Epigenetics Flashcards
What is meant by epigenetics?
Heritable modification of the phenotype without change in the genotype
How is cell identify defined?
By a unique transcriptional programme which is locked in place by epigenetic modifications
How do a liver and skin cell differ in a mouse?
They are all genetically identical but vary in phenotype
Define: genetics
Study of genetic material which determines the properties of inheritance and phenotype
Define: epigenetics
Study of modifications that alter the phenotype without altering the genotype
All cells from a common precursor share the same genotype. How does a liver cell become a liver cell, and a skin cell become a skin cell?
Through a unique transcriptional programme which is locked in place by epigenetic modifications
What is meant by the epigenetic code/ epigenome?
The set of epigenetic features that create different phenotypes in different cells
Give two key epigenetic mechanisms
1) DNA modifications - methylation
2) Chromatin modification - methylation, acetylation, phosphorylation, ubiquitination
On which residues does DNA methylation occur in:
1) prokaryotes
2) eukaryotes?
1) on adenine
2) on cytosine
In general, is DNA methylation reversible?
No, generally permanent
What are the two classes of DNA methyl’s enzymes?
Dnmt1
Dnmt3a/3b
What type of DNA is recognised by Dnmt1?
Hemi-methylated DNA
Which type of DNA methylation is mediated by Dnmt1?
After DNA replication
What is the purpose of Dnmt1?
Maintains the pattern of methylation through replication
What is the principal role of DNA methylation?
To switch off transcription LONG TERM
Why is most of the human genome methylated?
To switch off transcription of ‘junk’ DNA - i.e. to maintain genomic stability
What are the two lines of evidence for the importance of DNA methylation in keeping junk DNA silent and maintaining genomic stability?
1) 5-azacytidine: cytosine analogue that can’t be methylated. Dilutes DNA methylation through each round of cell division - loss of DNA methylation - activates endogenous retroviruses (i.e junk DNA)
2) Dnmt1 partial KO mice (HYPOMORPH) - develop aggressive T cell tumours due to genomic instability and Trisomy of chromosome 13
What is the difference between Dnmt1 and Dnmt3a/3b?
1) replication dependent
2) de novo
What percentage of CpG dinucleotides are methylated in the mammalian genome?
More than 50% (most)
How many cytidine residues are methylated in the mammalian genome?
3-4%
Cytidine is generally UNDER-represented in the mammalian genome. What does this mean?
Often undergoes spontaneous deaminated to thymidine - i.e. amine group is removed
Which residues are methylated in the human genome?
Cytidine - in CpG dinucleotides
Around how many CpG dinucleotides are lost due to cytidine under-representation? (i.e. deamination)
Most changes repaired: about 3-4 cytidines escape
Where will unmethylated CpGs be found?
Near active genes, as methylation is associated with silenced genes
What are CpG islands?
UNMETHYLATED CpGs
Where are CpG islands usually located?
5’-end of active genes (i.e. upstream)
Generally, how long are the regions containing CpG islands?
1-2 kb
What is the content of CpG islands?
> 50% CpG content
KEY feature of CpG islands?
CpG islands are RESISTANT to DNA methylation
What is the result of DNA methylation?
Long-term silencing
What type of genes are often surrounded by CpG islands?
House-keeping genes
Why are CpG islands often found by house-keeping genes?
Because they are constitutively active
In the course of differentiation, e.g. from stem cell to liver cell, how are other tissue-specific genes switched off?
Promoter methylation (e.g. of skin cell genes)
At what point will a gene start to be turned off?
> 50% methylation
How does DNA methylation silence genes?
Methyl groups act as binding sites for methylated DNA binding proteins (MBDs)
Which are the three main MBDs?
Mecp2, MBD2, MBD1
What is an MBD?
Methylated DNA Binding Proteins/domains
What is the NuRD?
Nucleosome Remodelling and Deacetylase complex
How does NuRD work?
MBD2 recognises whether gene is methylated - binds to methylated DNA, then recruits the rest of NuRD. Deacetylase activity of NuRD removes acetyl groups from lysines, which prevents transcription.
Mi2-Beta alters nucleosome positions. Silences transcription.
How is 5-methylcytosine changed into 5-HYDROXYmethylcytosine?
By TET1/ TET2
What are Tet1, Tet2 and Tet3?
Enzymes that add a hydroxyl group to the methyl group on cytidine to make 5-hydroxymethylcytosine
Where are the Tet enzymes predominantly expressed?
Brain and stem cells
What is the purpose of Tet enzymes?
They prevent recruitment of methyl binding proteins to the DNA
What are two proposed theories for the addition of a hydroxyl group to the methyl on 5-methycytosine?
1) may prevent binding of methylated-DNA binding proteins
2) stable intermediate in DNA demethylation process
What are the TET proteins?
Dioxygenases of the Ten-Eleven Translocation family. They are 5-methylcytosine oxidases that convert 5mC to 5hmC
Give a possible function of TET2
Role in generating iPS cells
What is the histone code hypothesis?
An epigenetic marking system using different combinations of histone modification patterns to regulate specific and distinct functional outputs of eukaryotic genomes
-i.e. it determines whether a gene is ON, OFF, or may be on in the future
How do the following modifications affect transcription:
1) lysine acetylation
2) lysine methylation
1) always ON
2) context-dependent; H3K4me3 = ON; H3K27me3 = OFF
What are the two different forms of chromatin?
Euchromatin and heterochromatin
Name 3 PTM histone modifications found in euchromatin
1) H3/H4 acetylation
2) H3K4me3
3) H3S10P
Name 3 PTM histone modifications found in heterochromatin
1) H3K27me3
2) H4K20me3
3) high DNA methylation
What charge do histone tails generally carry?
Positive due to lysine/arginine rich tails
When is Oct-4 inactivated?
During stem cell differentiation
Where does HDAC deacetylate lysines to repress transcription?
On H3K9
Where is heterochromatin found?
Centromeres Telomeres Repetitive elements Tissue-specific genes Intergenic regions Inactive X chromosomes
What is the effect of the following modifications on transcription: DNA methylation H3/H4 acetylation H3K4me3 H3K27me3 H4K20me3 H3S10P H3K9me3
1) DNA methylation - represses
2) H3/H4 acetylation - activates
3) H3K4me3 - activates
4) H3K227me3 - represses
5) H4K20me3 - represses
6) H3S10P - activates
7) H3K9me3
Name 3 modifications associated with active genes (euchromatin)
1) H3/H4 acetylation
2) H3K4me3
3) H3S10P
Name 3 modifications associated with repressed genes (heterochromatin)
1) DNA methylation
2) H3K27me3
3) H4K20me3
What does G9a-methylatransferase do?
Methylates H3 (H3K9me3)
Name two major epigenetic silencing machineries
G9a/GLP and Polycomb Repressive Complex 2 (PRC2; active subunit = Ezh2)
What is Oct-4?
A transcription factor
What is Oct-4 primarily responsible for?
Maintaining a stem cell as a stem cell
What must occur in order for stem cell differentiation to occur?
Oct-4 must be switched off
In a stem cell, Oct4 must be silenced in order for differentiation to occur. How does this happen?
1) transcriptional repressor binds- GCNF (germ cell nuclear factor)
2) repressor recruits HDAC - removes acetyl groups
3) gene repressed
4) ensure Oct-4 off permanently:
5) G9a methylates H3K9 (‘OFF’ mark)
6) H3K9me3 adds as recognition site for HP1 (heterochromatin protein 1)
7) HP1 recruits Dnmt3a/3b (DNA methytransferase 3a/3b)
8) Dnmt3a/3b methylates DNA
Once G9a has methylated H3K9, what happens?
H3K9me3 is recognised by HP1
What is HP1?
Heterochromatin Protein 1
How is HP1 recruited to DNA?
Recognises H3K9me3
How is Dnmt3a/3b recruited to the Oct-4 gene to allow methylation and subsequent silencing of Oct-4 to allow differentiation of stem cells?
It is recruited to H3K9me3 DNA via HP1
What is the first step of stem cell differentiation?
Binding of the transcriptional repressor GCNF, which recruits HDAC, which represses genes
What technique has been used to monitor histone modifications across the genome?
ChIP-seq
What is unique about some embryonic stem cell genes?
They are BIVALENT: they are poised, having a combination of off and on signals
What is ChIP-seq?
Chromatin immunoprecipitation to isolate the proteins bound to specific regions of DNA coupled to DNA sequencing to identify these regions
Name two powerful tools to identify genome-wide profiles of transcription factors, histone modifications, DNA methylation, and nucleosome positioning
ChIP-chip (ChIP followed by hybridisation to microarrays)
ChIP-seq (ChIP followed by high-throughput sequencing)
What makes ChIP-seq better than ChIP-chip?
It requires less DNA input, has expanded dynamic range, and is compatible with sample multiplexing
What happens to the genome in cancer?
Global genomic HYPOmethylation
How are TSGs silenced in cancer?
OCCASIONAL methylation of CpG Islands, e.g. in the promoter for the genes for p16INK4a and p14ARF (lead to dysregulation of the cell cycle)
What epigenetic marks do tumours lose?
H4K16Ac, and H4K20me3
Give two examples of diseases in which genomes are globally hypomethylated
1) cancer
2) autoimmune diseases, e.g. rheumatoid arthritis
Almost every disease type will have an epigenetic change. Give some examples
cancer, autoimmune diseases such as rheumatoid arthritis
What is the consequence of DNA demethylation?
Expression of junk DNA; increased plasticity of the genome
Give an example of drugs that target class I HDACs to treat certain cancers such as cutaneous T-cell lymphoma
SAHA
Romidepsin
Belinostat
