Lecture 14 - Epigenetics Flashcards
What processes regulate transcription and how do they do it?
- Acetylation and methylation of histones by influencing recruitment of transcription factors to target genes
What characteristic feature of tumours is related to epigenetic ?
Over expression or mutation of histone-modifying proteins
What are the future prospects of epigenetics ?
Drugs that target the epigenetic machinery are promising new anticancer agents
Epigenetic traits
are stable, heritable phenotypes resulting from changes to chromatin structure without alterations to DNA sequences
What is the difference between genetics and epigenetics ??
Genetics – Mutation of DNA > altered gene function > Altered phenotype
Epigenetic’s – alterations to chromatin structure > altered gene expression > altered phenotype
Through regulating chromatin structure, what do epigenetic mechanisms do ?
- control accessibility of target genes to incoming transcription machinery
- Directly control biochemical activity of transcription machinery
Epigenetic modifications
cause stable alterations to chromatin structure
and gene expression but do not change nucleotide sequence of DNA
Give examples of epigenetic modifications:
- Histone modifications – acetylation, methylation
- DNA methylation
- non-coding RNAs
Production of differentiated cells from proliferating progenitor cells involves selective transcription of specific genes which:
- suppress self-renewal
- promote lineage commitment
- activate one programme of cell differentiation and repress all others
Conrad Waddington
epigenetic landscape / journey all cells take through development
- intracellular signalling pathways
early development- cells lie in higher reaches not exposed to env then move through the landscape and succumb to various signals and adopt specific cell fates
sequential changes in transcriptome and proteome
Cancers can develop when ?
self-renewing stem/progenitor cells “get stuck” in the higher reaches of the epigenetic landscape
What causes epigenetic induced cancers ??
Chromatin in nucleus acquires tumour specific characteristics
Modification targeted to the N terminal tail of the histone
- covalent modifications
Covalent modification added to core histones
- Acetylation of lysines in core histone N-terminal tails
- Mono-, di-, tri-methylation of lysines and arginines in core histone N-terminal tails. Various residues modified by distinct enzymes
- Phosphorylation of serine 10 in Histone H3
General epigenetic change that causes cancer -
Modify way transcription machinery interact
What happens to lysines if acetylated??
they can no longer be methylated
Whats the difference between HATs and HMTs ??
Histone Acetyltransferases (HATs) can modify many different lysine residues
in core histones whereas histone methyltransferases (HMTs) exhibit exquisite
site-specificities - “Histone Code” Writers
EZH2
- Specific mark on histone 2 lysine 7 – implicated in cancer
If chromatin rich in lysine 27
transcriptionally inactive
What is the purpose of Acetylation of histones ?
histones creates binding sites for transcriptional
activation factors that contain a bromodomain – Histone Code Readers
Histone code readers
histones creates binding sites for transcriptional
activation factors that contain a bromodomain – Histone Code Readers
Histone code writers
Histone Acetyltransferases (HATs) can modify many different lysine residues
in core histones whereas histone methyltransferases (HMTs) exhibit exquisite
site-specificities - “Histone Code” Writers
Methylation of core histones can create binding sites for
(a) transcriptional repressors that contain a chromodomain
(b) transcription activators that contain a PHD zinc finger domain
Recruitment of chromodomain (H3K27)
repressor
or PHD finger domain (H3K4) activator
Epigenetic code
defining the parts of genome into active and inactive
building blocks of chromatin
Nucleosomes
Ed lewis
- discovere bi-thorax complex in flies
- discovered genes involved in epigenetics
Ed lewis - findings
- bithorax complex and anopaedia complex two homeotic complexes in drosophila
- Sex combs reduced gene in the anopaedia- bristle structure on foreleg in males used in matings
- discovered mutation of sex combs reduced
- mutaton carrys extra sex combs
- Loss of function mutations in the Drosophila Polycomb Group gene Enhancer of zeste [E(z)] cause the development of extra sex combs on mesothoracic (second pair) and metathoracic (third pair) legs - opposite phenotype to sex combs reduced phenotype
- evidence for widespread activity of the homeotic gene across the thorax
Enhancer of zeste codes for what
Histone 3 lysine methyl transferase
- involved in converting homeotic complex to a heavily transcription repressed locus - inactive in all parts of body apart from the first legs where there is expression of antenopaedia complex where sex combs reduced is expressed
- animals without enhancer of zeste- chromatin cannot be repressed so ubiquitously active - thats why when mutated it is expressed in all the other legs
Polycomb group of proteins
proteins that can generate or recognise repressive chromatin modifications - histone code writers and readers
- chromatin regulatory system
Code writer
H3-K27methylation mediated
by the Enhancer of zeste [E(z) /Ezh2]
component of PRC2
(Code Writer)
Code reader
Recruitment of PRC1 complex via
Polycomb (Pc) chromodomain-
containing component
Polycomb proteins & cancer
Polycomb family proteins highly expressed in cancers
Increased expression of EZH2 protein is a characteristic feature
of advanced Prostate Cancer
biopsies – stained with antibody for EZH2
-
PC cancer -
Increased expression of EZH2 is accompanied by increased H3K27methylation and decreased transcription of many genes in prostate cancer cells
EZH2
promotes proliferation and invasiveness of Prostate Cancer cells
KO – Lysine 27 methyl transferase – what happens to cells ability to proliferate ??
used a synthetic cell matrix and cultured cells with Luciferease SiRNA and EZH2 siRNA» shows cell proliferation significantly reduced in EZH2 siRNA
- inhibit enhancer of zeste function
- if cells invasive they well squeeze through the holes and migrate from one side of the matrix to the other and adhere
- EZH2 is required for the metastatic phenotype
Hallmarks of cancer - epigenetic
Increased expression of Polycomb Group proteins is a hallmark of
many different types of human cancer
what mutation can promote tumorigenesis
- give an example
Mutations in genes encoding histone modifying enzymes can promote tumourigenesis
Follicular lymphoma -mutation in chromatin modifying gene mutations - lysine methyl transferase commonly mutated
Therapy/Treatment prospects
Mutated forms identified to be hypersensitive to a class of small molecules Selective inhibitors being discovered that target EZH2 - but little effect on WT
Specific point mutations in EZH2 are highly associated with
malignant non-Hodgkin’s Lymphoma
Y641F A677G
These EZH2 mutant proteins are hyperactive and only catalyse
trimethylation of H3K27 (not able to cause mono- or di-methylation,
unlike wild-type EZH2)
Small molecule mechanism of action
EPZ005687 specifically blocks mutant H3K27 trimethylation by EZH2
in a dose-dependent manner
- detected by western blotting using ABs specific H3K27 me 3
lymphoma-specific EZH2 mutation
A677G catalyses exclusively
H3K27 trimethylation, which renders lymphoma cell proliferation
specifically sensitive to EPZ005687
- hypersensitive to concentrations of the small molecules
- cancer specific inhibitor of proliferation
- Wt there is no effect
How do you create Hybrid genes/ Hybrid proteins ?
Chromosomal translocations due to aberrant cross-overs between
non-homologous chromosomes can disrupt genes to create hybrid genes encoding hybrid proteins
What can cause leukaemia ?
examples :
Chromosomal translocations that disrupt HAT and HMT (chromatin modifying) genes can cause leukaemia
- Acute Myeloid Leukaemia - (HAT) MOZ - (HAT) CREBP fusion - Chr 8 and chr 16
-Acute Lymphoblastic leukaemia - (HMT)MLL–(HAT) CREBBP fusion
Chr11 and Chr16
AML
- Acute Myeloid Leukaemia - (HAT) MOZ - (HAT) CREBBP fusion - Chr 8p11 and chr 16p13
ALL
-Acute Lymphoblastic leukaemia - (HMT)MLL–(HAT) CREBBP fusion
Chr11q22 and Chr16p13
MLL fusion proteins
MLL fusion proteins drive the emergence of a leukaemic stem cell population by preventing differentiation of pluripotent haemopoietic stem cells and more committed progenitors
MLL structure
Leukaemogenic translocations involving the Histone Code Writer/Reader MLL
delete its PHD finger, its Transcriptional Activation region and its H3K4
methyltransferase (SET) domain, to create a fusion protein that retains the MLL
repressor domain
- contains a catalytic domain that allows it to make histone modifications
- interaction domain that allows it to bind HATS of which CBBP is n example
-PHD finger can bind to lysine 4 when methylated can methylate proteins on lysine 4 and recognise them for methylation events
MLL fusion protein structure
Loss of domains required for histone 4 methylation and recognition for methylation event
-suggestion that MLL exerts it leukaemogenic function by acting as a dominant negative inhibitor of its WT counterpart by compromising the ability of target gene to be stablished in active domain of chromatin
