Chromatin Flashcards
Describe histones
Core histones- H2A, H2B, H3, H4
Linker H1
They are small and positively charged
Core histones are highly conserved
What is the composition of chromatin?
DNA, protein and a small amount of RNA
There’s a 1:1 mass ratio of histones to DNA
Briefly describe the nucleosome
Made of 2 of each core histone and one linker histone
146bp of DNA in a left handed superhelix of 1.8 turns
Describe the structure of nucleosome
Two pairs of H3 and H4 heterodimerise in a histone ‘handshake’
The pair of diners form a tetramer at the centre of the histone in the shape of a horse shoe
Two heterodimers of H2A and H2B form and are located above and below the tetramer forming an optometric core
The DNA near the nucleosome is inaccessible for transcription so remodelling is require to switch the gene on
Flexible N-terminal tail allows signalling for chromatin remodelling
Describe some histone variants
H2A variants- H2AZ found on nearly all eukaryotes
H2AX found in histones near DSB, signals for repair proteins
MacroH2A is vertebrate specific- enriched in the inactivated X chromosome, identified the entire X chromosome as inactive
H2a.Bbd vertebrate specific- depleted in the inactivated X chromosome
H3 variants- H3.1, H3.2, H3.3 (few AA variance between them) H3.It- slightly lighter, Cenpa is associated with centromeres ie very highly packaged areas with no transcription
Describe H2AZ
A variant on histone H2A
Alters the interaction stability between H2A and H2B
Alters the interaction of the H2A/H2B dimer with the H3/H4 tetramer
ALTERS THE CANONICAL NUCLEOSOME
Often associated with transcriptionally active chromatin
Briefly describe functions of histone variants
Variants of H3 and H2A differentiate chromatin at centromeres, active genes and heterochromatin
Eg. H3.3 marks actively transcribed loci by replication independent nucleosome assembly
Epigenetically silenced chromatin can be enriched or depleted in many H2A variants
Describe level one DNA packaging
The nucleosome- four core histones + linker histone, 146bp + linker DNA (10-20bp depending on tissue)
1.8 left handed superhelical turns
Describe level 2 of DNA packaging
The 10nm fibre- beads on a string
Contains actively transcribing genes
Describe level 3 of DNA packaging
The 30nm solenoid
6 nucleosome per turn with H1 in the core, sitting at the diad access
Is conducive for transcription- needs remodelling into 10nm fibre
Describe level 4 of DNA packaging
300nm solenoid
Arranged in loops containing 60-100kb of DNA tethered by non-histone protein scaffold
The 30nm solenoid condenses further requiring more non-histone protein to keep the loops open
Genes are off
Describe level 5 of DNA packaging
700nm fibre
A coiled coil
The loops of chromatin to coil around an additional protein scaffold
Long term silencing of genes
How are the flexible N-terminal tails important?
Can be post-translationally modified
They can extrude from the the nucleosome and signal within the cytoplasm
They can communicate with other nucleosomes
What are different postranslational modifications?
Acetylation Methylation Phosphorylation ADP-ribosylation Ubiquitination
Describe acetylation to histone tails
Occurs on specific lysines at the amino terminal end of all four core histones
Eg. H4 at K5, K8, K12, K16
H3 at K9, K14, K18, K23, K27
Occurs by the action of Histone Acetyltransferase (HAT)
Reversed by Histone deacetylases (HDAC)
What is the result of acetylation of histone tails?
High levels of acetylation➡️ Increases accessibility of transcription factors to DNA by opening up chromatin
RNA Pol2 brings the HATs and HDACs to the chromatin which acetylates all four histones on the DNA to be transcribed and deacetylate chromatin that does not need to be transcribed
Are genes still acetylated during metaphase?
Yes, genes that need to be ‘on’ when the cell exits mitosis remain acetylated so the daughter cells keep their identity
Although heterochromatin is hypo acetylated and ‘off’ genes Eg. Inactive X chromosome, are characterised by very low levels of acetylation
Describe methylation of histone tails
Added by methyltransferases to specific lysines and arginines Lysine residues can be mono- di- or tri-methylated Arginines can be symmetric or asymmetric Occurs on H3, H4 and H1 only H3- lysine 4, 9 and 27 H4- lysine 20 H1- lysine 26 We have histone demethylases
What is the result of methylation of histone tails?
H3 di-methylated at K4 (+acetylation of core histones) is indicative of transcriptionally active DNA
Trimethylated at H3K9 signals for a protein called HP1 to bind which silences the chromatin- highly packaged- heterochromatin
High levels of H3K27 trimethylation + low levels of acetylation + DNA methylation makes chromatin inactive
What is the significance of the polycomb group?
Eg polymethyltransferase DNMT methylates DNA
2nd polycomb complex PRC1 binds and stops RNA Pol2 transcribing
+ Trimethylated H3K27 leads to permanent silencing of a gene
Describe phosphorylation of histone tails
Phosphorylated by kinases and de phosphorylated by phosphotases
Histones can be phosphorylated by O-phosphate linkage or N-phosphate linkage
At serine, threonine, tyrosine and lysines, histadines, arginines
Eg. H1 on 3 serine residues and 2 threonine residues
What is the importance of phosphorylation on H1?
H1 is located at the diad access, it keeps DNA wrapped around the nucleosome
Modifying it can loosen or tighten the DNA around the nucleosome
What are the conditions under which you find high levels of phosphorylation? How?
At mitosis on H3 and H1 and as cells are stimulated out of quiescence- only present on a small number of genes on H3 Combination of modification Opening- acetylation and phosphorylation -H3K9Ac -H3S10P -H3K27Ac Condensation- methylation and phosphorylation -H3K9Me -H3K27Me -H3S10/S28P
How are the signals that make DNA euchromatin or heterochromatin put on to the DNA?
Histone modifying enzymes eg. HATs or HMTs
Chromatin remodellers can remove nucleosomes from the DNA so transcription enzymes can bind
Histone modifications can recruit proteins such as Heterochromatin protein 1 that sits on the DNA to turn the gene off
What are ‘writers’, ‘readers’ and ‘erasers’ in the context of gene regulation?
Writers are histone modifying enzymes
Erasers are histone de-modifying enzymes
Reader domains recognise the epigenetic mark
The modifying proteins are targeted to promotors/enhancers
Name some structural components of the chromosome
Centromere- binds to kinetochores during mitosis, large region of satellite repeats
Specialised chromatin- CENPA variant of H2A
Telomeres- repeat bases at the ends of chromosomes to prevent loss of coding regions- end stability
How do you silence a gene for the long term?
Methylation of CpG Islands cytosine➡️methylcytosine
Associated with ‘house-keeping’ and ‘tissue specific’ genes
Repressors bind➡️ methylation➡️histone modification
Required for genomic imprinting, x-chromosome inactivation, repression of ‘parasitic’ DNA sequences
What is the x-chromosome inactivation mechanism?
Decision taken in the blastocyst stage A counting mechanism occurs and all but one random X chromosome is inactivated - repressive histone mods - DNA methylation - non-coding RNA
What is position effect variegation?
Position in the genome effects whether the gene is expressed
Closer to heterochromatin generally means that the gene expression is repressed
