Epigenetics Flashcards

Question

What are the three roles required for histone tail modifications?

Answer 1

Writers, Readers, Erasers Writers - enzymatic activities responsible for depositing the modification - e.g., histone acetyl transferase Readers - proteins that contain specific domain thats recognise (and binds to) the specific modified histone - e.g., bromodomain protein Erasers - enzymatic activities responsible for removing the modification - e.g., histone deacetylase (HDAC)

Answer 2

- Mediate transitions in the chromatin template - cis and trans effects E.g., - Manipulate the chromatin environment - by: - Altering charge of nucleosome - Loosening inter/intranucleosomal DNA histone interactions Or - directly regulate assembly of transcription machinery Or - directly exchange histone variants - histone exchange

Answer 3

Trans effects refer to binding of specific **proteins** that recognise the **specific histone tail modification** - done by **readers** - Involved in all aspects of chromatin regulation - transcriptional activation/repression Proteins that read histone modifications: -E.g., - TAFll-250 - transcription apparatus - Chromatin remodellers - SWI/SNF - Structural proteins that directly affect chromatin accessibility (HP1)

Answer 4

HATs are writers - involved in transcriptional activation - Catalyse histone tail acetylation - Different HAT subfamilies use diff catalytic strategies for acetylation

Answer 5

- Altered charge of nucleosome - acetylation of tail neutralises basic charge - Loosening of inter/intranucleosomal interactions - Acetylation of H4 tail inhibits compaction of 30nm fibres

Answer 6

Bromodomains (AA domain) that recognises **histone tail acetylation** - readers - Have unusual charge distribution - striped of acidic residues across top - Some bromodomains have HAT activity - i.e. attrated to acetylated histones and then deposit more acetylation once they are bound - leads to 'cascade' of HTA - e.g., Myc-TAFll-250 - recruits more TFIID; reinforces transcriptionally active state of promoter - Double bromodomains - spacing of binding pocket enables binding of two acetyl residues on same histone tail - e.g., TAFll-250 binds di-acetylated histone tails (K9 and K14)

Answer 7

HDACs are erasers - involved in transcriptional repression by removing histone tail acetylation - E.g., Mad1 - interacts with Sin3 protein - interacts with HDAC - clinical relevance

Answer 8

- Transcription factors control **histone acetylation** - E.g., Myc (activator; recruits HAT) and Mad(repressor; recruits HDAC) bind to the same sequence - 'target' for regulation - So - in proliferating cells Myc is highly expressed; in differentiating cells - Mad is highly expressed - Both are critical for regulating the switch between cell proliferation to differentiation - alterations in relative levels - lead to cancer

Answer 9

Pioneer transcription factors independently interact with their DNA-binding site when in the context of a nucleosome - establishing competence for gene expression Mechanisms: - Directly modulate chromatin structure - Facilitate binding of other transcription factors - Recruit HAT/HDAC and chromatin remodellers - to facilitate environment for transcription

Answer 10

- Result in gene activation/repression - Use facultative vs constitutive heterochromatin - Mechanisms of reinforcement - active/inactive states - Interactions of adjacent HTMs

Answer 11

- Regulate transcriptional activation - and formation of constitutive and facultative heterochromatin - Each methylation site has very specific function - degree of methylation is critical - HTMs reinforce active/inactive chromatin states - Different readers recognise methylated histone tails - e.g., chromo/PHD domains

Answer 12

- HTAs - more sites occur on **all four histones**; HTMs - fewer sites - mainly on H3 and H4 - HTAs **alter charge** of nucleosome; HTMs do not - they **alter hydrophobicity** of tail - HATs catalyse HTA; histone methyltransferases catalyse methylation - Methylation = gene silencing; acetylation = gene activation

Answer 13

PEV results when a gene normally in euchromatin (transcriptionally active) is spread with heterochromatin via translocation/rearrangement - It is easier to get heterochromatin spread if there is less general transcriptional activity (that may oppose) - Studies in Drosophila - using white gene - led to identification of Su(Var)3-9/HP1

Answer 14

- Su(Var)3-9 encodes a **histone methyltransferase** - **H3K9** is substrate for Su(Var)3-9 - Methylated H3K9 col-localises with **HP1** - at centromeres - Spread of **heterochromatin** - gene silencing - HP1 **chromodomain** selectively recognises methylated K9 histone H3 - Highly specific interaction - no other chromodomains bind H3K9

Answer 15

HP1 - **heterochromatin protein** - Chromo domain - binds **methylated H3K9** - Chromoshadow domain - mediates **homodimerisation** and interaction with **Su(Var)3-9**

Answer 16

- Would abolish heterochromatin - reduce gene silencing - Would abolish interaction with methylated H3K9

Answer 17

- HP1 and H3K9 histone methyltransferase mediate the spreading of constitutive heterochromatin - Forms model for heterochromatin spread - Methylated H3K9 is recognised by HP1 - which interacts with Su(Var)3-9 - which deposits further methylation - cascade

Answer 18

- HP1 is removed from heterochromatin during mitosis - to allow for proper segregation

Answer 19

H3K trimethylation and H3K36 methylation regulate initiation and elongation stages of transcription H3K4 trimethylation - initiates RNA polymerase - set1 histone methylase - start site - Provides binding site for 'reader' - that contain a PHD finger/chromodomain H3K36 - elongates RNA polymerase - recruits set2 histone methylase

Answer 20

- Established by short-lived transcription factors that generate HOX gene expression patterns - that define the morphology of each segment - Maintained by Trithorax and Polycomb group proteins

Answer 21

Mechanisms of repression: - Chromatin compaction - Exclusion of remodellers - Hold RNA polymerase at start site

Answer 22

**Place repressive histone modifications at promoters (CpGs mainly)** - Two complexes: PRC1/PRC2 - Both contain tri-methylated H3K27 - PRC2 - has component that methylates H3K27 - Polycomb proteins **suppress** the expression of **HOX genes** - Therefore polycomb proteins and H3K27 mediate spreading of facultative **heterochromatin**

Answer 23

Chromatin with marks of both transcriptional **activation (euchromatin)** and of **facultative** **heterochromatin** - Genes not expressed because **polycomb** proteins pause RNA polymerase at their promoters - Coexistence of active (**H3K4me3**) and repressive (**H3K27me3**)

Answer 24

- **Regulate stem cell pluripotency** and lineage commitment - Upon differentiation - polycomb complexes are displaced - and bivalent chromatin resolved into active (H3K4me3) or repressed (H3K27me3) states

Answer 25

Critical role in shaping the 3-D organisation of the genome - Bind to proteins such as CTCF

Answer 26

CTCF is a critical transcription factor (zinc-fingered protein) - Binds to insulator elements - for the formation of insulated domains - directs formation of insulated neighbourhoods - critical in regulating individual genes -Binding only occurs if DNA recognition site is unmethylated - DNA binding inhibited by CpG methylation - Also act as an 'architectural' protein - - Critical in genomic imprinting - relevant in cancer

Answer 27

DNA methylation occurs at CpG sequences - carried out by DNA methyltransferases (DNMTs) 1. De novo methylases - methylates unmethylated templated - important in early development 2. Maintenance methylases - methylates hemi-methylated template - important following DNA replication

Answer 28

Demethylation is important in **early development** - By active enzymatic demethylation (**TET**) enzymes- sperm - Or passively - by dilution following DNA replication - oocyte

Answer 29

1. Initially, both are heavily methylated 2. After fertilisation - both are demethylated: - Male genome (sperm) is '**actively**' demethylated (TET enzyme) - Female genome (oocyte) is '**passively**' demethylated (dilution - **slower**) 3. Both genomes are then remethylated - BUT - imprinted genes are NOT affected - they maintain their original status

Answer 30

CpG islands represent gene clusters of **unmethylated C-G dinucleotides** **- Majority of CpGs are methylated - except CpG islands** - Associated with **transcriptional activity** - Found at the **promoters** of housekeeping genes

Answer 31

- Inactive x-chromosomes - Some imprinted genes

Answer 32

Inappropriate methylation of CpG islands - silences genes - Can cause silencing of tumour suppressor genes - cancer

Answer 33

Cfp1 keeps CpG islands **unmethylated** - contributes to the formation of an active chromatin configuration at gene promoters - Cfp1 - interacts with Set1 histone methylase - writer for H3K4me3 - **prevents de novo methylation** - KDM2A (histone methylase) - erases H3K36me - leads to histone **acetylation** - enhanced recruitment of basal transcription apparatus

Answer 34

1. Interference of transcription factor (CTCF are sensitive to methylation status) 2. Via recruitment of proteins that interact with methyl-C - methyl-CpG binding proteins

Answer 35

- Both create a repressive chromatin structure **MeCP2** - recruits **histone deacetylase** (removes acetyl groups) - repressive chromatin structure **MBD1** - recruits **methylase** that writes histone H3K9me3 - repressive chromatin structure

Answer 36

- Distinct regions of the nucleus occupied by individual chromosomes - Activated genes can move outside of their territory

Answer 37

A. Chromosome territories - Individual interphase chromosomes occupy small portions of the nucleus B. TADs (Topologically Associating Domain) - chromosomes are partitioned into megabase sized TADs - that have high intradomain 'interaction' frequencies C. Insulated neighbourhoods - TADs are divided into insulated neighbourhoods - formed by a CTCF-CTCF homodimer - using cohesin - 3 genes (200 kb)

Answer 38

- TADs are spatially separated by functional state - even from same/different chromosome - TADs with same functional state are found close together - 'Active' TADs - interior - associated with nuclear speckles - 'Inactive' TADs - periphery of nucleolus - At boundaries - TADs can contain insulated domains that are different compartments - active/inactive

Answer 39

During differentiation - Correlated with an altered epigenetic state - and with transcriptional activation

Answer 40

DNA sequence that blocks the ability of an enhancer to activate a gene when located between them

Answer 41

- Two bound CTCF mediate formation of loop - bind at 'anchors' - asymmetric/directional - using cohesin - Enhancer can only activate gene if its contained within the same loop - TADs are nested loops of insulated neighbourhoods

Answer 42

- Deletion or mutation of anchor - leads to aberrant gene activation - cancer - CTCF - some of most mutated transcription factor binding sites in cancer

Answer 43

- Can be organised via different combinations of loops - that dictate which genes are co-regulated - See schematic

Answer 44

- **Architectural proteins** - act as bridges via multivalent interactions - LBD1, YYC, PRC1 - **Liquid-liquid phase** separation - mediated by intrinsically-disordered regions of proteins with weak interactions

Answer 45

- By contributing to a phase separation mechanism - Leads to the formation of discrete regions of heterochromatin in the nucleus - H3K9me3 - drives liquid-liquid phase separation - leads to compartmentalisation of constitutive heterochromatin - TRIM28 - HP1 scaffold protein - abundant in heterochromatin

Answer 46

Genomic imprinting refers to the process whereby two alleles of a given gene (i.e. paternal & maternal) are rendered non-equivalent - i.e. - where one is expressed and the other is silent

Answer 47

- Mice experiments - embryo cell transfers - Showed that both parental genomes are required for proper development - and that some alleles are 'marked' or 'imprinted so that only one allele is expressed - 'silent' allele = 'imprinted' allele

Answer 48

Chimeric embryo = embryo containing a proportion of all-male or all-female cells - All-female cells: small body, large brain - All-male cells: large body, small brain

Answer 49

- Some are tissue-specific - e.g., brain - Not always 'all or none' - 'noncanonical imprinting) Imprinting genes are found in clusters - controlled by: - Imprinting centres (ICs) - Differentially methylated regions (DMRs) - sites methylated on one of the two parental alleles - Enhancer-blocking mechanisms - Long-coding RNA (IncRNA) - including antisense transcripts

Answer 50

Example of imprinting control via an imprinting centre (IC) Maternal allele: IC binds to CTCF - creates insulated neighbourhood around H19 and enhancer - Prevents enhancer interacting with IGF2 - so IGF2 is off; H19 is on Paternal allele: IC is methylated; prevents CTCF binding - so larger insulated loop enables enhancer to activate IGF2 - Methylation of IC silences adjacent H19 gene; IGF2 is expressed in paternal genome - contributing to larger body size in chimeric mice with all-male cells

Answer 51

Mediated by antisense RNA - Igfr2 encodes protein that degrades IGF2 - imprinted in opposite direction to IGF2 - i.e. from maternal genome Airn - non-coding RNA whose promoter is located in an intron of Igfr2 gene - Airn and Igfr2 are in antisense orientation Maternal allele - airn expression silenced - as IC is methylated Paternal allele - IC is unmethylated; allows airn IncRNA experssion - Airn RNA silences Igfr2 (antisense orientation)

Answer 52

- Placental biology & embyronic growth (paternal - promoters - Igf2, Peg1, Peg2; maternal - repressors - Igfr2, Gnas) - Postnatal processes - regulation of brain, behaviour, metabolism, behaviour (e.g., Peg1/Mest)

Answer 53

- Describes the opposing interests of the maternal and paternal genomes Maternal - suppress fetal growth - more equal distribution to all offspring Paternal - increase embyronic growth - also alter behaviour - hypothalamus

Answer 54

- Waves of methylation/demethylation are important - set patterns of genomic imprinting - After fertilisation, methylation is stripped off maternal/paternal genomes - but imprinted genes are UNAFFECTED - Methylation is re-established in blastocyst - imprinted genes remain

Answer 55

DNA methylation patterns are established by DNMT3A. Patterns are very different for males/females - Males - sperm methylation - acquired at intergenic sequences and transposon elements - Females - oocyte methylation - occurs later; is acquired in the bodies of actively transcribing genes - includes intragenic CpG islands

Answer 56

- Differentially methylated regions regulate enhancer-blocking mechanisms or regulate expression of antisense transcripts

Answer 57

- ICs contain TGCCGC - ZFP57 and ZNF445 bind to middle C; recruit KAP1 cofactor and DNA methyltransferases - They act to retain methylation - even in presence of demethylation in the early zygote - CpG islands have higher probability of containing TGCCGC - as oocyte methylation is preferentially found in intragenic CpG islands - sites have higher probability of retaining methylation - Hence - more differentially methylated regions involved in imprinting are in the female genome

Answer 58

Primary ICR methylation: is acquired during the process of germline methylation - that survives demethylation in early zygote - Maternal: >20 methylated ICRs - intragenic; found at CpG island promoters; often act as antisense promoters of IncRNA transcripts - that silence genes in cis - Paternal: 3 ICRs; all intergenic; often act as enhancer blockers (insulators) - fewer regions as less regions are protected from demethylation process

Answer 59

- Secondary DMRs (differentially methylated regions) arise within imprinted gene clusters - Arise as a consequence of imprinted expression of noncoding transcripts or passively following transcriptional repression - E.g., igfr2 promoter becomes methylated on paternal allele because it is not expressed; IGF2 gene on maternal allele is methylated

Answer 60

Due to an alteration in the methylation of the DNA - Lead to genomic imprinting disorders

Answer 61

Cis effects: mutations that influence the overlying chromatin structure - CpG methylation in germline; CTCF organisation in somatic cells Trans effects: result from mutations that directly or indirectly affect proteins involved in an aspect of chromatin biology - CpG methylation in germline (proteins that affect chromatin - e.g., remodellers); enzymes in methylation (IDH) in somatic cells - e.g., remodellers, histone variants, histone modifying enzymes

Answer 62

- When a gene that is normally active, becomes inappropriately silenced - so total loss of expression from both alleles - Or when the gene that is meant to be silenced becomes inappropriately activated - gives overexpression of gene

Answer 63

- Mutation is on maternal allele - so is silent (has no effect) - Imprints are reset in gametes of parents - male and female offspring will be phenotypically normal - but contain mutation on maternal allele (silent) - Imprints reset again in their gametes, so: - One male gamete has the mutation - so male offspring have 50% of inheriting the disease phenotype (as it is not silent on the paternal allele - But female gametes will not have the disease phenotype - they pass mutation silently

Answer 64

Sister syndromes - Prader-Willi - Neurodevelopment disorders - affecting 5- to 6- Mb deletion in 15q11-q13 - Critical gene (UBE3A -in brain - so imprinting occurs in brain) PWS: - Arises due to loss of paternal genes - leading to doubling of UBE3A expression AS: - loss of expression of maternally-derived cluster - AS genes are regulated by an antisense RNA (normally antisense RNA is not expressed; UBE3A/APT10A are expressed from maternal allele) - So - loss of AS IC methylation on maternal allele causes antisense RNA to be expressed that silences the maternal allele = loss of expression of UBE3A

Answer 65

BWS: large offspring syndrome - Involving two gene clusters on chromosome 11: IGF2/H19 and KCNQ1 - ICR1/2 regulate clusters Normally: ICR1: - Maternal allele - CTCF binds ICR1 (insulator); blocks enhancer - no interaction with IGF2 (silent); H19 is expressed - Paternal allele - methylation of ICR1 blocks CTCF binding; insulator activity removed - allowing IGF2 expression; H19 repression ICR2: - Maternal allele - methylation of ICR2 prevents expression of antisense RNA (KCNQ1OT1) - allowing expression of domain 2 genes - CDKN1C normally expressed - Paternal allele - ICR2 is unmethylated; ICR2 promoter produces anitsense RNA (KCNQ1OT1) - blocking expression of domain 2 genes (including CDK inhibitor - CDKN1C) IN BWS: Overexpression of IGF2 or loss of function of CDKN1C - Both cases can be caused by genetic/epigenetic mechanisms

Answer 66

IGF2 and CDKN1C are involved in growth regulation - associated with increased tumour risk - So tumours show preferntial loss of 11p15 region = loss of CDKN1C (maternal) or expression of IGF2 (paternal)

Answer 67

- Opposite effect to BWS - associated with growth retardation - Most frequently: epigenetic demethylation of paternal ICR1 = decrease expression of IGF2 growth factor - Caused by biallelic expression of H19 and decreased expression of IGF2

Answer 68

- Regulators of DNA methylation machinery - Histone modifying enyzymes - Chromatin remodellers - e.g., FHS

Answer 69

- FHS is caused by a mutation in **chromatin remodeller** - **deposits histone variant H2A.Z.2** in AT-rich enhancers of genes involved in cranial neural crest cell migration and differentiation - Causes craniofacial features, bone growth delay, language defects - Investigated using Xenopus model - knockdown of SRCAP

Answer 70

- Common inherited intellectual disability - Associated with methylation of an expanded CGG repeat in CpG island in the 5' UTR of the FMR1 gene = silencing

Answer 71

Stochastic or environmental/physiological: - Global methylation - Hypomethylation of oncogenes - Altered methylation of imprinting centres - Hypermethylation of insulators (CTCF sites) - Hypermethylation of CpG islands (tumour suppressor genes)

Answer 72

Cis effects: overlying chromatin structure - CTCF binding site mutation Trans effects: directly/indirectly affect proteins that influence chromatin organisation - Writers of CpG methylation - Enzymes that influence DNA methylation - IDH - Proteins that directly affect chromatin - histone modifying enzymes, remodellers, histone variants *Many mechanisms can have the same outcome E.G., Insulator function of CTCF binding can be affected by genetic mutation (primary cis genetic) or aberrant methylation of CTCF binding site sequence that occurs stochastically (epigenetic) or due to mutation in IDH (primary trans genetic)

Answer 73

**Global hypomethylation** - loss of methylation over large region - open chromatin structure - genome instability - oncogene activation **Localized hypermethylation** - short stretches of increased methylation - often at CpG islands - silencing of tumour suppressor genes **Mutation** - methylated DNA is prone to mutation - inefficient correction of deaminated methylated DNA

Answer 74

- Deamination of C to U is frequent; repaired via repair pathway - BUT: deamination of methyl-C leads to Thymidine - which is not efficiently corrected - Methyl-C is a hotspot for C to T transitions in cancer genomes - Methyl-C is more readily bound to chemical carcinogens - increases rate of UV-induced mutation - Methylation increases chance of UV-induced mutation

Answer 75

Due to global hypomethylation - Causes decompaction of chromatin - increasing fragility/instability - Leads to increased recombination and aneuploidy (abnormal no. of chromosomes) - Results in regions becoming inappropriately activated - that normally silenced - can activate nearby proto-oncogene or activate repeats and transposons

Answer 76

**At CpG islands** - Most gene promoters have CpG islands - that are transcriptionally active (unmethylated) throughout development - So CpG island promoter methylation can lead to silencing of tumour suppressor gene - loss of function = cancer - CpG-island promoter methylation occurs at an early stage in tumourigenesis - E.g., RASSF1A (p16INK41) **At genomic insulator sequences** - Play key role in altering 3-D structure of chromatin in cancer - Methylation of CTCF-binding site at anchor of insulated loop - can open loop - and inappropriately activate nearby genes - In imprinted gene clusters - this can alter imprinted gene expression

Answer 77

- Becasue DNA methylation changes occurs early; can be used as 'markers' for early diagnosis - prevention - Later stages - accumulation of methylation hits (alterations) can be markers for prognosis and treatment

Answer 78

- Hypermethylation at gene promoters can silence tumour suppressor genes or genes involved in maintaining genome integrity - e.g., DNA repair pathway - Hypermethylation is as common as a mechanism of inactivation through DNA mutation - Many tumour suppressor genes are involved in hereditary predisposition to cancer - i.e. inheritance of mutant germline allele

Answer 79

Germline mutations - CpG sites are methylated in the germline - So methylation status is passed onto all cells of the body - Can arise from DNA variant - that can lead to increased predispostion to DNA methylation E.g., variant in mismatch repair gene MLH1 - Germline mutation in MLH1 - causes epigenetic silencing - via methylation - cancer prone families

Answer 80

LOI - from biallelic expression (both alleles) or complete silencing of an imprinted gene - Observe loss/gain of methylation at ICs E.g., IGF2/H19 imprinted gene cluster - If maternal IC is methylated - no CTCF binding; enhancer activates IGF2 (insulated domain lost); overexpression of IGF2 - IC in this gene cluster is very susceptible to de novo methylation during cancer progression

Answer 81

- Genomic imprinting plays a key role as a natural tumour suppressor mechanism - And that loss of imprinting (LOI) predisposes to cancer

Answer 82

- Alterations in DNA methylation machinery increases methylaton across many genomic regions - CpG island methylation phenotype (CIMP) - Can arise due to overexpression of de novo DNA methyl transferase or mutations in TET/IDH (mutually exclusive)

Answer 83

- Isocitrate dehydrogenase (IDH) mutations (gain-of-function) - neomorphic activity - Mutant IDH needs heterodimeric activity to inhibit alpha-KG - which forms D-2-HG (TCA cycle) - D-2-HG is competitive inhibitor for enzymes that require alpha-KG for function - e.g., TET demethylases; histone demethylases - So - mutant IDH prevents demethylation; increases DNA methylation throughout genome; increases histone methylation

Answer 84

IDH increases methylation throughout the genome - So methylation prevents CTCF binding (but not at all sites - only where CpG recognition sequences are) - Pinpointed PDGFRA gene - next to CTCF sites affected by IDH mutants - PDGFRA gene (brain signalling) SO: IDH mutant gliomas are characterised by high levels of DNA methylation - at CTCF binding sites at genomic insulators (PDGFRA gene)- because D-2-HG formed by mutant IDH prevents demethylation - competitive inhibitor

Answer 85

- Genetic disruption (e.g., point mutations) at CTCF binding sites are preferntially found in boundary elements - Mutation of CTCF boundary sites - disrupts normal formation of insulated loop; activates adjacent oncogenes - Strongly selected for in development of cancer (common)