L10 - Chromatin Modifications Flashcards
Characteristics of epigentic inheritance
Change to the chromatin structure
Stable changes to gene expression
Erased in the germ line
Reversible
Characteristics of genetic inheritance
Change to the DNA base sequence
Can be repaired if recognised (e.g. nucelotide excision repair) if not recognised then they are permanent
If in a somatic cell (passed into all progeny)
If in a germ cell passed into all of the cells of the offspring
Describe the metaphor for the epigentic landscape
Like a ball (early ES cell) rolling down a hill
Series of valleys (fates_ and obstacle which the cell must overcome to reach its sepcialised cell fate
How many BP of DNA wrap around a histone
144 bp
Wrapped twice
How many subunits of the histone
8
What are the N terminal tails of the histone octomer
How do these project
2 of each: H2A H2B H3 H4
Project into free space which allows them to be modified
Describe the characteristics of closed chromatin
Concdensed
Topology makes it hard for RNA pol 2 to bind
Histones are closely associated to the DNA
Describe the characteristics of open chromatin
Sometimes dissociated from the DNA
RNA pol II able to bind
What are the 3 modifications which can be made to the chromatin
Acetylation of lysines
Mono, di, tri methylation of lysines and arginines
Phosphorylation of serines
Acetylation and methylation can both occur to which AA
Can these both occur at the same time
Lysine
No these are chemical modifications
Describe the side chain of lysine
4(CH2) - NH3
Describe how acetylation of lysine occurs
What is the name of this new structure
Final N -H(C(CH3)O)
Aceyl lysine
What enzymes add acetyl groups to lysines
What is the specificity of these enzymes
Histone acetyltransferases
Can add acetyl groups to many different lysines
What enzyme methylated lysines
What is the specificity of these enzymes
Histone methyltransferases
Exhibit equisite site specificity
What lysines/argines can be methylated of histone tail H3
H3K4 H3K9 H3K27 H3R17
What are some types of histone acetyltranferases
CREB binding protein
PCAF GCNS
What are the two code erasers (for methylation and acetylation)
Histone deacetylases
Histone demethylases
What are the two code wirters (for methylation and acetylation)
Histone acetyltransferase
Histone methyltransferase
Does acetylation cause activation or inactivation
All acetylations are activators
Does methylation cause activation or inactivation
Depends which residue is methylated
Two residues that can be methylated to activate the chromatin
H3K4 H3R17
Two residues that can be methylated to INactivate the chromatin
H3K9 H3K27
How does acetylation cause transcriptional activation
Creates a binding site for proteins with a bromodomain
Describe the bromodomain transcription activator
An epigenetic code reader
Affinity for bidning is higher when there are many resides which have been modified
What does methylation of core histone N terminal tails create binding sites for
Transcription repressors which contain chromodomaisn
Transcriptional activators which contain a PHD zinc finger domain
DEPENDS ON WHICH RESIDUE IS METHLYATED
How do transscription activator proteins act in combination with a chromatin remodelling complex
Selective nucelosome remodelling
Selective histone remodelling
Selective histone replacement
How do transscription activator proteins act in combination with a histone modifying enzyme
Selective histone modifcations causing the recruitment of code writers and code readers
What are the ways in which transcriptional repressors work in chromatin
Competitive binding with the activator for the DNA
Masking of the activation surface
Direct interaction with general transcription factors
Recruitment of chromatin remodelling complexes
Recruitment of histone deacetylases
Recruitment of histone methyltransferases
What is the polycomb group of proteins
Polycomb repressive complexes (PRC)
Includes proteins which are able to generate/read repressive chromatin modificaitons
What type of the system in the polycomb (PRC)
Histone code reading and code writter
Are chromodomains associated with transcription activation or inactivation
Inactivation
PRC2
Makes the mark triggering transcriptional repression
PRC1
Maintains the repressed state (chromodomain)
What does PRC2 require inorder to be able to function
What is the action of this component
Requires EZH2 (enhancer of zeste) This is what actually methyaltes (e.g. H3K27)
Once PRCR and EZH2 has performed the methylation of the H3K27 what occurs
Recruitment of PCR1 with polycomb chromodoation (code reader) formation of silent/repressed chromatin
How can cytosine be modified
What enzyme is responsible for this
Can be methylated DNA methyltransferases (DNMTs)
What is the interaction between DNMT and histone methyltransferase EZH2 `
Physical interaction
Mutually reinforce each others effects
What are transcriptionally inactive promoters rich in
Methylated CpG dinucleotides
What binds to methylated CpG dinucleotides
Methyl CpG binding protein 2 (MECP2)
Interact with the histone deacetylases and and histone methyltransferases in order to transform acetylated nucleosomes to methylated nucleosomes
Why is X inactivation required
In males XY, there is only one dose of X linked genes, important that this is conserved in female cells
What determines which X chr is silenced
One in each cell is silenced in each somatic cell in the early embryo - ONCE DONE THIS IS PERMANENT
RANDOM
The silencing decision is then
Propagated clonally
Calico cats are exlusively
Female
Descirbe how the calico cat shows evidence of X inactivation
Fur colour on X chromsome
Heterozygous for fur colour - random which X chr get silenced in the embryo so random pigmentation (orange/black) of the fur
Describe the mechanism by which one X chromsome is silenced
Synthesis of non-coding RNA - (xist RNA) from the X inactivation centre (XIC) of the Chr destined for inactivation
Xist RNA binds to the X chr in cis and promotes chromatin condensation which spreads from the XIC in both directions
Xist RNA also recruits histone modififying enzymes and other poly comb components leading to H3K27 AND H3K9 methylation of core histone N-temrinal tails also promotes deacetylation
What forms as a result of X inactivation
A barr body - region of highly condensed X chromosome at the periphery of the nucleus of female somatic cells
What maintains the expression of the active X
Unknown ammount of an unknown autosomal activator
What can also effect DNA methylation
Dietary components (e.g. a diet high in methyl donors)`
Describe the Agouti gene
Codes for yellow fur and obesity - constitutively high expression of the agouti protein
What element does the agouti gene contain
IAP element which strongly activates the gene but is also highly sensitve to DNA methylation
How can the w/t mouse phenotype be restored
Diet high in methyl donors causes methylation of that gene and a rescue of the phenotpye
What is the key difference between genetic and epigenetic modifications
Genetic alterations occur to the DNA sequence directly and permanently affect gene expression. Epigenetic alterations occur to chromatin structure and act to modulate gene expression. These do not alter the DNA sequence and are reversible
Epigenetic modifications facilitate stable changes in gene expression which may persist or the life of the cell of organism but are erased in the germ line, T or F
F – whilst the statement is true, epigenetic modifications are transferred to the progeny these cells can then decide whether to remove the epigenetic change
How did Conrad Waddington describe how cells differentiate due to epigenetic changes
Cells traverse and epigenetic landscape of various differentiation possibility which gradually restricts cell fates
What structures are referred to as the building blocks of chromatin
Nucleosomes
What is significant about the tails of core histones
Nucleosome core histones have N-terminal lysine rich tails which project radially from the core. These can be reversibly covalently modified
Which residues are commonly acetylated by histone acetyltransferases
Lysine residues
Histone methyltransferases mono, di or trimethylated which amino acids within the histone tails
Lysine and arginine
Acetylation and methylation of core histone tails can occur simultaneously, T or F
F – methylation and acetylation are mutually exclusive and are competing modifications
Histone acetyltransferases can modify many different lysine residues, T or F
T
Histone methyltransferases can modify many different lysine or arginine residues, T or F
F – histone methyltransferase exhibit exquisite site specificities
Enhancer of zeste is one enzyme that methylates a lysine residue. What position in the polypeptide chain does it act
EZH2 methylates lysine 27
What is the role of EZH2 in development
EZH2 is a gene required to repress Hox gene expression in a specific anterior-posterior fashion
Histone methyltransferases act as regulators of gene transcription and are uniquely site specific, T or F
T
What is meant by histone code writers
Histone methyltransferases are histone code writers, they act as an additional code on top of the genetic code i.e. epigenetic to
What are the names of the enzymes that reverse histone acetylation and methylation respectively
Histone deacetylase and histone demethylase
Reversibility of the acetylation and methylation of histones accounts for what attribute of epigenetic changes
Means that they can be removed – aren’t permanent
Lysine acetylation is an indication of what
Transcriptionally active genes
Methylation can denote transcriptionally active or inactive genes depending on the loci of the residue. Determine whether methylation of lysine 4, 9, 27 and arginine 17 denote transcriptional activation or repression
Arginine 17 and Lysine 4 – transcriptionally active. Lysine 9 and 27 – transcriptionally inactive
How does methylation and acetylation of histone influence transcription
Acetylation and methylation marks in general create binding sites for transcription factors
Specifically what is the effect of histone acetylation on gene transcription
Acetylation of histones creates binding sites for transcriptional activation factors that contain a bromodomain. Histone Acetylation is associated primarily with transcriptionally active promoter sequences.
Will genes that are more transcriptionally active show higher or lower levels of acetylation
Higher
Methylation of core histones creates binding sites for transcriptional repressors that contain what kind of domain
Bromodomain
Methylation of core histones creates binding sites for transcriptional activators that contain what kind of domain
PHD finger domains
Which type of transcription factors will bind to methylated lysine 4 and arginine 17 residues
Transcriptional activators containing PHD fingers
Which type of transcription factors will bind to methylated lysine 9 and 27 residues
Transcriptional repressors containing bromodomains
Transcriptional activators recruit ATP-dependant chromatin remodelling enzymes, what four ways can these enzymes act to upregulate gene transcription
Selective histone octamer and whole nucleosome remodelling, selective histone removal and replacement and/or by recruiting code writers and readers
In general transcription activators promote RNA polymerase II recruitment, T or F
T
What ways can transcriptional repressors act to decrease gene transcription
Compete for activator binding sites, prevent bound activators from functioning, keep transcriptional activators and transcription machinery away from the start site and also reverse effects of transcriptional activators by creating dense transcriptionally inert chromatin. Finally, they can also recruit enzymes such as histone demethylases and deacetylases as well as methyltransferases
Which residue does enhancer of zeste act on and what type of modification is it
Enhancer of zeste methylates lysine 27
Explain the role of enhancer of zeste in hox gene repression and the involvement of polycomb repressive complexes
Enhancer of zeste is the catalytic subunit that acts as part of a complex that represses hox gene expression. PRCs contains chromodomains and methylate lysine 27 loci toproduce global derepression of hox genes
As well as histones, DNA can also be modified by methylation. When and how does this occur
There is a close functional relationship exists between transcriptionally repressive histone methylation and corresponding DNA methylation on cytosine bases. The addition of methyl groups to cytosine residues is mediated by DNA methyltransferases (DNMTs). Transcriptionally inactive promoters are frequently rich in methylated CpG dinucleotides
Explain what is meant by X-chromosome inactivation
X-chromosome inactivation is a process that occurs in mammals and acts as a dose compensation mechanism that equalizes the levels of X-chromosome derived gene products in males and females. One X chromosome copy is silenced in each somatic cell during early development of female embryo
The X-chromosome copy that is silenced is determined by maternal gene expression in the fertilised oocyte, T or F
F - Initial selection of the chromosome for silencing is random
How are the progeny of the cell that silences one of the X chromosomes in somatic female cells affected
The silencing decision is propagated clonally and all progeny of each cell in which the silencing decision was taken inherit the same silenced X chromosome
Explain how X-chromosome inactivation accounts for tortoiseshell or calico cats
Calico cats are exclusively female. They are heterozygous for two coat pigment alleles, black and orange. Early in development in progenitor cells either orange or black alleles are inactivated. Where black pigment allele containing X-chromosomes are switched off, all progenitors will produce fur orange in colour. Where orange pigment allele containing X-chromosomes are switched off, all progenitors will produce fur black in colour.
Why are regions on the face, neck, bely and white still white in calico cats, despite X-chromosome inactivation
These regions don’t contain pigment cells
Explain the molecular mechanism of X-chromosome inactivation
X-chromosome inactivation involves the synthesis of a non-coding RNA known as Xist from the X-inactivation centre (XIC) on the chromosome destined for inactivation. Xist RNA binds to the X chromosome and acts as a recruitment signal to promote the formation of silent chromatin. This is achieved by recruitment of histone modifying enzymes and other Polycomb Group components and leads to the Histone-3 lysine 27 and H3K9 methylation of core Histones in X chromosome chromatin
Explain how H3K27 and H3K9 leads to the formation of a Barr body
Lysine 9 and 27 methylation along the entire length of the X chromosome leads to the entire silencing of the chromosome and its shunting to the periphery of the nucleus. This leads to the production of a Barr Body, a highly condensed inactive X chromosome at the periphery of the nucleus of every female somatic cell
