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Epigenetics Flashcards

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1
Q

Epigenetics Definition

A

study of any potentially stable and ideally heritable change in gene expression or cellular phenotype that occurs without changes in the base sequence

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2
Q

What is chromatin and what is the model used to describe it

A

Chromatin is a complex of DNA with its associated proteins

  • Bead on string model is used - beads are nucleosomes
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3
Q

What is contained within in nucleosome?

A

Octamer of core histones (two each of H2A, H2B, H3 and H4)
- 147 bp wrapped around twice

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4
Q

Two key features of the core (canonical) histones

A
  1. Highly conserved between species
  2. Amount of DNA wrapped around them is always the same
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5
Q

Structure of core histones (domain/tail)

A
  • Histone-fold domain: directs specific interactions between histones - regulates formation of bead
  • N-terminal tails - unstructured/basic tails - where charge resides - can become post-translationally modified - e.g., methylated/acetylated
    (Play critical in modifying chromatin structure - determine how tightly chromatin is packed and its association with other proteins)
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6
Q

Organisation of nucleosome

A
  • H3:H4 dimers form tetramers (H2A:H2B don’t)
  • Tails stick out of nucleosome - (not required for formation of nucleosome core structure)
  • H1 binds to linker between nucleosomes (linker highly variable)
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7
Q

Nucleosome core particle formation:

A
  • +ve AAs face outwards towards -ve charged phosphate backbone
  • Dyad located in centre - centered on H3-H4 tetramer
  • Various interactions between histones and DNA
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8
Q

Surface features of nucleosomes

A
  • Acidic patch contributed by H2A and H2B
  • Histone H4 tail - important in modulating chromatin higher order structure - interacts with acidic patch of neighbouring nucleosome
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9
Q

States of chromatin compaction

A
  1. Accessible euchromatin - transcriptionally active
  2. Restricted heterochromatin - transcriptionally inactive
    - Constitutive - all the time (e.g., both alleles)
    - Facultative - some settings (e.g., on one of the X chromosomes in females)
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10
Q

How is chromatin structure regulated? (4 ways)

A
  • Histone variants
  • Chromatin remodelling
  • Histone tail modification
  • DNA methylation
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11
Q

Two non-covalent mechanisms for the alteration of chromatin

A
  • Incorporation of histone variants
  • Chromatin remodelling complexes
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12
Q

Why is transcription of a gene within chromatin a challenge?

A

Because RNA polymerase cannot access the gene due to its wound up structure

  • Need to ‘open up’ structure and ‘reset’ it after the polymerase has gone through
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13
Q

3 Features of core (canonical histones)

A
  1. Found in all cell types (highly conserved)
  2. Encoded in gene clusters that are tightly regulated
  3. Synthesised in S-phase and deposited behind the replication fork
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14
Q

What is the role of CAF-1?

A

CAF-1 (Chromatin assembly factor-1)

  • Assists the assembly of chromatin - facilitates H3-H4 incorporation
  • Interacts with replication processivity clamp (PCNA)
  • Does replication-coupled assembly - incorporates the canonical histones
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15
Q

4 Features of histone variants

A

Histone variants (non-canonical histones)

  • Present in smaller amounts than canonical histones
  • Generally less well conserved - however H3.3 is highly conserved (3 differences between H3-H3.3 precent replication-independent assembly of H3)
  • Synthesised in interphase - inserted via exchange canonical histones (replication-independent assembly)
  • Most common variants are of H2A and H3
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16
Q

What is replication independent assembly?

A
  • RI assembly is the process by which histone variants are incorporated into chromatin
  • Large molecular machine (remodeller or RNA polymerase) partially unravels nucleosome during transit - disrupts nuclesome
  • E.g., H3.3 is incorporated - replacing H3 - using specific chaperones
  • RI significantly changes chromatin structure
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17
Q

What are the roles of histone variant H3.3? (3 roles)

A
  • H3.3 is associated with active chromatin - is incorporated into actively transcribed regions where nucleosomes are lost - it ‘plugs holes’ - done by RI assembly
  • Plays role in epigenetic memory - remembers gene expression pattern through multiple generations
  • Plays role in germline - H3.3 is a mediator of totipotency in the zygote
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18
Q

What are the key features of H2A.Z and H2A.B?

A

H2A.Z & H2A.B - both associated with transcriptional activation

H2A.Z - localized to gene promoters - prevents inappropriate silencing of genes (prevents DNA methylation) - if you loose this - cancer
- Also provides epigenetic memory during mitosis - e.g., marks gene for rapid reactivation following cell division

H2A.B - involved with looser chromatin structure - enhance transcription - at start sites

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19
Q

What effect does MacroH2A have?

A

Opposite effect to H2A.B at X chromosome

  • Associated with transcriptional repression of the inactive X chromosome
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20
Q

What is the role of chromatin remodelling activities? And what are some examples?

A

Remodelling activities shift the nucleosome to make it more accessible
- Nucleosome assembly, mediate chromatin access and histone exchange

E.g., SWI/SNF - transcriptional activation; mediates local disruption of chromatin
- NURD (CHD class) - transcriptional repression; oppose SWI/SNF at same promoter; mediate nucleosome invasion of regulatory DNA
- INO80 - catalyses exchange between canonical histone H2A and variant H2A.Z - facilitating transcriptional activation

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21
Q

5 classes of nucleosome remodeller

A

All classes contain an ATPase domain - and are defined by their other specific domain

  • SWI/SNF Family - HSA - binds actin related proteins
  • ISWI Family - SANT/SLIDE - nucelosome interaction
  • CHD Family - Chromo domains - mediate chromatin interaction by binding to methylated Lysine residue on histone tail
  • INO80 Family - HSA - binding actin related proteins
  • Bromo - recognition of acetylated lysine residue on histone tail
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22
Q

What is the importance of remodelling activities?

A
  • Individual remodellers may function in a diversity of tissue-specific complexes - e.g., SWI/SNF complexes work in many species
  • Remodelling is critical in maintaining a permissive chromatin environment in self-renewing stem cells - and regulating differentiation
    Both: - Exit from self-renewing state - to allow multi-lineage commitment; formation of heterochromatin - silence of pluripotency genes
    AND - nuclear programming - remodellers needed to reverse process - reactivate pluripotency genes
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23
Q

How is differential gene expression in different cell types achieved?

A
  • The presence of different trans-acting transcription factors in diff cell types

AND/OR:

  • Differences in the accessibility of these factors to their binding sites in DNA - determined by high-order organisation of chromatin
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24
Q

Where do histone tails lie? And where do post-translational modifications occur?

A
  • Histone tails protrude from nucleosome
  • Post-translational modifications occur on all four histone tails (H2A, H2B, H3 and H4)
  • Acetylation - on all four tails - on lysine
  • Methylation - mainly on H3 and H4 - mainly lysine, some arginine
  • Some residues can have multiple modifications; some adjacent sites of modification show ‘cross talk’
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25
Q

What are the three roles required for histone tail modifications?

A

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)

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26
Q

What do histone modifications do?

A
  • 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

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27
Q

What are trans effect?

A

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)

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28
Q

What are histone acetyl transferases (HATs)?

A

HATs are writers - involved in transcriptional activation

  • Catalyse histone tail acetylation
  • Different HAT subfamilies use diff catalytic strategies for acetylation
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29
Q

What does histone tail acetylation (HTA) lead to?

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

What are bromodomains?

A

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)
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31
Q

What are histone deacetylases (HDAC)?

A

HDACs are erasers - involved in transcriptional repression by removing histone tail acetylation

  • E.g., Mad1 - interacts with Sin3 protein - interacts with HDAC - clinical relevance
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32
Q

How do transcription factors regulate gene expression at gene promoters, and what is the importance of Myc/Mad?

A
  • 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
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33
Q

What is the role and mechanisms of pioneer transcription factors?

A

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
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34
Q

Why are histone tail modifications complexed?

A
  • Result in gene activation/repression
  • Use facultative vs constitutive heterochromatin
  • Mechanisms of reinforcement - active/inactive states
  • Interactions of adjacent HTMs
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35
Q

What is the role of histone tail methylation (HTM)?

A
  • 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
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36
Q

What are the key differences between HTA and HTMs? (3 differences)

A
  • 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
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37
Q

What is position effect variegation (PEV)? And how has it been studied?

A

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
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38
Q

What is Su(Var)3-9, and how does it interact with HP1?

A
  • 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
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39
Q

What is HP1, and what is the function of its different domains?

A

HP1 - heterochromatin protein

  • Chromo domain - binds methylated H3K9
  • Chromoshadow domain - mediates homodimerisation and interaction with Su(Var)3-9
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40
Q

What would mutations in HP1 chromodomain cause?

A
  • Would abolish heterochromatin - reduce gene silencing
  • Would abolish interaction with methylated H3K9
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41
Q

What key role that HP1 plays with regards to heterochromatin?

A
  • 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
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42
Q

What happens to heterochromatin during mitosis - why is it significant?

A
  • HP1 is removed from heterochromatin during mitosis - to allow for proper segregation
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43
Q

What HTM patterns correlate with gene activation rather than repression? Examples

A

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

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44
Q

How are HOX genes established and maintained?

A
  • 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
45
Q

How do polycomb proteins suppress HOX genes?

A

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

46
Q

What are polycomb proteins and what do they do?

A

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
47
Q

What is bivalent chromatin and why are they not expressed?

A

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)
48
Q

What do bivalent chromatin and polycomb proteins do together?

A
  • Regulate stem cell pluripotency and lineage commitment
  • Upon differentiation - polycomb complexes are displaced - and bivalent chromatin resolved into active (H3K4me3) or repressed (H3K27me3) states
49
Q

Role of genomic insulator elements

A

Critical role in shaping the 3-D organisation of the genome

  • Bind to proteins such as CTCF
50
Q

What is CTCF and what does it do?

A

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
51
Q

What are the two types of DNA methylase? How do they differ?

A

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
52
Q

When is demethylation important, and how are methyl groups removed?

A

Demethylation is important in early development

  • By active enzymatic demethylation (TET) enzymes- sperm
  • Or passively - by dilution following DNA replication - oocyte
53
Q

How do the methylation patterns compare between oocyte and sperm DNA?

A
  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)
  1. Both genomes are then remethylated - BUT - imprinted genes are NOT affected - they maintain their original status
54
Q

What are CpG islands, and where are they found?

A

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
55
Q

When are CpG islands found to be methylated?

A
  • Inactive x-chromosomes
  • Some imprinted genes
56
Q

How does CpG island methylation lead to cancer?

A

Inappropriate methylation of CpG islands - silences genes

  • Can cause silencing of tumour suppressor genes - cancer
57
Q

What is the role of Cfp1 and KDM2A with regards to CpGs?

A

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
58
Q

How does methylation of CpG sites contribute to the formation of a repressive chromatin state?

A
  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
59
Q

What are the roles of MeCP2 and MBD1?

A
  • 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

60
Q

What are chromatin territories?

A
  • Distinct regions of the nucleus occupied by individual chromosomes
  • Activated genes can move outside of their territory
61
Q

What is the 3-D structure of chromatin - (A, B, C)?

A

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)

62
Q

How are TADs separated? And where are ‘active’/’inactive’ TADs found?

A
  • 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
63
Q

When can genes switch from inactive to active compartments?

A

During differentiation

  • Correlated with an altered epigenetic state - and with transcriptional activation
64
Q

What are insulators?

A

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

65
Q

How does an insulated neighbourhood form a TAD?

A
  • 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
66
Q

Why are anchors for loops important in cancer

A
  • Deletion or mutation of anchor - leads to aberrant gene activation - cancer
  • CTCF - some of most mutated transcription factor binding sites in cancer
67
Q

Why are insulated neighbourhoods complexed in organisation?

A
  • Can be organised via different combinations of loops - that dictate which genes are co-regulated
  • See schematic
68
Q

What other types of interactions are there in the formation of chromatin?

A
  • 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
69
Q

How do histone modifications regulate chromatin compartmentalisation?

A
  • 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
70
Q

What is genomic imprinting?

A

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
71
Q

Where did the first experiments come from that showed genetic imprinting?

A
  • 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
72
Q

What is a chimeric embryo, and what do maternal/paternal chimeric mice display?

A

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
73
Q

Features of imprinted genes, and how they are controlled

A
  • 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
74
Q

How is imprinted cluster containing IGF2 and H19 controlled? And how does it differ for maternal and paternal alleles?

A

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
75
Q

What mechanism is used for imprinting control of Igfr2 gene cluster? Detail for alleles

A

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)

76
Q

What roles do imprinted genes play in biology?

A
  • 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)
77
Q

What is the ‘parental conflict hypothesis’, and what do maternal/paternal imprinted genes act to do?

A
  • 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

78
Q

How are imprinted genes affected during development?

A
  • 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
79
Q

How are DNA methylation patterns established in the germline? And how do they differ for males/females?

A

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
80
Q

What are the advantages that differentially methylated regions provide for ICs?

A
  • Differentially methylated regions regulate enhancer-blocking mechanisms or regulate expression of antisense transcripts
81
Q

How is methylation maintained in ICs?

A
  • 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
82
Q

What is primary ICR methylation? And how does it differ for maternal/paternal genome?

A

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
83
Q

What are secondary DMRs?

A
  • 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
84
Q

How do epigenetic defects contribute to chromatin-related diseases

A

Due to an alteration in the methylation of the DNA

  • Lead to genomic imprinting disorders
85
Q

What are the two different categories of genetic defects, and how do they differ?

A

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

86
Q

When does a disease phenotype occur in imprinted genes?

A
  • 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
87
Q

Summary of the unusual patterns of inheritance for imprinted genes
(using example of a silent mutation on the maternal allele)

A
  • 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
88
Q

What are Prader-Willi syndrome (PWS) and Angelman syndrome (AS), what is the critical gene, and how do disorders appear?

A

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

89
Q

What is Beckwith-Weidemann syndrome (BWS), what gene(s) are involved, and how is it caused?

What is normal, and what happens in the case of the disorder?

A

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

90
Q

How does BWS increase tumour risk?

A

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)

91
Q

What is Silver-Russel Syndrome, and what is it associated with?

A
  • 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
92
Q

How do in trans disorders affect chromatin structure? (3 ways)

A
  • Regulators of DNA methylation machinery
  • Histone modifying enyzymes
  • Chromatin remodellers - e.g., FHS
93
Q

How is Floating Harbor Syndrome caused, and how has it been investigated?

A
  • 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
94
Q

What is Fragile X syndrome, and what is it associated with?

A
  • Common inherited intellectual disability
  • Associated with methylation of an expanded CGG repeat in CpG island in the 5’ UTR of the FMR1 gene = silencing
95
Q

By what mechanisms can the primary epigenetic changes that can cause cancer? (5)

A

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)
96
Q

What genetic changes can influence cancer development (cis and trans effects)

A

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)

97
Q

What 3 general ways can DNA methylation changes result in cancer, and how?

A

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

98
Q

Why are C-T transitions characteristic of cancer genomes?

A
  • 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
99
Q

Why is there reduced methylation in cancer genomes?

A

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

100
Q

Where can DNA hypermutation be associated with cancer?

A

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

101
Q

Why is methylation critical in tumour progression?

A
  • 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
102
Q

What is the relevance of hypermethylation at gene promoters with cancer?

A
  • 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
103
Q

What are constitutional epimutations, and give an example?

A

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

104
Q

How does Loss of Imprinting (LOI) arise in Cancer?

A

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

105
Q

What did mouse models prove about the importance of genetic imprinting in cancer?

A
  • Genomic imprinting plays a key role as a natural tumour suppressor mechanism
  • And that loss of imprinting (LOI) predisposes to cancer
106
Q

How does DNA hypermethylation arise at CpG islands?

A
  • 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)
107
Q

How do IDH mutations contribute to cancer?

A
  • 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
108
Q

What effect does IDH have on insulator boundaries?

A

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

109
Q

How are CTCF boundary elements affected in cancer?

A
  • 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)

Advanced Genetics (4 decks)

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