Toward a molecular understanding of yeast silent chromatin : roles for H4K16 acetylation and the Sir3 C-terminus

Discrete regions of the eukaryotic genome assume a heritable chromatin structure that is refractory to gene expression. In budding yeast, silent chromatin is characterized by the loading of the Silent Information Regulatory (Sir) proteins (Sir2, Sir3 and Sir4) onto unmodified nucleosomes. This requires the deacetylase activity of Sir2, extensive contacts between Sir3 and the nucleosome, as well as interactions between Sir proteins forming the Sir2-3-4 complex. During my PhD thesis I sought to advance our understanding of these phenomena from a molecular perspective. Previous studies of Sir-chromatin interactions made use of histone peptides and recombinant Sir protein fragments. This gave us an idea of possible interactions, but could not elucidate the role of histone modifications in the assembly of silent chromatin. This required that we examine nucleosomal arrays exposed to full length Sir proteins or the holo Sir complex. In Chapter 2, I made use of an in vitro reconstitution system, that allows the loading of Sir proteins (Sir3, Sir2-4 or Sir2-3-4) onto arrays of regularly spaced nucleosomes, to examine the impact of specific histone modifications (methylation of H3K79, acetylation of H3K56 and H4K16) on Sir protein binding and linker DNA accessibility. The “active” H4K16ac mark is thought to limit the loading of the Sir proteins to silent domain thus favoring the formation of silent regions indirectly by increasing Sir concentration locally. Strikingly, I found that the Sir2-4 subcomplex, unlike Sir3, has a slight higher affinity for H4K16ac-containing chromatin in vitro, consistent with H4K16ac being a substrate for Sir2. In addition the NAD-dependent deacetylation of H4K16ac promotes the binding of the holo Sir complex to chromatin beyond generating hypoacetylated histone tails. We conclude that the Sir2-dependent turnover of the “active” H4K16ac mark directly helps to seed repression. The tight association of the holo Sir complex within silent domains relies on the ability of Sir3 to bind unmodified nucleosomes. In addition, Sir3 dimerization is thought to reinforce and propagate silent domains. However, no Sir3 mutants that fail to dimerize were characterized to date. It was unclear which domain of Sir3 mediates dimerization in vivo. In Chapter 3, we present the X-ray crystal structure of the Sir3 extreme C-terminus (aa 840-978), which folds into a variant winged helix-turn-helix (Sir3 wH) and forms a stable homodimer through a large hydrophobic interface. Loss of wH homodimerization impairs holo Sir3 dimerization in vitro showing that the Sir3 wH module is key to Sir3-Sir3 interaction. Homodimerization mediated by the wH domain can be fully recapitulated by an unrelated bacterial homodimerization domain and is essential for stable association of the Sir2-3-4 complex with chromatin and the formation of silent chromatin in vivo

Regulating repression : roles for the Sir4 N-terminus in linker DNA protection and stabilization of epigenetic states

The Gasser laboratory is supported by the Novartis Research Foundation and the EU training network Nucleosome 4D. SK was supported by an EMBO long-term fellowship, a Schrodinger fellowship from the FWF, and the Swiss SystemsX.ch initiative/C-CINA; HCF by an EMBO long-term fellowship.Silent information regulator proteins Sir2, Sir3, and Sir4 form a heterotrimeric complex that represses transcription at subtelomeric regions and homothallic mating type (HM) loci in budding yeast. We have performed a detailed biochemical and genetic analysis of the largest Sir protein, Sir4. The N-terminal half of Sir4 is dispensable for SIR-mediated repression of HM loci in vivo, except in strains that lack Yku70 or have weak silencer elements. For HM silencing in these cells, the C-terminal domain (Sir4C, residues 747-1,358) must be complemented with an N-terminal domain (Sir4N; residues 1-270), expressed either independently or as a fusion with Sir4C. Nonetheless, recombinant Sir4C can form a complex with Sir2 and Sir3 in vitro, is catalytically active, and has sedimentation properties similar to a full-length Sir4-containing SIR complex. Sir4C-containing SIR complexes bind nucleosomal arrays and protect linker DNA from nucleolytic digestion, but less effectively than wild-type SIR complexes. Consistently, full-length Sir4 is required for the complete repression of subtelomeric genes. Supporting the notion that the Sir4 N-terminus is a regulatory domain, we find it extensively phosphorylated on cyclin-dependent kinase consensus sites, some being hyperphosphorylated during mitosis. Mutation of two major phosphoacceptor sites (S63 and S84) derepresses natural subtelomeric genes when combined with a serendipitous mutation (P2A), which alone can enhance the stability of either the repressed or active state. The triple mutation confers resistance to rapamycin-induced stress and a loss of subtelomeric repression. We conclude that the Sir4 N-terminus plays two roles in SIR-mediated silencing: it contributes to epigenetic repression by stabilizing the SIR-mediated protection of linker DNA; and, as a target of phosphorylation, it can destabilize silencing in a regulated manner.Publisher PDFPeer reviewe

A Subset of Human Bromodomains Recognizes Butyryllysine and Crotonyllysine Histone Peptide Modifications

SummaryBromodomains are epigenetic readers that are recruited to acetyllysine residues in histone tails. Recent studies have identified non-acetyl acyllysine modifications, raising the possibility that these might be read by bromodomains. Profiling the nearly complete human bromodomain family revealed that while most human bromodomains bind only the shorter acetyl and propionyl marks, the bromodomains of BRD9, CECR2, and the second bromodomain of TAF1 also recognize the longer butyryl mark. In addition, the TAF1 second bromodomain is capable of binding crotonyl marks. None of the human bromodomains tested binds succinyl marks. We characterized structurally and biochemically the binding to different acyl groups, identifying bromodomain residues and structural attributes that contribute to specificity. These studies demonstrate a surprising degree of plasticity in some human bromodomains but no single factor controlling specificity across the family. The identification of candidate butyryl- and crotonyllysine readers supports the idea that these marks could have specific physiological functions

Structural basis for the role of the Sir3 AAA+ domain in silencing: interaction with Sir4 and unmethylated histone H3K79

The Sir2/3/4 complex is a key component of the Sir gene silencing machinery in S. cerevisiae. This structure–function study examines the unique folding structure of the Sir3 AAA+ domain and provides insight into new interaction surfaces between Sir3 and Sir4 and into its chromatin-binding properties

Sir4C is not sufficient for silencing at telomeres.

<p>A) Telomeric silencing was monitored by a Tel7L::<i>URA3</i> reporter gene (GA503, GA5809, GA5822) expressing the indicated proteins from pRS, including the SIR4N-C fusion. Growth on plates containing 5 nM rapamycin (rapa) was also monitored. B) Relative mRNA levels of three different subtelomeric genes and <i>HML-ALPHA1</i> were measured using QPCR. Bars represent averages of biological triplicates, data represent mean value ± s.e.m. C) Scheme of the <i>HM</i> loci and telomeres analyzed, indicating additional recruiting elements and distances of promoters from nucleating elements.</p

Sir4C can form a stable and active SIR complex in a recombinant system.

<p>A) SIR complexes as indicated were purified form co-infected insect cells. 1 µg of each complex was run on a SDS-PAGE and visualized by Coomassie staining. B) Purified Sir2–Sir3–Sir4 and Sir2–Sir3–Sir4C complex were incubated with histone octamers acetylated at H4K16 with or without the essential cofactor NAD. The deacetylation reaction was stopped after various time points by the addition of sample buffer and monitored by immuno blotting for H4K16^ac and H3, for equal loading. C, D) Sir2–Sir3–Sir4 or Sir2–Sir3–Sir4C complexes were analyzed by density gradient sedimentation. Fractions were run on 4–12% NuPAGEs Novex Bis-Tris Gels and stained with Sypro Ruby dye. Intensities of Sir2, Sir3 and Sir4 full length proteins were quantified (QuantityONE) and plotted in line graphs. The asterisk in D) indicates a Sir4 degradation band that runs very closely to Sir3.</p