Epigenetics Flashcards
What is a gene composed of?
Promoter, 5’ and 3’ untranslated region, exons/introns, Start codon (ATG) and stop codon
What factors effect gene expression?
Promoter, enhancers, repressors, transcription factors, mutations and gene duplications
Why is the nucleus important in gene expression?
Each cell (except blood cells) contain a copy of the genome - in humans it’s 4billion bp and it needs to fit in a space 6microns wide:
Packaging is important: Versatility to ensure ability to switch on/off genes; express certain genes immediately; 24,000 genes and not all expressed in certain cell types - “spatiotemporal regulation”
Nucleus provides: compartmentalisation, organisation and regulation of DNA
How is the genome organised in the nucleus?
DNA is never naked… present in the form of chromatin which is made up of nucleosome complexes: 146bp of DNA wrapped around a histone octamer twice.
Histones: Proteins, H2A, H2B,H3, H4
How is gene expression regulated?
Info on: Methods, transcription factors, chromatin….
Methods:
- Housekeeping genes (constitutively expressed genes): encode for proteins required for cell maintenance
- Certain genes are permanently switched off in certain cells (e.g. enzymes in neurons)
- Genes that need to be switched on/off in response to external stimuli
Activation and repression of transcription factors is not enough for gene expression regulation - have many targets
Thus, specificity achieved through chromatin structure:
Heterochromatin: repressed, tightly wound (inactive) state
Euchromatin: loose and easily accessible (active) state
Definition of epigenetics?
Inheritable methods of manipulating gene expression without changing the primary structure of DNA
What does epigenetics regulate?
Types and facts
Epigenetics modifies the structure of chromatin - between repressed (inactive) and lose (active)
- repression of parasitic sequences
- genomic imprinting
Heritable; create permanent or temporary changes; established during early development
How does DNA methylation occur? Enzymes….
Where does it occur and frequency?
Enzymatic addition of a methyl group to a cytosine residue = 5-methylcytosine (5-mC)
Dnmt3a and Dnmtb: catalyse de novo methylation
Dnmt1: maintenance methylation
- Established in embryogenesis
- Occurs within a CpG sequence: 60-80% of mammalian CpG sequences are methylated
- 50% of genes have ‘CpG Islands’ (unmethylated and active)
Example of necessary DNA methylation? (Disease)
Parasitic DNA silencing
17% of cells contain LINE1 sequences (long interspersed nuclear elements). 80-100 copies still active
- LINE1 is normally methylated
- Hypomethylated LINE1 is found in cancer and correlates with poor prognosis
How does methylation cause gene repression?
What happens without protein
Methylation itself doesn’t cause repression - instead changes Cytosine landscape
MeCP2 (methyl CpG binding protein 2) has high affinity to methylated CpG sequences and represses chromatin structure
Loss of ‘functional’ MeCP2 results in Rett Syndrome- random mutation in 1, methyl binding domain and 2, transcriptional repression domain.
Restoration of MeCP2 rescues mouse models
What is DNA hydroxymethylation?
Correlations?
Where and when is it established?
What is the purpose?
Tet enzyme oxidises methyl group in 5-mC to produce 5-hydroxymethylcytosine (5-hmC)
Correlated to neural activity (new neural circuits shape 5-hmC profile) and gene activation
Occurs postnatally in all tissue and cell types but primarily in CNS - neurons
Changes the landscape, allowing binding of other subsets of proteins:
- MeCP2 has high affinity to 5-mC during embryogenesis: Gene Repression
- MeCP2 has high affinity to 5-hmC postnatally: Gene Activation
How is DNA demethylation achieved?
Doesn’t happen directly - as methylation is energetically stable:
Achieved through tet-mediated modification of methyl group
Each step alleviates gene repression
Eliminates epigenetic modification
2 types of non-CpG methylation?
CpA: postnatally (during primary phase of synaptogenesis) in neurons and catalysed by Dnmt1: GENE ACTIVATION
adenosine methylation: in RNA modification, postnatally
Detecting methylation…
Single gene methods?
1) methylation sensitive restriction enzyme digestion:
Used to analyse methylation status of Cytosine. Enzymes only cut unmethylated cytosine. Then, PCR methylated cytosine to amplify product.
Limitation: only works with short sequences
2) Bisulphite conversion/sequencing:
Denature DNA and treated with ‘Sodium Bisulphite’ - this turns unmethylated cytosine into uracil, methylated cytosine remains protected.
PCR products, this turns U into T.
Then sequence products to find mismatched CG/AT pairs…. mismatch reveals unmethylated sites.
Detecting methylation…
Genome-Wide method?
Digest and denature fragments before incubating with an antibody against 5-mC
Can then perform immunoprecipitation…
followed by either:
- hibridisation to micro array
- OR sequencing
Resulting product is a ‘methylome’
What are histones?
Lineage, and types.
Uses and how does it interact with DNA
Protein complexes…
Evolutionary conserved: not in prokaryotes but in archea (though modification process is different)
- Complex is a histone octamer: 2x H2A, 2x H2B, 2x H3, 2x H4.
- H1 acts as a linker histone, separate from nucleosomes - slightly different amino acid sequence and modification sites
Tails are positively charged: allowing for interaction with DNA
8) facts about histone modification?
1) histones have tails that protrude from octamer - sites for post transcriptional modification
2) It’s more dynamic than DNA methylation - adding fine tuning of expression… more responsive to environmental stimuli
3) Established during embryonic development, but can change through life span
4) Histone modification otherwise known as the ‘histone code’ which can be read by cells
5) Modifications can compete for same site (e.g methylation and acetylation of H3K9)
6) Histone modification and DNA methylation work cooperatively
7) Modifications can be additive - with different functions (e.g. H3K9me1, H3K9me2, H3K9me3, can all be found in cell at same time)
8) Histone tails can be binding sites which recruit proteins; or can themselves modify the nucleosome
What are 4 types of histone modification?
1) Phosphorylation
2) Methylation
3) Acetylation
4) Glutarylation
Describe in detail what happens in methylation (example of cross talk) and acetylation?
What, why, how?
Short description on phosphorylation and glutarylation?
Acetylation:
The enzymatic addition of an acetyl group to the lysin residue of the histone tail - which neutralises its positive charge, reducing the amino side chains affinity for DNA, thus loosens from nucleosome
-Catalysed by HAT ( histone acetyl transferase and KAT
-Purpose: Gene expression
Methylation:
-Can either express or repress genes depending on histone
Example: H3K9me3…. 1) KAP1 protein (enigmatic master regulator of genome’ recruits SETB1 to methylate DNA while recruiting HDAC (histone deacetylase) acetylate histone (example of cross talk)
2) HP1a can then be recruited to form constitutive heterochromatin, as well as, Dnmt3 can be recruited to methylate DNA and maintain heterochromatin
Phosphorylation:
Histones monitor health of genome and are used as signals to repair pathway proteins
Glutarylation:
Novel histone modification of H4K91 - which is enriched upstream of highly expressed genes. Down-regulation associated with mitosis.
How to analyse histone modification?
1) Single Cell: immunofluorescence ( imaging and quantification using confocal microscopy)
2) Western blotting ( to understand proportion of modified histones - can be normalised using total histone level)
3) CHIP: identify histone modification signatures to infer functions of the genomic region
Coordination of histone modification leads to?
Histone modification can influence the ‘deposition’ of other hosted marks by;
- Steric hinderance (Stop due to chemical structure) ‘of another reaction’
- decreased/increased affinity of binding of transferases ‘for another modification’
This can cause amplification of epigenetic signal
Or, multiple modifications may be needed to activate a specific pathway
What does chromatin remodelling do?
Makes DNA accessible to RNA polymerase in genes which switch one/off in response to stimuli
Histone modification complexes are: Swi/Snf
Cause histone displacement
3 facts about developmental epigenetics?
1) Monozygotic twins are genetically identical, but epigenetically different
2) inheritability: mothers phenotype has important consequences in establishment of markers
3) Epigenetic modifications are huge drivers of phenotype
