chromatin structure and histone code

Question 1

what are histones?

Answer 1

proteins responsible for the first level of packaging

Question 2

what are nucleosomes?

Answer 2

→DNA wrapped around 8 core histones is called a nucleosome.
→octamer core is made up of a pair of H2A, H2B, H3 and H4 histones.
→the positively-charged N-terminal tails are outside the octamer core.

Question 3

what are the 4 levels of chromatin folding?

Answer 3

→Histones are proteins responsible for the first level of packaging, creating nucleosomes.
→7-fold packaging

→Nucleosomes fold further into 30nm fibers called solenoids (beads on a string)
→6-fold packaging

→ The solenoid fibres fold into 80-100 nm fibres,
→ 3-fold packaging

→the solenoid fibers coil to form the mitotic chromosome.
→ 10,000-fold packaging.

Question 4

how much DNA is inside a cell?

Answer 4

6ft

Question 5

what are the 4 components of DNA?

Answer 5

→DNA
→histone proteins
→non-coding RNA
→non-histone proteins

Question 6

what does compaction of nucleosomes involve?

Answer 6

→ linker histones (eg. H1)
→interactions of histones tails with adjacent nucleosomes
→ binding of packing proteins to histone tails (this plays a vital role in gene access)

Question 7

what happens to the histones during transcription?

Answer 7

→nucleosomes must be removed from the DNA in front of the polymerase, and replaced behind the polymerase.

→Histone remodelling factors are enzymes that remove and replace nucleosomes.

Question 8

what is the difference between heterochromatin and euchromatin?

Answer 8

EUCHROMATIN:
→lightly staining areas of chromatin
→ rich in genes
→made up of nucleosomes, but not dense, higher order packaging

HETEROCHROMATIN
→darkly staining areas of chromatin
→few genes
→dense, higher order of packaging of nucleosomes

Question 9

what is constitutive heterochromatin?

Answer 9

→ remains condensed throughout the cell cycle and development.

→contains highly repetitive sequences that are not transcribed and play a role in chromosome structure.

Question 10

what is facultative heterochromatin?

Answer 10

→contains genes that are not expressed in that cell type.

→ DNA is as tightly packaged as constitutive heterochromatin, but it may be packaged as heterochromatin in other cell types.

Question 11

what determines whether nucleosomes are euchromatin or heterochromatin?

Answer 11

→chemical modification of lysine residues on histone tails:
→ acetylation
→methylation

Question 12

how do chromosomes appear when histones are extracted?

Answer 12

→They don’t completely fall apart.
→ they appear as long DNA loops attached to a scaffold of tightly bound proteins.
→each loop may have a different degree of chromatin compaction.
→The scaffold isolates the chromatin in one loop from the next loop.
→one loop may have open chromatin and active genes, while the neighbouring loop may be tightly packed as heterochromatin.

Question 13

how is chromatin structure studied?

Answer 13

→DNAse digestion.
→DNAse I cuts double-stranded DNA.
→Histone binding protects the DNA from DNAse digestion.
→there are DNAse I sensitive sites (HSS):
→sequences of DNA without histones
→it may be naked DNA or binding transcription factors
→ cut by very brief digestion with DNAse I
→found in promoters and enhancers

Question 14

how do proteins fit in DNA if it is so tightly packed?

Answer 14

→transcription factors open up the chromatin structure. → they recruit basal transcription factors.

Question 15

how do the transcription factors open up the chromatin structure?

Answer 15

→The transcription factors recruit chromatin modifying enzymes via a nuclear coactivator (NCoA) or corepressor (NCoR).

Question 16

what are the four different chromatin modifying enzymes and what are their functions?

Answer 16

HISTONE ACETYL TRANSFERASES (HATs):
→acetylate lysine residues on histones, which leads to unpacking of chromatin

HISTONE METHYL TRANSFERASES (HMTs):
→methylate lysine residues on histones, which leads to compaction of chromatin

HISTONE DEACETYLASES (HDACs): 
→ de-acetylate histones 

DEMETHYLASES (DMs):
→de-methylate histones

Question 17

how does the thyroid hormone receptor work?

Answer 17

→ (TR) thyroid hormone receptor binds to the thyroid response element (TRE) on the DNA.

→triggers the recruitment of the histone modification enzymes HDAC and DM via the N-CoR (which causes demethylation).

→A T3 (thyroid hormone) bonds to the TR.

→the histone modification enzymes HAT and HMT are recruited via the N-CoA (which causes acetylation, ie. the unpacking of DNA)

Question 18

what is the histone code?

Answer 18

→a set of histone modifications that determine whether the chromatin is open or closed

Question 19

how are histone marks read?

Answer 19

→Histone ‘marks’ (modifications) are read by binding proteins.

→Related domains are found in multiple code reading proteins.

Histone ‘marks’ aren’t read in isolation:
→there are multiple lysine residues on each histone
→there are multiple modifications
→ac, me1, me2, me3, etc.
→there are other histone modifications, eg. phosphorylation of serine ‘Code readers’ are protein complexes that read the combination of marks.

Question 20

are methylation and acetylation the same?

Answer 20

→The methylation seems more complex than the acetylation.

→ While the methylation of some lysine residues causes chromatin condensation, the methylation of other lysine residues causes chromatin decondensation.

→The effect may also vary if the residue is mono-, di- or tri-methylated.

Question 21

what are two examples of marks for promoters and enhancers?

Answer 21

→ promoters are strongly enriched in H3K4me3

→ active enhancers are enriched for H3K4me1