compate the repressive marks H3K9me3 vs H3K27me3

Answer 62

H3K9me3 - strongest form of repression - STRUCTURAL HP1 binding creates 'locked' chromatin state HP1 binds and dimerises -> recruits proteins for further H3K9me3 = pulls nucleosomes together and recruits DNMTs -> positive feedback DNA methylation is a permanent mark for long term repression H3K9me3 harder ro remove -> small number of proteins (KDMs) = permanent heterochromatin H3K27me3 - 'softer,reversible repression' PRC1/2 repression is DEVELOPMENTAL not STRUCTURAL = marks genes that need to be off in ONE cell BUT NOT ANOTHER = repression must be reversible Does not recruit DNA methylation machinery = dynamic repression to regulate gene expression during development

Question 63

what do we mean when we say H3K9me3 is stuctural and H3k27me3 is developmental

Answer 63

Structural - chromatin is silenced for structural/genome integrity - telomeres, centromeres = repression is peramanent and hard to reverse - KDM small subset H3K27me3 silencing is dynamic, reversible, and context-dependent = genes OFF in one cell but may need to be ON later = PRC1 binds chromatin less tight than HP1 can be outcompeted by strong transcription factor

Question 64

describe the removal of the 'reversible/developmental' PRC1/2 represion by transcriptiuon factors

Answer 64

UTX is a KDM-family demthylase = removes H3K37me3 from lysines taking away CBX binding site of PRC1 = primary trigger for PRC1 eviction UTX is recruited by strong transcription factors = NF-kB or Hox = Oxidatively demythlates back to H3K27 TFs recruit HATs (CBP/p300) = opens chromatin weakening PRC1 SAMs polymerisation -> 'daisy chain breaks' = blocks methylation by acetylting H3K27

Question 65

Link chronic inflammation to cancer with NF-kB

Answer 65

Direct affects of NF-kB binding to NF-kB genes: - NF-κB binds - Recruits UTX - Removes H3K27me3 locally - Adds H3K27ac (via p300/CBP) - Opens chromatin - Transcribes inflammatory/survival genes = direct and expected effect UTX also demthyates surrounding area at enhancers and PRC1 repressed sites = not JUST where NF-kB binds Chronic inflammation can erode H3K27me3 repression AROUND NF-kB = activate developmental genes that should be repressed = de-differentiation = cells become less differentiated and stem-like = cancer