Chromatin

Question 1

Describe histones

Answer 1

Core histones- H2A, H2B, H3, H4
Linker H1
They are small and positively charged
Core histones are highly conserved
They have flexible end terminal regions that interact and communicate between DNA and nucleosomes.

Question 2

What is the composition of chromatin?

Answer 2

DNA, protein and a small amount of RNA

There’s a 1:1 mass ratio of histones to DNA

Question 3

Briefly describe the nucleosome

Answer 3

Made of 2 of each core histone and one linker histone

146bp of DNA in a left handed superhelix of 1.8 turns

Question 4

Describe the structure of nucleosome

Answer 4

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

Question 5

Describe some histone variants

Answer 5

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

Question 6

Describe H2AZ

Answer 6

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

Question 7

Briefly describe functions of histone variants

Answer 7

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

Question 8

How are the flexible N-terminal tails important?

Answer 8

Can be post-translationally modified
They can extrude from the the nucleosome and signal within the cytoplasm
They can communicate with other nucleosomes

Question 9

What are different postranslational modifications?

Answer 9

Acetylation
Methylation
Phosphorylation
ADP-ribosylation
Ubiquitination

Question 10

Describe acetylation to histone tails

Answer 10

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)

Question 11

What is the result of acetylation of histone tails?

Answer 11

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
HATs are in front of Pol II and HDACs behind to make the path mono-directional.

Question 12

Are genes still acetylated during metaphase?

Answer 12

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

Question 13

Describe methylation of histone tails

Answer 13

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
Active chromatin has high levels of H3K4Me
To silence, H3K9 boundary elements are Me, signalling to HP1 to bind and condense into heterochromatin
To turn off pluripotency, H3K27 is Me by PRC2 and made irreversible by PRC1

Question 14

What is the result of methylation of histone tails?

Answer 14

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

Question 15

What is the significance of the polycomb group?

Answer 15

Eg polymethyltransferase DNMT methylates DNA
2nd polycomb complex PRC1 binds and stops RNA Pol2 transcribing
+ Trimethylated H3K27 leads to permanent silencing of a gene

Question 16

Describe phosphorylation of histone tails

Answer 16

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
H1 P releases the tight bind around dyad access and allows transcription.
Highest level of P during mitosis and immediate early genes.

Question 17

What is the importance of phosphorylation on H1?

Answer 17

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
S17, 169, 185 T134, 151

Question 18

What are the conditions under which you find high levels of phosphorylation? How?

Answer 18

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

Question 19

How are the signals that make DNA euchromatin or heterochromatin put on to the DNA?

Answer 19

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

Question 20

What are ‘writers’, ‘readers’ and ‘erasers’ in the context of gene regulation?

Answer 20

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

Question 21

Name some structural components of the chromosome

Answer 21

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

Question 22

How do you silence a gene for the long term?

Answer 22

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

Question 23

What is the x-chromosome inactivation mechanism?

Answer 23

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

Question 24

What is position effect variegation?

Answer 24

Position in the genome effects whether the gene is expressed

Closer to heterochromatin generally means that the gene expression is repressed