W7 Chromatin structure and histone code

Question 1

Histones packaging

Answer 1

Proteins responsible for the 1ST LEVEL OF PACKAGING
DNA + HISTONES = NUCLEOSOMES
NUCLEOSOME structure increases DNA packaging 7-fold

Question 2

Nucleosomes packaging

Answer 2

Pack themselves in fibers of 30nm constituting the 2nd LEVEL OF PACKAGING
Increases packaging 6-fold

Question 3

30nm fibres packaging

Answer 3

30nm fibers pack themselves into 80-100nm fibers constituting the 3rd LEVEL OF PACKAGING
Increases packaging 3-fold

Question 4

Mitotic CS packaging

Answer 4

The 4th LEVEL OF PACKAGING is represented by the mitotic chromosome.
Represents 10000-fold packaging

Question 5

Chromosomes

Answer 5

Consist predominantly of:
DNA
Histone proteins
Non-histone proteins
Non-coding RNA

Question 6

Nucleosomes

Answer 6

First level of chromatin packing is the nucleosome

Histones assemble to form an octamer core:

• DNA is wrapped around the histone core
• 2 molecules each of histones H2A, H2B, H3 and H4
• Note N-terminal tails outside the octamer core

Electron micrograph
“beads on a string” appearance

Question 7

Higher order structure

Answer 7

When chromatin is extracted at physiological salt concentration, much of it appears as a 30nm thick fibre

The fibre is actually made up of nucleosomes tightly packed together

The 30nm fibre can be further compacted to form 80-100nm fibres

Question 8

Compaction of nucleosomes to form higher order structures involves

Answer 8

Linker histones (e.g. H1, long c- but short N-terminal tails)

Interaction of histone tails with adjacent nucleosomes

Binding of packing proteins to histone tails (post translational modifications)

Question 9

Chromatin structure is not static

Answer 9

During transcription, or DNA replication, nucleosomes must be removed from the DNA in front of the polymerase, and replaced behind the polymerase

Question 10

Histone remodelling factors

Answer 10

Enzymes that remove and replace nucleosomes

Question 11

Euchromatin

Answer 11

Lightly staining areas of chromatin
Rich in genes
Made up of nucleosomes, but not dense higher order packaging

Question 12

Heterochromatin

Answer 12

Darkly staining areas of chromatin
Few genes
Dense higher order packaging of nucleosomes

Question 13

Facultative heterochromatin

Answer 13

Contains genes not expressed in that cell type
DNA tightly packaged as heterochromatin
But may be packaged as euchromatin in other cell types

Question 14

What determines whether nucleosomes are packed as euchromatin or heterochromatin?

Answer 14

One key level of control- chemical modification of lysine residues in histone tails
Acetylation
Methylation
and others

Question 15

There are other levels of structure in chromatin

Answer 15

Chromosomes treated to extract histones and most non-histone proteins
Don’t completely fall apart
Appear as long DNA loops attached to a scaffold of tightly bound proteins

Question 16

Loops and chromatin domains

Answer 16

There is evidence that suggests
Each loop may have a different degree of chromatin compaction
The scaffold isolates the chromatin in one loop from the next loop
So one loop may have open chromatin and active genes
The neighbouring loop could be tightly packed as heterochromatin

Question 17

Methods to investigate chromatin structure- DNAse digestion

Answer 17

DNAse I cuts double stranded DNA

Histone binding protects DNA from DNase digestion

Question 18

DNAse I sensitive sites (HSS)

Answer 18

Sequences of DNA without histones

May be naked DNA, or binding transcription factors

Cut by very brief digestion with DNAse I

Found in promoters and enhancers

Question 19

If DNA is tightly packaged in higher order chromatin structures, how can all these proteins get at the DNA?

Answer 19

First TF opens up chromatin structure

Then recruits basal transcription factors

Then transcription happens

TFs recruit chromatin modifying enzymes
Via a nuclear coactivator (NCoA, transcriptional coregulatory protein) or corepressor (NCoR)

Question 20

Corepressor (NCoR)

Answer 20

Protein = TCP

Contains nuclear receptor interacting domains

Recruits histone deacetylases to DNA promoter regions

Question 21

Histone modification- acetylation

Answer 21

Loss of +ve charge

Heterochromatin:
- Histones largely unacetylated

Expressed genes in euchromatin:
- Many lysine residues of histones are acetylated

Question 22

Histone acetyl transferases (HATs)

Answer 22

Acetylate lysine residues on histones

Lead to unpacking of chromatin

Question 23

Histone deacetylases (HDACs)

Answer 23

De-acetylate histones

Lead to compaction of chromatin

Question 24

Thyroid hormone receptors

Answer 24

Thyroid hormone receptor (w/activation domain, DNA binding domain w/2 zinc fingers and ligand binding + dimerisation domain)

Thyroid response element (TRE)

T3 binds to TR so can now stabilise basal transcription complex

Question 25

Histone modification- methylation

Answer 25

Histone tails are methylated by histone methyl transferases (HMTs)

Demethylated by histone demethylases (HDMs)

A lysine residue can be mono- di- or tri-methylated

Question 26

Histone modification- methylation (part 2)

Answer 26

Methylation story appears more complex than acetylation story

Methylation of some lysine residues causes chromatin condensation

Methylation of other lysine residues causes chromatin decondensation

Effect may also vary if residue is mono-, di- or tri-methylated

Question 27

Methylation example

Answer 27

Trimethylation of histone H3 lysine at position 9 (H3K9me3) associated with heterochromatin
Monomethylation of histone H3 lysine at position 9 (H3K9me1) usually associated with active chromatin

Question 28

“Histone code”

Answer 28

Histone “marks” are read by binding proteins
Related domains are found in multiple code reading proteins e.g.
Bromodomain proteins bind to acetylated lysines
Tudor domain and chromodomain proteins read lysine methylation

Question 29

Histone “marks” not read in isolation

Answer 29

Multiple lysine residues on each histone
Multiple modifications- ac, me1, me2, me3
Other histone modifications e.g. phosphorylation on serine residues

Question 30

“Histone code” evidence for…

Answer 30

Evidence for “code readers”- protein complexes that read combinations of marks

Question 31

Marks for promoters and enhancers

Answer 31

Promoters strongly enriched for H3K4me3

Active enhancers enriched for H3K4me1