L7, Epigenetics Flashcards
1
Q
Definitions of epigenetics: (Particularly molecular definition)
A
- Inheritance of any change in gene function that does not involve a change in DNA sequence
- Persists in absence of the initiating signal
- Molecular: Chemical modification of histones and DNA because these are required for or contribute to a heritable change in gene expression
2
Q
Reminder: Nucleosome structure
A
- 10nm diameter
- 8 core histone proteins with DNA wound around 2x
- Tails interact with other nucleosomes to compact DNA -> typical site for modification (K, R, S, P residues)
3
Q
Chromatin modifications: Effects and usefulness
A
- Affecting chromosome accessibility
- Inherited -> stable mitotically speaking but still reversible changes
- ncRNAs (e.g. Xist)
4
Q
Main types of epigenetic modification:
A
- Modifications of DNA
- Modification of histones
- Assembly of protein structures on DNA
5
Q
Properties of heterochromatin:
A
- Highly condensed; generlly not transcribed
- Typically low gene density
- Replicates late in S phase
- Often localised to nuclear periphery
- No meiotic recombination is possible in these regions
- Typically heavily methylated with characteristic PTMs of histones
- Either constitutive (all cells the same) or facultative (e.g. Inactive X)
6
Q
How is CpG methylation enacted? How may it be reversed?
A
- DNA modification
- De novo methylase adds methyl group to cytosine (does not affect base pairing) e.g. DNMT3a/b
- Perpetuation methylase add Me to hemi-methylated strand e.g. DNMT1
- Methyl group is removed by Demethylase enzymes
7
Q
What is the function of CpG islands?
A
- Typically found in promoter regions of mammalian genes
- Leads to constitutive expression as the action of DNTs meaning they tend not to get methylated
- Weaker, less dense CpG sequences can be regulated so can be either methylated or unmethylated
- This is a heritable change
8
Q
How is heterochromatin created after replication?
A
- Created by self-assembling complexes, part of which remain bound after replication
- This allows the bound proteins to recruit more subunits to daughter chromosomes
9
Q
Well-characterised sites on H3 tail for modification:
A
- Mono, di or tri methylation of H3K9 and H3K27
- K = Lysine
- H3K4 demarcates euchromatin
10
Q
What types of proteins regulate epigenetic marks?
Give example
A
- Writers (histone acetyl transferases, serine/threonine kinases, PRMT, HKMT)
- Readers (proteins with chromo- domains etc)
- Erasers (histone deacetylase, protein phosphatase , deiminase, amine oxidase hydroxylase)
11
Q
Readers for acetylation vs methylation:
A
- Acetylation recruits Bromo-domain containing proteins, facilitating euchromatin -> ACTIVE
- Methylation recruits chromo-domain containing proteins -> can vary but generally leads to heterochromatin -> transcriptional repression
12
Q
Example: Formation of heterochromatin
A
- Typically coupled events, often with positive feedback loops in action
- Eraser: HDAC removes Ac from H3K14
- Writer: Allows SUV39H1 to trimethylate H3K9
- Reader: HP1 binds H3K9me3 -> able to propagate condensing signal due to self aggregation
13
Q
Heterochromatin protein 1 (HP1): Key domains and interactions
A
- Chromodomain: binds to specific methylated lysines (K), often associated with transcriptional silencing
- Linker domain
- Chromoshadow domain: facilitates interaction with itself and other proteins -> thus able to self-aggregate, propagating condensing signal -> also interacts with DNMT1 and H3K9 methyltransferase (positive feedback loop)
14
Q
+ Further feedback loop examples (slide 20)
A
- Pericentric heterochromatin
- Repression via HDAC, Sin3A and MeCP2
- Further repression…
15
Q
+ How is the spread of heterochromatin limited?
A
Barrier regions -> physically halting positive feedback loops
- e.g. Nucleosome depletion
- e.g. Nucleosome turnover
- e.g. Opposing PTMs
- e.g. PTM-mediated eraser recruitment
16
Q
What are the polycomb group proteins?
A
- First identified in drosophila -> mutants allow expression of homeotic genes which are usually repressed
- Pc-G proteins maintain repressed state
- Able to perpetuate repression through cell divisions
17
Q
How do Pc proteins function?
A
- Pc is a prototype for ~15 Pc-G proteins
- Function in large complexes, which form Polycomb response elements (~10kbp), maintaining repression throughout development
- PRE provides nucleation centre for complex binding and repression spread either side of itself
18
Q
How are PREs established?
A
- PRC2 (writer) trimethylates H3K27 -> recruitment of PRC1
- PRC1 (reader and writer) binds H3K27me3, then monoubiquitylates histone H2A on lysine 119
19
Q
How is X-inactivation carried out? (Overview)
A
- Dosage compensation creating facultative heterochromatin
- Depends on X inactivation centre locus (Xic) on X chromosome
20
Q
How is X-inactivation carried out? (Mechanistic)
A
- Both chromosomes initially express Xist from the Xic locus (unstable lncRNA) -> coats chromosome, repressive
- In one cell, Tsix (antisense) is expressed -> Xist degradation
- More Xist in other cell, which coats it and drives heterochromatin formation
- Stable Xist expression -> PRC1 and PRC2 recruitment -> hypermethylation and hypoacetylation -> inactive (Barr body)
21
Q
+ 3 broad groups of PcG proteins:
A
- PRC1 and PRC2 as in lectures
- Polycomb repressive Deubiquitinase
22
Q
+ What 3 key processes are PcG proteins essential in?
A
- Embryonic development
- Stem cell differentiation
- Tissue homeostasis -> PcG misregulation in humans gives rise to various cancers
23
Q
+ Specific roles of the 3 PcG complex groups; Include the catalytic subunit for each:
A
- PRC1: E3 ubiquitin ligases -> monoubiquitinate H2AK119 (using RING1 protein)
- PR-DUB: deubiquitinates H2AK119 (catalytic subunit: Calypso)
- PRC2: methyltransferase -> H3K27 methylation (1/2/3x) (actually made up of 2 distinct complexes, PRC2.1 and 2.2); multifaceted catalytic lobe
24
Q
+ What are SIRs? Example in yeast:
A
- Silent information regulator
- Silencer elements are recognised by sequence-specific DNA-BPs that recruit Sir1, 2, 3, 4
- Sir2 is a NAD+-dependent histone deacetylase
- Complex interaction between the 4 proteins results in gene silencing
- System is unique to yeast
25
Q
+ What is the role of SPEN during X inactivation?
A
- Xist recruits SPEN which recruits HDAC3
- Ensuing histone deacetylation triggers recruitment of various silencing machineries including that of PRC1 and 2
