Chromatin and TFs

Question 1

Chromatin

Answer 1

DNA complexed with histone proteins.

Two basic states (visible under light microscope): Heterochromatin and euchomatin

Question 2

Heterochromatin

Answer 2

• Tighter (darkly stained)

- Not transcribed (generally inactive)

Question 3

Two kinds of interphase heterochromatin

Answer 3

a) Constitutive heterochromatin

b) Facultative heterochromatin

Question 4

Constitutive Heterochromatin

Answer 4

Always heterochromatic in interphase and in all cells.

Usually repeating in sequence, non coding

Examples: chromatin at centromeres, telomeres

Question 5

Facultative heterochromatin

Answer 5

Heterochromatic in interphase in some cells but not others.

Ex: inactive X (same X is not inactive in all cells)

*Most of the DNA that is inactive during interphase is euchromatic NOT heterochromatic

Question 6

Mitotic chromatin

Answer 6

All chromatin is heterochromatic (tight) during mitosis and meiosis

Question 7

Which types of chromatin (hetero v eu) are replicated?

Answer 7

ALL chromatin (both hetero and euc) are replicated in S)

-Hetero is not genetically active = not transcribed, but IS replicated)

Question 8

What is Euchromatin?

Answer 8

• Looser (less condensed, stains lightly)
• capable of genetic activity (transcription)
• normal state of most DNA during interphase
• transcribable but not necessarily being transcribed now. (DNA must be euch to be active but not all euch is active)

*MOST interphase DNA is euchromatic, whether it is transcribed or not

Question 9

How does chromatin appear in the EM?

Answer 9

Low salt: “beads on a string”

Physiological salt: 30 nm fiber

Question 10

Treatment of Chromatin with DNase

Answer 10

Little nuclease: ladder sequence of multiples of 200 bps. (easily cuts about once per 200 bp)

Lot of nuclease: resistant core of around 145 bps (repeating structure)

Question 11

Nucleosome core

Answer 11

about 145 bps of 200bps is a bead

about 55 bps remaining is a linker sequence

Question 12

Linker

Answer 12

Linker DNA has one site every 200 bps that is relatively unprotected and readily cut by DNase.

Question 13

Histone 1 (H1)

Answer 13

One molecule per nucleosome (~ every 200 bps) plus linker…(?)

Low salt (or digestion of linker DNA) removes H1: H1 is on outside of bead, more easily removed

Question 14

How can chromatin fixture be changed?

Answer 14

1) Modification of tails

2) nucleosome sliding

Question 15

Modification of histone tails

Answer 15

Regulatory function: modification affects folding of chromatin and binding to regulatory proteins, consequently they affect the activity of genes.

(“Tails” means either end: carbox or amino)

Question 16

Nucleosome sliding

Answer 16

Remodelers (proteins) allow RNA and DNA polymerase to progress along DNA by sliding one or a few nucleosomes at a time along the DNA

Question 17

What are the stages of nucleosome folding?

Answer 17

1. Nucleosomes: DNA+ histones, about 1/7 original lengths of DNA. “10nm fiber”
2. 30 nm fiber: chain of nucleosomes folded back onto itself (supercoils)
3. Loops of 30 nm fiber into 300 nm fiber
4. Higher order of folding –> –> heterochromatin

Question 18

Describe the structure of “Loops” of Nucleosomes

Answer 18

30 nm fiber forms about 300 nm in diameter (1/750 original length). Different sections are tighter/looser. Individual loops are stretched out (prob beads on a string stage) when actually transcribed. Loops bay be units of transcription or potential transcription.

Question 19

30 nm fiber

Answer 19

chain of nucleosomes folded back onto itself (supercoils) forming 30 nm fiber.
Fiber has about 6 beads/turn = 1/42 length of DNA. Need tails of histones and H1 to form 30 nm fibers and higher orders of folding

Question 20

What are the higher orders of folding for chromatin?

Answer 20

a) chromatids: folds back on self to form fibers of ~700 nm across (per chromatid)
b) at metaphase (TIGHTEST): 1/15000 - 1/20000 original lengt. (Chromosone is about 4-5 microns long but each chromatid contains about 74 mm of double helical DNA.

Question 21

Histone modificaiton

Answer 21

• helps tighten up or loosen chromatin.

- ‘tails” affect higher order of structure via many different modifications

Question 22

Histone tail phosphorylation

Answer 22

decreases transcription (e.g. at mitosis

Question 23

Histone tail acetylation

Answer 23

increases transcription

Question 24

Methylation

Answer 24

can increase (at H3K4)
or decrease (at H3K27) transcription

Question 25

Phosphorylation of H1

Answer 25

occurs at start of M and is reversed at end of M

• changes in kinase and phosphatase activity occur during the cell cyle
• alters state of histones and folding of chromatin in parallel with changes in lamins

Question 26

Experimental Procedure for Testing the State of Euchromatin

Answer 26

1) Treat chromatin with DNase (differentially degrade DNA in active vs inactive chromatin)
2) Remove protein to leave DNA
3) Examine DNA: use probe to test DNA for state of genes of interest and see if the genes were degraded or not

Question 27

Hypersensitive Sites (HSS)

Answer 27

Only sections of euchromatin that are not in regular nucleosomes:

• 10x more sensitive to degradation by DNase than average euchromatin. Degraded by very lo amts of DNase.
• correspond to regulatory, not coding, regions (in areas of transcription)
• different in different tissues: different genes are turned on (transcribed) in each cell. DNA is same in almost all cells but only the genes that are “turned on” in that cell type will have HSS.

-DNA is still not naked! (bound to regulatory proteins)

Question 28

Why are HSSs so sensitive to DNase?

Answer 28

1 view) TFs (reg proteins) have replaced histones and the other proteins don’t protect the DNA as well as histones do.
2nd view) histones are still present in addition to reg proteins. histones are much more loosely attached to the DNA than in normal nucleosomes, so the DNA is not as well protected as in ordinary nucleosomes.

Question 29

Cis-acting regulatory element

Answer 29

affect only the nucleic acid molecule on which it occurs. Usually this is a DNA sequence that binds some regulatory protein

Question 30

Trans-acting regulatory element

Answer 30

affects target nucleic sequences anywhere in the cell. The regulatory sequence codes for a regulatory molecule (usually a protein) that binds to a target, usually a DNA sequence.

Question 31

Diagram a eukaryotic gene ready to be transcribed. Show: basal promoter, activator, RNA polymerase, enhancer, basal transcription factors, and the mediator proteins.

Answer 31

(`---------/enhancer/------...
 (             {activator}
(             {mediator}
(             [RNA pol]
 (            {A][B][C][D}*
  (_------/basal prom/xx-tscribed-gene...

*A,B,C,D are basal transcription factors