L7 : Polycomb Repression, Xi, Imprinting Flashcards
How was polycomb repression discovered?
Discovered in Drosophila as negative regulators of HOX genes
Name originates from defective polycomb repression resulting in multiple sex combs (bristle-like structures) on all 3 rather than 1 pair of legs
Briefly describe PcG
Gene silencing by PcG mainly through regulation of chromatin structure, including modification of histones
PcG comprises two polycomb repressive complexes: PRC1 and PRC2
What is the role of PcGs and TrxGs?
Maintains specific expression programs throughout lifespan
Polycomb group:
- Repress expression after departure of initial repressor
Trithoprax group:
- Maintains expression of genes after departure of initial activator
- Act antagonistically to PcGs to activate gene expression
Explain polycomb repressive complex 2 (PRC2)
Acts through H3K27me2 or 3 (trimethylation of Lys27)
- Associated with chromatin compaction
- Transcriptional repression
What are the key components of PRC2 and their roles?
Core catalytic unit:
Ezh1/2 (enhancer of zeste 1/2)
- Catalysis of H3K27 di/tri methylation
Stabilisation and activation:
EED
(embryonic ectoderm development)
- Required for Ezh1/2 activation
SUZ12
(suppressor of zeste 12)
- Required for Ezh1/2 activation
RbAp46/48
- Stabilises complex
- Required for full enzymatic activation
Accessory proteins:
JARID2
(Jumanji and AT-rich interaction domain 2)
- Preference for GC-rich DNA
PCL and ARBP2
(polycomb like proteins and adipocyte enhancer binding protein 2)
- Assist recruitment of PRC2 to DNA/histones
Explain polycomb repressive complex 1 (PRC1)
Acts through monoubiquitination of H2A 119
- Affects chromatin compaction
- Contributes to transcriptional repression
How does composition of PRC1 change dynamically?
Composition, targeting and activity of PRC1 changes dynamically during embryonic development, cell differentiation, in cancer
Note: PRC1 composition more variable than 2
What are the key components of PRC1 and their roles?
RING1 A/B Ub ligase
- with either BMI1, MEL18, NSPC1
CBX
- bind to H3K27me3 (product of PRC2 catalysis) via chromodomains
HPH1/2/3
- involved in PPIs in polycomb complex
X,Y,Z
- denote additional proteins with undetermined functions
How does PRC1 action affect transcription?
- Mono ubiquitination of H2A K119
- Prevents association of chaperone which temporarily removes histone octamers in nucleosomes during transcription
- Persistence of histone octamers inhibits transcription by Pol2
- Results in transcriptional repression
Describe the canonical model for repression by polycomb complexes
- Recognition and binding of repressor molecules activates PRC2 component E(z)
- Trimethylation of H3K27 as repressive marker
- CBX of PRC1 complex recognises K27me3, and binds
- Further compacts DNA through ubiquitination of K119 and remodelling of chromatin
- Genes not accessible and transcription inhibited
Compare canonical and non-canonical PRC1
Always have RING1 Ub ligase but other components can vary
Canonical has lower H2AK119Ub catalysis and PRC2 dependent recruitment by H3K27me3
What is X-inactivation?
Dosage compensation
One X chromosome inactivated through action of ncRNA
Induces alterations in histone acetylation
Which X-chromosome is inactivated?
In mammalian embryos, X-inactivation is random
In extraembryonic tissues, paternal X-chromosome preferentially inactivated
What is Xist?
Xist is long non-coding RNA
Tsix is antisense repressor of Xist
- Transcribed until down regulation of Tsix
- Both chromosomes X activa and transcribing Tsix until switched off
What is the mechanism for initiating X-inactivation (XCI) by lncRNA?
INITIATION
1. Biallelic Tsix prevents loading of RepA-PRC2 and initiation of XCI
2. Two events enable Xist expression during cell differentiation
- Induction of Jpx activation
- Monoallelic loss of Tsix on Xi, allowing RepA-PRC2 to load
3. Xist cotranscriptionally recruits PRC2
- YY1 protein binds Xi nucleation centre, but is blocked from binding Xa
4. Xist-PRC2 complex cotranscriptionally loads onto YY1-based nucleation centre
PROPAGATION
5. From nucleation centre, Xist-PRC2 spreads in cis-limited fashion (only the chromosome it was produced on) to 150 strong Polycomb station
- In turn, spreads repressive histone mark H3K27me3 via 3000-4000 moderate polycomb sites
- Chromatin condenses and transcription/gene expression silenced
MAINTENANCE (Xist independent)
6. Xi condenses into Barr bodies and is late replicating
7. Hypermethylation of promoters (CpG islands) by Dnmt3b
Note: Around 25% genes escape Xi
What is genomic imprinting?
Form of epigenetic gene regulation resulting in expression from a single allele in a parent of origin dependent manner
- Transcriptional inactivation relying on DNA methylation
Approx 30 imprinted genes in mice and humans (comprise ~1% genome)
What is the effect of imprinting on H19 and IGF2 gene expression?
H19
Maternally expressed and paternally suppressed
IGF2
Paternally expressed and maternally suppressed
- Promotes foetal and placental growth
How does methylation control H19 and IGF2 expression?
Both share imprinting control region (ICR)
- Regulates access to downstream enhancer region
Unmethylated maternal allele
- CTCF insulator protein binds ICR
- Blocks enhancer from activating IGF2
- H19 expressed, IGF2 silenced
Methylated paternal allele
- CTCF cannot bind
- Enhancer activates IGF2
- IGF2 expressed, H19 silenced
What are examples of imprinting defects for IGF2 and H19?
Beckwith-Wiedemann syndrome (BWS)
- IGF2 expressed from both alleles
- H19 silenced
-> Overgrowth from excess IGF2 growth signalling
Silver-Russell syndrome (SRS)
- H19 expressed from both alleles
- IGF2 silenced
-> Leads to restricted growth from lack of IGF2
What is the epigenetic status at different stages of cellular development?
- Totipotent
- Global DNA demethylation - Pluripotent
- Only Xa
- Global repression of differentiation genes and promoter hypomethylation - Multipotent
- X inactivation
- Repression of lineage specific genes and promoter hypermethylation - Unipotent
- Derepression of Polycomb silenced lineage genes
How can cells be reprogrammed to an earlier state?
Achieved by ectopic retroviral expression of 4 TFs
Yamanaka factors (OSKM):
- Oct4 (octamer-binding TF 4)
- Sox2 (SRY-box2)
- Klf4 (Kruppel like factor 4)
- c-Myc (avian myelocytomatosis viral oncogene homolog)
What are characteristics of iPSCs (induced)?
- Self-renewal
- Pluripotency
- ES cell morphology
- Demethylation and reactivation of pluripotency genes
- X-chromosome reactivation
- Telomerase activity
- Loss of G1 checkpoint
How do epigenetic signatures change during reprogramming into iPSCs?
Mouse fibroblasts can be reprogrammed to pluripotency
- Epigenome is different according to whether pluripotent or differentiated
- iPSCs show morphology and growth characteristics of ESCs
