L4 : Xi and Xist Regulation Flashcards
Why regulate chromatin?
Structure can be altered to regulate transcription and provide epigenetic memory of transcriptional state
How can chromatin be regulated?
- DNA methylation
- Histone modification
- Nucleosome remodelling
What makes up the RNA component of chromatin?
- Pre-mRNA and other nascent non-coding RNAs (ncRNAs)
- snRNAs (involved in processing rRNAs)
- RNAs from repeat elements (ef. SINEs, LINEs)
- Some mature long non-coding RNAs (lncRNAs)
What is a Barr body?
Female (XX) mammalian cells contain extra nuclear body not present in male cells
Barr body identified as one X chromosomes present in compacted state
Why is X-inactivation important?
Provides dosage compensation and equal expression of X chromosome between sexes
Which X-chromosome is inactivated in human embryos?
In inner cell mass (embryo) - randomly silences either paternal or maternal
In extraembryonic tissue (placenta) - paternal is preferentially inactivated
What is epigenetic mosaicism and an example?
Arises from random X-inactivation in female mammals
- Some cells express genes from paternal while others express from paternal X
Generally offers protection females from X-linked diseases
Example: tortoise shell cat fur coat
What role does Xist RNA play in X inactivation?
Xist is gene located in X inactivation centre
- Active only in cells with 2+ X-chrs
- Expressed from X-chr that will become silenced
- Produces non coding RNA that coats X-chr and its initiates inactivation
- Xi centre also contains other ncRNAs to help regulate Xist expression
What occurs to chr when Xist is transcribed from X inactivation centre?
- Xist RNA spreads along X chr which becomes gradually compacted
- Xist forms a cloud of RNA around inactive X
How does Xist RNA associate with the X chr?
Requires RNA binding proteins
- HNRNPU tetheres Xist to chromatin
- CIZ1 maintains association
Why may repeat elements within Xist RNA be important?
Contains several conserved repeat elements
Thought to contribute to Xist function through forming structures
How was it shown that Xist interacts with chromatin modifiers?
- UV crosslinking (formaldehyde) within cells
- Purification of Xist RNA with biotinylated oligonucleotides
- Proteins co-purifying with Xist RNA identified by mass spec
What is an example chromatin remodeller DIRECTLY recruited by Xist?
SHARP (SPEN) binds Xist A repeats (identified by MS)
- Necessary for Xist mediated transcriptional repression (exclusion of RNA Pol II)
- Recruits HDAC3 (component of SHARP), which removes acyl groups from histones, promoting repressed state
Why are Polycomb proteins significant?
Essential for development and cell differentiation (also maintenance of stem cells) in all multicellular organisms
Polycombs frequently dysregulated in cancer
What are the 3 types of polycomb repressive complexes?
vPRC1 (variant)
PRC2
cPRC1 (canonical)
Function together to compact and repress chromatin
How does Xist recruit polycomb complexes?
- Xist binds variant PRC1 through hnRNPK (indirect recruitment of PRC1)
- Variant PRC1 ubiquitinates H2A at K119, creating H2AK119ub1
- Mark is recognised by JARID2 (subunit of PRC2)
- PRC2 then methylates H3 at K27, creating H3K27me3
- Methyl mark is bound by canonical PRC1 via the CBX chromodomain
- Reinforcing gene silencing
What are the subsequent epigenetic changes of Xi?
DNA methylation and H3K9 methylation
- Blocks transcription by preventing binding of TFs and chromatin regulators (eg. CTCF and MLL)
- Recruits methyl-binding proteins (MBP) and associated HDACs
How do the Xa and Xi chromosome fold?
Exhibit different pattern of folding as a result of Xist action
- Xi heavily modified with repressive genetic marks, compacting chromatin
- Xa maintains more open and accessible structure to support gene expression
How do CTCF and cohesin shape higher order chromatin structure
- CTCF binds insulator elements, creating boundaries between chromatin loops
- Cohesin (ring-like molecule) extrudes DNA into loop until its hits pair of CTCF-bound insulator elements
- CTCF recognise specific DNA sequence that forms base of loop
- CTCF binding to DNA is inhibited by DNA methylation, in turn controlling higher-order chromatin structure
What is genomic imprinting and how does it work?
Limits gene expression to only paenral or maternal allele
In humans, over 200 genes known to be imprinted
Inactive allele is silenced by DNA methylation in sperm or egg and this pattern is maintained after fertilisation
What are the proposed reasons for imprinting?
Still unknown but may reflect evolutionary conflect between sexes
- Paternally expressed genes tend to promote growth
- Maternally expressed genes promote balanced provision of resources
How can disorders arise from imprinting and example?
Disruption by genetic mutations or epigenetic changes
Example: Prader-Willi syndrome of chromosome 15 can be the result of imprinting (genes normally expressed from paternal copy are not present or silenced)
- Hyperphagia, intellectual disability, hypotonia in infancy, behavioural issues
How do imprinting control regions guide genomic imprinting?
Depends on allele-specific DNA methylation of ICR
- Maternally methylated ICRs correspond to promoters (often of ncRNAs)
- Paternally methylated ICRs tend to be insulators or enhancers
What is an example of how genomic imprinting is controlled?
11p15.5 locus
Contains IC1 at H19 gene promoter and IC2 at KCNQ1OT1 gene promoter
Maternal
- IC1 lacks 5mC = expression of H19
- IC2 has 5mC = silencing of KCNQ
Paternal
- IC1 has 5mC = block H19 expression
- IC2 lacks 5mC = antisense ncRNA KCNQ transcription
