L2 : Chromatin Dynamics Flashcards
What is the structure of nucleosomes?
Histone octamer is core component
Histone tails very accessible and can be heavily modified
What is the histone code hypothesis?
Combinations of histone PTMs modulate chromatin and carry epigenetic information that regulates genome function
Specific proteins can recognise these modifications
Describe histone acetylation, effect, enzymes?
- Acetyl marks nucleosomes with more neutral charge
- Reduces strength of interaction
- Open chromatin leads to more transcription
Lysine acetyltransferases (KATs)
Lysine deacetylases (KDACs)
Where does methylation commonly occur?
H3 K4, K9, K27, K36
H4 K4
Compare histone methylation and acetylation?
Both occur mostly at Lys residues of histone tails
- Acetylation neutralises histone charge
- Can add only 1 acetyl group
- Methylation retains histone +ve charge
- 3 degrees of methylation
How is histone methylation important in health and disease?
Linked to range of cancers, neurodegenerative diseases, autoimmune diseases
How was drosophila used to discover histone methyltransferases?
Studied heterochromatin formation using position effect variegation (PEV)
- Gene becomes variably silenced depending on position in genome
- White = red eyes
- No white = white eyes
Genetic screen for factors affecting heterochromatin formation
- Identified key locus Su(var)3-9
- Showed dosage dependent effect of Su(var)3-9 on PEV (3 copies = white)
How did they test Su(var)3-9 for methylation activity?
- Purify Su(var)3-9 from human cells
- Incubate with SAM (14C) and histones
- Check for activity in fluorogram using radioactive labelling
How did they test where this methylation occurs on Su(var)3-9?
Histone?
1. Radioactive labelling with various histone substrates
2. Only H3 showing much activity
AA position?
1. Mutate histone Lys position to identify which is being methylated
2. K9 mutation removes activity
Su(var)3-9 targets H3K9 for methylation
What are the key domains and functions of SUV3-9?
Chromodomain - for binding chromatin me2/3
SET domain - methyltransferase enzyme activity
H3K9 methylation recruits heterchromatin protein 1 (HP1) which acts to repress chromatin
How were demethylases discovered?
- Biochemical fractionation of nuclear extracts
- Used radioactively labelled histone H3K36me with 14C
- Detected release of 14C-formaldehyde, indicating demethylation
- Further separation by size (gel filtration) and charge (anion exchange)
- Mass spectrometry to identify protein FBXL11
- FBXL11 contains JmjC domain containing histone demethylase 1A (JHDM1A)
How does JHDM1A work?
Specific to H3K36me2 (dimethylation)
Produces formaldehyde as byproduct
Shown using western blots
How were methylation reader proteins discovered?
Stable Isotope Labelling by/with Amino acids in Cell culture (SILAC)
- Incubate medium with light (12C/14N) or heavy (13C/15N) lysine/arginine nuclear extracts
- IP with peptides of H3 labelled modified methyl or unmodified
- Mass spec to identify difference between modified and unmodified peptide
- Showed specific set of proteins interacting with H3 methylation marks
- Key protein found was major basal TF complex TFIID
What experiment showed TFIID binding?
- Taf3 was expressed in bacteria for binding studies
- Peptides with different histone mods attached to beads
- Incubated with Taf3
- Elution and gel stain to detect binding
How does TFIID interact with histone modifications to regulate transcription?
- Taf3 directly interacts with trimethylate histone marks
- Interaction helps recruit TFIID to gene promoters
- Promotes transcription initiation
How to investigate where histone methylation takes place?
ChiP-seq and CUT&RUN
- Use sequencing approach to identify location of methylation mark
- Crosslink chromatin and fragment
- Antibody can recognise chromatin modification
- Extract DNA and performing sequencing
What did the metagenome plot of H3K4me3 and K36me3 show?
- H3K4me3 peaks around TSS
- H3K36me3 peaks at 3’ end of gene
How are H3K4 and H3K36 methylation linked to transcription and lncRNA?
Recruited by RNAPII CTD modification
- CTD Ser5p is initiating RNAPII
- CTD Ser2p is elongating RNAPII
Used as gene signature for discovery of lncRNA (approach revealed >1600)
What is the function of H3 K4 and K36 methylation? What do they recruit?
H3K4 recruits HAT
- Example: me3 found at promoters of actively transcribed genes, recruits NuA3
H3K36 recruit HDAC
- Example: me3 found within gene bodies of active genes, recruits Rpd3S
- Prevents inappropriate (cryptic) transcription within gene bodies
What is the function of Ime1?
Master regulator for entry into meiosis
(haploid cells cannot enter meiosis)
What is IRT1 and its role?
lncRNA transcribed in IME1 promoter, specifically in haploid cells
Essential for IME1 repression in haploid cells
How is methylation linked to IME1 expression?
