Advanced Molecular Genetics-Biology 566 Yeast Silencing
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Chromatin structure

Figure F09-35. Hiearchical folding of chromatin. (Lodish et.al., 2000)

Nucleosome Structure

Fig F09-30A. Nucleosome structure. (Lodish et al., 2000).

Yeast Chromasome organization of mating type cassettes

Chromosome for yeast matin type showing the silent HMR and HML cassettes coding for the a and alpha mating types respectively. The active Mat loci is also shown. The telomeres are also silenced.

Nature of silenced Chromatin

-akin to heterochromatin

-N-terminal tails of histones H3 and H4 are hypoacetylated

-the structural proteins of the silenced chromatin are the Sir proteins

-the Sir (silent information regulator) proteins form an ordered compact structure that is restrictive to transcription.

Figure 1. The organization of the silent domains in yeast. (Rusche et al., 2003). ORC binds ACS site, Rap1p binds to RAP1 site, and Abf1p binds ABF1 site.

Fig. 2.20. Folded structure of the end of the chromosome (telomere). Rap1p binds at Rap1 site and recruites Sir proteins.

-additionally genes near telomeres are can also be silenced

-factors in silencing HMR, HML and Telomeres are almost the same and turn off all transcription

-Sir2 only needed to silence rDNA. Reduces transcription of marker genes.

Proteins used in silencing

Sir2p

-members of Sir2p family are NAD+ -dependent deacetylases.

-1 NAD+ used per acetyl group removed.

-Sir2p activity is weak and probably need to function as a complex.

Sir4p

-no known enzymatic activities, structural role.

-associates with silencer-binding proteins.

-binds deacetylated tail of H3 and H4.

-required for association of Sir2p and Sir3p with silencer or telomeres.

-Associates with an immobile peripheral component of nucleus.

-interacts with Dis1p/Ris1p used in recombination during mating type switching.

-targets Ty5 transposon integrase to silenced domains.

-interaction with ubiquitin hydrolases, Dot4p and Ubp3p, which may act at different targets.

-can be phosphorylated (regulation mechanism?).

Sir3p

-no known enzymatic activities, structural role.

-binds to both nucleosomes and DNA.

-binds deacetylated tail of H3 and H4.

-can be phosphorylated (regulation mechanism?).

Sir1p

-contributes but is not essential for silencing

-acts in the establishment of silencing by facilitating the assembly of the other Sir proteins at the silencer.

Histones

-actively transcribed regions associated with acetylated histones.

-silenced regions associated with deacetylated histones.

-histones in turn lead to further chromatin modifications that untimately dictate particular expression states. i.e., through methylation of DNA.

Initiation of Silencing

-HMR-E has binding site for ORC, Rap1p, and Abf1p,

-Rap1p, and Abf1p are two of the most common transcriiption activators in S. cerevisiae.

-ORC is the orgin recognition complex for initiation of DNA replication.

-mutations in two of the three sites result in loss of silencing.

-juxtaposition of three protein may provide a site for Sir protein binding.

-arrays of multiple Rap1p binding sites recruit Sir proteins to the telomeres.

-Sir1p binds directly to Orc at silencer but does not spread along silenced region. Can activate Sir assembly when tethered directly (Orc not necessary) .

Sir4p recruited to silencer by Sir1p and Rap1p.

Sir2p probably comes to silencer with Sir4p complex.

Sir3p recruited to silencer by Sir4p and binds Sir4p, Rap1p and perhaps Abf1p.

Spreading of Silencing

-Once all three Sir proteins are recruited to a silencer, cooperative interactions enable them to spread throughout the silent locus with Sir3p and Sir4p binding to to the deacetylated tails of histones H3 and H4.

-Sir2p deacetylates adjacent histones and creates high affinity sites for the binding of Sir3p and Sir4p.

(sidelight) mutations in proteins that acetylate or methylate histones can cause the titration of Sir proteins into active chromosomal regions at the expense of histone association at silenced regions. Also modifications in supporting proteins can impact activity (Rad6p ubiquitination of H2B).

Maintenance of Silenced chromatin

Maintenance = silencing within cell cycle.

Inheritance = perpetuation of silenced state through DNA replication.

Epigenetic Inheritance = Heritability of silenced or active state in otherwise identical cells.

Factors fall into three categories:

- Stuctural proteins. (Sir3 & Sir4) Mutations can cause suboptimal assembly which easily falls apart.

- Silencers and silencer binding proteins. Need one silencer protein to maintain silencing.

- Chromatin assembly factors. Mutations in chromatin assembly factor Cac1p results improperly assembled or modified nucleosomes.

Two major mechanisms for maintenance of silenced/active states:

1- Self-reinforcing marks recruit chromatin modifiers that re-establish silenced or active chromatin state.

- Marking chromatin by DNA methylation.

- Marking by random distribution of modified H3/H4 histones between replicated chromosomes.

2- Recruiting chromatin silencing modifiers or chromatin activation modifiers to localized replication complex. Evidence for associations with PCNA.

Relationship between silencing and repression.

Sum1p is a repressor. Sum1-1p differes by 1 amino acid substitution and silences in the absence of Sir2, Sir3, and Sir4 proteins by recruiting other proteins to spread silencing.

How does silencing inhibit transcription?

Observed:

- TBP and Pol II both present at HMR a1 promoter.

- Sir2p might deacetylate a transcription factor. (mammalian Sir2p deacetylated p53).

- Sir proteins might influence by direct interactions (polycomb complex, interaction with TAFs).

- Sir modifications of nucleosomes to make them less likely to dissociate and clear the transcription complex's conversion to transcription or elongation path. (block dissassembly of nucleosome).

rDNA Silencing

Requires Sir2p but not Sir3p or Sir4p.

Artificial situation.

Perhaps Pol I specific interactions prevent Pol II transcription (UAF of Pol I inhibits Pol II).

Silencing and Chromatin metabolism

Life span is measured as the number of daughters that a mother cell can produce

Observations:

- As cells age, mother cells divide more slowly, increase in volume, and have decreased soliencing at the HM loci.

- Daughter cells from old mothers have short life spans, but those daughters' daughters have normal life spans.

Indicate that an aging factor, ERCs, accumulates in and is prefferentially inherited by, mother cells over time.

One aging factor is rDNA circles, preferrentially retained in mother cells.

Sir2p affects the rate of formation of ERCs

In young cells Sir2p localized in nucleolus and Sir3p and 4p localized in periphery. In older cells Sir3p relocalizes to the nucleolus and requires active Sir2p but not Sir4p. Results in titrating of Sir proteing from silenced areas.

Metabolism influenced by Sir2p and NAD+.

-