Lecture 21 - Chromatin sorting and histone code

Question 1

DNA Packaging (4)

Answer 1

Chromosomes –> Chromatin
DNA + Histones –> Nucleosomes (1 LEVEL) —> 30 nm fibre (2 LEVEL) –> Increases packaging to 3rd fold (3 LEVEL) –> mitotic chromosome (4 LEVEL)..

Question 2

Chromosomes (5)

Answer 2

Consists predominantly of: DNA, Histone proteins, Non-histone proteins, Non-coding RNA.
In interphase almost all DNA packages at 1st level (nucleosomes).

Question 3

Nucleosomes (7)

Answer 3

DNA is complexed with histones made into chromatin.
DNA double helix wound into 8 histone subunits (2A, 2B, 3 and 4) - histone octate.
N-terminal chains outside the octamer core (histone tails- positively charged aa) interacts with -vely charged phosphate groups in DNA. Electrostatic attraction,
Compacting the DNA by a factor of 6. Histone 1 binds to the outside and to linker DNA.
Basic building block of chromatin.
Multiple nucleosomes are coiled together, resulting in a fibre called chromatin, which is then condensed to a 30nm chromatin fibre.
Then looped and coiled more (into 80-100 nm fibres), using other proteins to form the chromosomes, which only form when cells are dividing.

Question 4

Compaction of nucleosomes to form higher order structures (3)

Answer 4

o Linker histones (e.g. H1)
o Interaction of histone tails with adjacent nucleosomes.
o Binding of packing proteins to histone tails.

Question 5

Chromatin structure is not static (2)

Answer 5

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

Histone remodelling factors - Enzymes that remove and replace nucleosomes.

Question 6

Chromatin structure - Euchromatin (3)

Answer 6

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

Question 7

Chromatin structure - Heterochromatin (3)

Answer 7

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

Question 8

Chromatin structure - Facultative heterochromatin (3)

Answer 8

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

Question 9

Chromatin structure - What determines whether nucleosomes are packed as euchromatin or heterochromatin? (4)

Answer 9

One key level of control- chemical modification of lysine residues in histone tails. e.g. acetylation, methylation, phosphorylation, ubiquitinylation and others.

Question 10

Chromatin structure - Loops and chromatin domains (4)

Answer 10

o Each loop may have a different degree of chromatin compaction.
o The scaffold isolates the chromatin in one loop from the next loop.
o So, one loop may have open chromatin and active genes.
o The neighbouring loop could be tightly packed as heterochromatin.

Question 11

Methods to investigate chromatin structure- DNAase digestion [3,4]

Answer 11

• DNAase I cuts the double stranded DNA.
• Histone binding protects DNA from DNAase digestion.
• DNAase I sensitive sites (HSS)
o Sequences of DNA without histones.
o May be naked DNA, or binding transcription factors.
o Cut by very brief digestion with DNAase I.
o Found in promoters and enhancers.

Question 12

RTF (Regulatory Transcription Factors) (4)

Answer 12

1. TFs bind promoter (and enhancers).
2. Recruit general transcription factors.
3. Assemble initiation complex.
4. Which recruits RNA polymerase.

Question 13

If DNA is tightly packaged in higher order chromatin structures, how can all these proteins get at the DNA? (4)

Answer 13

• First TF opens up chromatin structure  Then recruits basal transcription factors  Transcription
• TFs recruit chromatin modifying enzymes via a nuclear coactivator (NCoA) or corepressor (NCoR).

Question 14

Histone modification- acetylation (9)

Answer 14

• Heterochromatin –> Histones largely unacetylated.
• Expressed genes in euchromatin –> Many lysine residues of histones are acetylated.
• Histone acetyl transferases (HATs)
o Acetylate lysine residues on histones
o Lead to unpacking of chromatin
• Histone deacetylases (HDACs)
o De-acetylate histones
o Lead to compaction of chromatin
• e.g. Thyroid hormone receptors. Thyroid response element (TRE) - AGGTCANNNNAGGTCA.

Question 15

Histone modification- methylation (7)

Answer 15

• Histone tails are methylated by histone methyl transferases (HMTs).
• Demethylated by histone demethylases (HDMs).
• A lysine residue can be mono- di- or tri-methylated.
• Methylation of some lysine residues causes chromatin (de)condensation.
• Effect may also vary if residue is mono-, di- or tri-methylated.
o Trimethylation of histone H3 lysine at position 9 (H3K9me3) associated with heterochromatin.
o Monomethylation of histone H3 lysine at position 9 (H3K9me1) usually associated with active chromatin.

Question 16

Histone code (9)

Answer 16

• Histone “marks” are read by binding proteins.
• Related domains are found in multiple code reading proteins e.g.
o Bromodomain proteins bind to acetylated lysines.
o Tudor domain and chromodomain proteins read lysine methylation.
• Histone “marks” not read in isolation.
o Multiple lysine residues on each histone.
o Multiple modifications- ac, me1, me2, me3
o Other histone modifications e.g. phosphorylation on serine residues.
• Evidence for “code readers”- protein complexes that read combinations of marks.

Question 17

Marks for promoters and enhancers (2)

Answer 17

• Promoters strongly enriched for H3K4me3.

* Active enhancers enriched for H3K4me1.