L3 : Chromatin Modifications Flashcards
Compare euchromatin and heterochromatin?
Heterochromatin = densely packed and highly condensed
Transcriptionally inactive (silenced genes)
Euchromatin = loosely packed and less condensed
Transcriptionally active (expressed genes)
What is the nucleosome structure?
Octamer core of eight histone proteins (H2A, H2B, H3, H4) + linker H1
Diameter = ~11 nm
What is the function of the nucleosome?
Restricts access of transcription proteins to the DNA
Numerous contacts between histones and DNA minor groove
How can gene expression be regulated through altering DNA-histone interactions?
- Non canonical histones
- Histone PTMs
- DNA methylation
- Binding site for TFs/ chromatin remodelling factors
How can variant histones alter DNA-histone interactions?
Exchange of canonical histones with variants (small change in AA seq can change interactions)
During nucleosome assembly or exchange
What are the main types of post-translational histone modifications
Mainly occurs on flexible tails
- Methylation by HMTs
- Acetylation by HATs
- Phosphorylation by kinases
- Ubiqutination by Ub ligase
How can histone PTMs control gene expression?
Through affecting higher order chromatin structure by recruiting chromatin-remodellers
Or signalling with other protein complexes
What is the histone code hypothesis?
Combinatorial pattern of PTMs occurring on histone tails serve as binding sites for specific proteins
To regulate chromatin structure and gene expression
What is histone methylation?
Addition of methyl groups donated from S-adenosylmethionine (SAM) catalysed by methyltransferase
Side chain more bulky (proton -> methyl)
What is the mechanism of histone methylation?
- Methyl group on activated methionine
- Nuclear attack from N on Lys
Slow turnover but can be reversible
What types of methylation can residues have?
Arginine
- me1, me2s, me2a (symnetrical or asymnetrical)
Lysine
- me1, me2, me3
Histidine
- me1 (rare)
Each have different effects within the cell
Give examples of histone marks which are mutually exclusive?
- Dimethylation of H3R2 prevented by H3K4me3
- H3R2me2a prevents H3K4 methylation
- Phosphorylation of H3S10 prevents H3K9
What are some of the most studied methylation sites?
Lys = H3K4, H3K27, H3K36, H4K20
Arg = H3R2, H3R8, H3R17, H3R26
- H3K27me3 associated with repressed chromatin
- H3K79me2 important for cell cycle regulation
What is the mechanism of histone acetylation?
Acetyl from acetyl coA, nucleophilic attack from lysine to acetyl coA
- Can only ever install one acetyl group
- Causes change in charge (+ve amine group)
What is an example of histone acetylation?
Example: Xi chromosome almost absent of acetylated H3 and H4 whereas histones incorporated onto Xa are abundantly acetylated
How is the traditional model of how acetylation affect chromatin?
- Acetylation of core histones at Lys neutralise +ve charge
- Reduces interactions between DNA and histone proteons
- Weakens interactions between nucleosomes and promotes decondensation of chromatin
What is another model of how acetylation affects chromatin and an example?
Provides a marker for recognition by other proteins
Example: conserved bromodomain found in SWI/SNF (chromatin remodelling complex) and other TFs
What is interesting about domain structure in histone modifying enzymes?
Multiplicity of non-enzymatic domains allows some sort of positive feedback
- Catalytic HAT domain present with eg. bromodomain
- Acetylation attracts HATs, leading to more acetylation activity
- Combination of domains reveals further layers of regulation
What are the two types of histone acetyltransferases and their roles?
Type A:
- Localised in nucleus
- Acetylates nucleosomal histones
Type B:
- Localised in cytoplasm
- Most likely acetulates newly synthesised histones prior to nucleosome assembly
- Newly synthesised H4 acetylated at (K5, K12) not (K8, K16)
What are 3 major HAT families and what differs between them?
GNAT, MYST, p300/CBP
Each has different acetylation site preferences
What are the classifications and key features of HDACs?
- Grouped into 4 classes, each with multiple isoforms
- Most HDACs in humans are zinc dependent
- Most function in complexes with other factors
- Class III not zinc-dependent so not generally considered HDAC
Give an example and exception of HDACs working in multi-protein complexes?
Example: DNA-bound repressors recruit sin3A-HDAC1/2 complex to chromatin, leading to transcriptional repression
Exception: HDAC8 is catalytically active in isolation
What is the proposed mechanism of Class I - HDAC8?
Active site performs nucleophilic attack on acetyl group of Lys
- Thermodynamically favourable (-ve deltaG)
- No energy supply required, only catalysis
What are 2 ways HDAC8 can be regulated?
PTM: phosphorylation at S39
- Not in active site
Allosterically: Monovalent cations (K+)
- At least 1 activating K+ binding site
- Binding of K+ highly correlated with activity of enzyme
- Dependent on conc of K+
