M140 Lecture 2, Part 1, Heterochromatin, Heterochromatin Formation, Chip

Front 1

Features of Heterochromatin, differential staining of chromosomal structures

Back 1

- in interphase itself (not interphase and then metaphase), there exists darkly staining DNA and lightly staining DNA - this must correspond to heterochromatin and euchormatin because heterochromatin is more condesnsed - and has a higher density of DNA and can absorb more stain.
- Heterochromatin is found at the nuclear periphery, in between nuclear pores
- it often contains repeated sequences
- It represses adjacent genes epigenetically (meaning it occurs in some cells, but not others, even though they both contain identical DNA sequences)
- found in centromere, telomere sequences in complex eukaryotes.

Front 2

Yeast and heterochromatin

Back 2

- Saccharomyces cerevisiae
- easy to do genetics
- first eukaryotic organism to be abe to be transformed/ transfected
- its chromosomes are too small to see by light microscopy, but there are molecular clues for heterochromatin: - near telomeres, silent mating loci.: if certain genes are placed near these, they are epigenetically repressed.

Front 3

Telomeric Position Effect (TPE)

Back 3

- the URA3 gene converts 5FOA to 5Fu ( a toxic compound)
- When URA3 is at its normal position, it does this conversion and the cells are sensitive to 5FOA.
- but when it is positioned (transposed) near the telomere, it is repressed, and it is resistant to 5FOA.
- this effect is not gene specific
- this is epigenetic repression and differs from regular gene repression because it occurs only in some cells, but not in other genetically identical cells.
- also it is not promoter specific - repressive complex spreads to adjacent genes.
- you can mutagenoze the cells with URA next to telomere to identify genes involved in the repression of URA3 at TEL.

Front 4

Components of the Telomeric Heterochromatin in yeast

Back 4

- Rap1
- Sir 2
- Sir 3
- Sir 4
- (the telomeric sequence)

Front 5

Rap 1

Back 5

- 14-16 Rap 1 molecules recognize and interact with the telomeric (repeated) DNA of about 300 bp.
- this starts the heterochromatin formation process.

Front 6

Sir 3

Back 6

- interacts with unacetylated H4 K16 preferentially.
- if it is acetylated, it won't bind to it.

Front 7

Sir 2

Back 7

- deactylates H4 K16, allowing Sir 3 to bind to it.

Front 8

Chromatin Immunoprecipitation (ChIP)

Back 8

- a way to determine where proteins bind in chromatin (not necessarily DNA because the proteins might be bound to other chromatin associated proteins which are bound to DNA)
1. cross-link protein to chromatin using formaldehyde
2. isolate and shear chromatin mechanically (sonication) or using enzymes) --> get it 300-1000bp in length - you don't want it to be any greater than this because then you won't be narrowing down where you protein binds
3. Add antibody specific for the protein of interest
4. Immunoprecipitate
5. Reverse cross-link with heat to get rid of protein
6. Do PCR in order to figure out if the sequence you think the protein binds at is actually present (you must know sequence of gene to get primers - you need to have an educated guess of where the protein binds)
- you can also label DNA and hybridize to microarray to see where in genome the DNA belongs to (and the protein binds).

Front 9

ChIP results for Sir2, 3, and 4

Back 9

- you can design primers that bind to sequences that are different distances from the telomeres
- you do ChIP with anti-Sir 2, 3, and 4 and do PCR with those primers to see are those sequences still present. In other words, you can determine how far from telomere the Sir proteins bind to the chromatin - how far do they spread?
- It turns out that all three spread the same distance from telomere
- as the distance from the telomere gets greater, the relative amount of precipated DNA decreases
- by 2.8kb, the Sir proteins really don't bind to the chromatin

Front 10

How do you identify where in genome the chromatin in acetylated?

Back 10

1. isolate and shear chromatin
2. Add antibody specific for acetylated N-terminal histone tail. Antibody would have to be made by injected peptide with lysine attached to acetyl group in bunny.
3. Immunoprecipitate
4. Release immunoprecipitated DNA and assay by PCR --> to test if your gene is acetylated, you can add primers for that gene and see if it is amplified in the immunoprecipitated DNA.

Front 11

The Big Picture of Heterochromatin Formation and Spreading

Back 11

1. Rap1 binds to telomere, and recruits Sir4. This is known by doing ChIP with anti-Sir4.
2. Sir2 binds to Sir4 (we know that Sir2 directly doesn't bind to Rap1 because you can do ChIP with Sir4 knockout), and deactlylates H4 K16AC
3. Sir3 binds deactylated K16 (to prevent re-acetylation?), and this process continues or spreads
4. It stops when
a.
b. presence of Sas2, which acetylates H4 K16 --> therefore, there exists a competition between acetylation by Sas2 and deacytlation by the sir proteins.
- In conclusion, to get heterochromatin formation, you need deacetylation

Front 12

How does epigentic repression work by heterochromatin acting on genes transposed to telomeric region?

Back 12

- when genes are transposed to heterochromatin region, they become covered by Sir proteins - can't be transcribed.

Front 13

Yeast telomeric foci

Back 13

- there are 32 telomeres for 16 chromosomes in yeast
- but when you use fluorescent probe that binds to yeast telomeres, you get only 7-8 foci - meaning that the telomeres are interacting.
This could be because
1. this would allow more repression
2. would allow sharing of the Sir proteins, which would be found at high concentrations at the these foci.

Front 14

Why are heterochromatin concentrated at nuclear periphery?

Back 14

- concentration of Sir proteins is high there

Front 15

How do euchromatin and heterochromatin differ structually in more complex than yeast?

Back 15

- Heterochromatin has H3-K9 methylated -->gene repression
- Euchromatin has H3-K6 methylation --> gene activity.

Front 16

Histone Modifications

Back 16

- histones are one of the most post-translationally modified proteins --> several residues on each histone in the octamer are modified.

Front 17

Sir 3 homolog in metazoans (multi-cellular, as opposed to protozoans)

Back 17

- Sir 3 in yeas interacts with deacetylated H4 K16 lysine to help form heterochromatin
- In metazoans, the protein HP1 (Heterochromatin protein 1) chromodomain interacts with METHYLATED H3 lysine 9 to help form heterochromatin

Front 18

How does heterochromatin in more complex eukaryotes initiate?

Back 18

- You start with with un-methylated nucleosomes which have DNA repeats
- these DNA repeats increase the number of RNA that can fold back on itself (hence heterochromatin involves simple sequence DNA) --> this is because if you have AAAATTTT a bend can form between the 4th A and the 1st T and dsRNA hairpin can be created - this dsRNA is recognized by Dicer and RDRP(RNA Dependent RNA Polymerase) and is cleaved to ~22 nt siRNAs
- Ago1 (part of RITS complex) binds these siRNAs and can then bind the repetitive DNA.
- then HP1 is recruited, which then binds HDAC1 and HMTase. Then the H3-K9AC is deacytelated (by HDAC1) and then methylated (HMTase)- since it becomes methylated, more HP1 can bind, and the heterochromatin formation can spread.
- therefore, if you include repetitive DNA into genome, it becomes heterochromatin.