Chromatin and TFs Flashcards
Chromatin
DNA complexed with histone proteins.
Two basic states (visible under light microscope): Heterochromatin and euchomatin
Heterochromatin
- Tighter (darkly stained)
- Not transcribed (generally inactive)
Two kinds of interphase heterochromatin
a) Constitutive heterochromatin
b) Facultative heterochromatin
Constitutive Heterochromatin
Always heterochromatic in interphase and in all cells.
Usually repeating in sequence, non coding
Examples: chromatin at centromeres, telomeres
Facultative heterochromatin
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
Mitotic chromatin
All chromatin is heterochromatic (tight) during mitosis and meiosis
Which types of chromatin (hetero v eu) are replicated?
ALL chromatin (both hetero and euc) are replicated in S)
-Hetero is not genetically active = not transcribed, but IS replicated)
What is Euchromatin?
- 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
How does chromatin appear in the EM?
Low salt: “beads on a string”
Physiological salt: 30 nm fiber
Treatment of Chromatin with DNase
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)
Nucleosome core
about 145 bps of 200bps is a bead
about 55 bps remaining is a linker sequence
Linker
Linker DNA has one site every 200 bps that is relatively unprotected and readily cut by DNase.
Histone 1 (H1)
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
How can chromatin fixture be changed?
1) Modification of tails
2) nucleosome sliding
Modification of histone tails
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)
Nucleosome sliding
Remodelers (proteins) allow RNA and DNA polymerase to progress along DNA by sliding one or a few nucleosomes at a time along the DNA
What are the stages of nucleosome folding?
- Nucleosomes: DNA+ histones, about 1/7 original lengths of DNA. “10nm fiber”
- 30 nm fiber: chain of nucleosomes folded back onto itself (supercoils)
- Loops of 30 nm fiber into 300 nm fiber
- Higher order of folding –> –> heterochromatin
Describe the structure of “Loops” of Nucleosomes
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.
30 nm fiber
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
What are the higher orders of folding for chromatin?
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.
Histone modificaiton
- helps tighten up or loosen chromatin.
- ‘tails” affect higher order of structure via many different modifications
Histone tail phosphorylation
decreases transcription (e.g. at mitosis
Histone tail acetylation
increases transcription
Methylation
can increase (at H3K4) or decrease (at H3K27) transcription
Phosphorylation of H1
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
Experimental Procedure for Testing the State of Euchromatin
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
Hypersensitive Sites (HSS)
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)
Why are HSSs so sensitive to DNase?
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.
Cis-acting regulatory element
affect only the nucleic acid molecule on which it occurs. Usually this is a DNA sequence that binds some regulatory protein
Trans-acting regulatory element
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.
Diagram a eukaryotic gene ready to be transcribed. Show: basal promoter, activator, RNA polymerase, enhancer, basal transcription factors, and the mediator proteins.
(`---------/enhancer/------...
( {activator}
( {mediator}
( [RNA pol]
( {A][B][C][D}*
(_------/basal prom/xx-tscribed-gene...
*A,B,C,D are basal transcription factors
