SIR–nucleosome interactions: Structure–function relationships in yeast silent chromatin

doi:10.1016/j.gene.2013.05.088

Gene

Volume 527, Issue 1, 15 September 2013, Pages 10-25

https://doi.org/10.1016/j.gene.2013.05.088 Get rights and content

Highlights

•
Histone PTMs regulate chromatin structure and assembly of yeast silent chromatin
•
Sir2-dependent turnover of H4K16ac actively reinforces assembly of silent chromatin
•
Protein contacts within and between adjacent SIR complexes are crucial to silencing
•
Variegation and regulation of subtelomeric silencing respond to environmental changes

Abstract

Discrete regions of the eukaryotic genome assume a heritable chromatin structure that is refractory to gene expression, referred to as heterochromatin or “silent” chromatin. Constitutively silent chromatin is found in subtelomeric domains in a number of species, ranging from yeast to man. In addition, chromatin-dependent repression of mating type loci occurs in both budding and fission yeasts, to enable sexual reproduction. The silencing of chromatin in budding yeast is characterized by an assembly of Silent Information Regulatory (SIR) proteins—Sir2, Sir3 and Sir4—with unmodified nucleosomes. Silencing requires the lysine deacetylase activity of Sir2, extensive contacts between Sir3 and the nucleosome, as well as interactions among the SIR proteins, to generate the Sir2–3–4 or SIR complex. Results from recent structural and reconstitution studies suggest an updated model for the ordered assembly and organization of SIR-dependent silent chromatin in yeast. Moreover, studies of subtelomeric gene expression reveal the importance of subtelomeric silent chromatin in the regulation of genes other than the silent mating type loci. This review covers recent advances in this field.

Graphical abstract

Introduction

The packaging of DNA into nucleosomes and the interaction of the nucleosomal fiber with transcription modulators, regulate promoter accessibility and gene expression. Recent structure–function studies have yielded insights into the molecular details of gene silencing, and in no organism is this more advanced than in Saccharomyces cerevisiae. Here we focus in particular on the yeast-specific Silent Information Regulator (SIR) complex and its contacts with nucleosomes, which control transcription both at silent mating type loci and in subtelomeric domains.

Section snippets

Structural aspects of the chromatin fiber

Silent chromatin reflects an integrated assembly of non-histone and histone proteins into a chromatin structure that represses promoter activity independent of the specific promoter sequence. To understand gene silencing it is first necessary to describe the structure of the nucleosomal core particle (NCP), which lies at the core of silent chromatin. A canonical NCP is composed of 147 base pairs (bp) of DNA wrapped around a histone octamer in 1.65 superhelical turns. The highly basic core

Chromatin regulates gene expression

Gene expression results from both the binding of transcription factors to regulatory sequences and changes in nucleosomal organization in these control regions. This latter involves the post-translational modification (PTM) of histone tails and core residues, the exchange of histone variants, and the shifting and eviction of nucleosomal themselves. Here we summarize evidence showing that nucleosomes and their positioning information alone can influence transcriptional efficiency, before we

Silent chromatin in S. cerevisiae

Silent chromatin in yeast occurs at the subtelomeric regions (Gottschling et al., 1990) and at silent homothallic mating-type loci (HM), HML and HMR (Rine and Herskowitz, 1987). It requires the loading onto the chromatin fiber of the Silent Information Regulator (SIR) proteins Sir2, Sir3 and Sir4 (Fig. 2). These three proteins form a heterotrimeric Sir2–3–4 complex—the SIR complex—which together with histones, constitutes the essential silencing machinery in budding yeast (Cubizolles et al.,

Outlook

It is clear that the combination of genetics, biochemistry and structural work has put the SIR system of gene repression at the forefront of model systems for the study of chromatin-mediated gene repression. It remains to determine the structure of the SIR-nucleosomal complex and to define the transition from “SIR binding” to “silencing” in molecular terms. Indeed, the precise step in the transcription process that is impaired by the SIR complex is still debated. Nonetheless, a wealth of

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgments

The Gasser laboratory is funded by the Swiss National Science Foundation, the NCCR Frontiers in Genetics, the SystemsX RTD CINA, various EU FP7 programs, and the Novartis Research Foundation. We thank colleagues in the laboratory for critical reading of this review and for a stimulating environment. We thank David Shore, and Nicolas Thomae for comments on earlier versions of the text and Laurent Rueff for artistic help. SK also thanks EMBO and FWF for support.

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Citation Excerpt :
H2Bub has also been implicated in heterochromatin protection in S. pombe, where acetylation of the H2B ubiquitin ligase subunit Brl1 by Mst2 regulates H2Bub levels and protects euchromatic genes from heterochromatic silencing by small RNAs [51]. H4K16ac in S. cerevisiae is a classic example of ‘anti-silencing’, which corrals heterochromatin factors into silent regions of the genome to protect active gene transcription from silencing [52]. Even though S. cerevisiae and S. pombe use very different heterochromatin machinery, the functional parallels between scH4K16ac and spH3K14ac are becoming apparent and point to a critical role of specific acetyl marks as repellents in the protection of euchromatin.
Eukaryotic genomes are segregated into active euchromatic and repressed heterochromatic compartments. Gene regulatory networks, chromosomal structures, and genome integrity rely on the timely and locus-specific establishment of active and silent states to protect the genome and provide the basis for cell division and specification of cellular identity. Here, we focus on the mechanisms and molecular machinery that establish heterochromatin in Schizosaccharomyces pombe and compare it with Saccharomyces cerevisiae and the mammalian polycomb system. We present recent structural and mechanistic evidence, which suggests that histone acetylation protects active transcription by disrupting the positive feedback loops used by the heterochromatin machinery and that H2A and H3 monoubiquitination actively drives heterochromatin, whereas H2B monoubiquitination mobilizes the defenses to quench heterochromatin.
Stabilization of Sir3 interactions by an epigenetic metabolic small molecule, O-acetyl-ADP-ribose, on yeast SIR-nucleosome silent heterochromatin
2019, Archives of Biochemistry and Biophysics
Citation Excerpt :
The silent heterochromatin of the budding yeast Saccharomyces cerevisiae provides an excellent system for epigenetic studies. In vivo genetics and in vitro biochemical studies have demonstrated that the silent information regulator (Sir) proteins, Sir2, Sir3, and Sir4, are required to establish and maintain silent heterochromatin domains at the telomeres and the mating type loci [8–15]. The Sir proteins form the SIR complex that is capable of nucleosome binding and is important for silent heterochromatin assembly [16–19].
In Saccharomyces cerevisiae, Sir proteins mediate heterochromatin epigenetic gene silencing. The assembly of silent heterochromatin requires histone deacetylation by Sir2, conformational change of SIR complexes, and followed by spreading of SIR complexes along the chromatin fiber to form extended silent heterochromatin domains. Sir2 couples histone deacetylation and NAD hydrolysis to generate an epigenetic metabolic small molecule, O-acetyl-ADP-ribose (AAR). Here, we demonstrate that AAR physically associates with Sir3 and that polySir3-AAR formation has a specific and essential role in the assembly of silent SIR-nucleosome pre-heterochromatin filaments. Furthermore, we show that AAR is capable of stabilizing binding of the Sir3 BAH domain to the Sir3 carboxyl-terminal region. Our data suggests that for the assembly of SIR-nucleosome pre-heterochromatin filament, the structural rearrangement of SIR-nucleosome is important and result in creating more stable interactions of Sir3, such as the inter-molecule Sir3-Sir3 interaction, and the Sir3-nucleosome interaction within the filaments. In conclusion, our results reveal the importance of AAR, indicating that it not only affects the conformational rearrangement of SIR complexes but also might function as a critical fine-tuning modulatory component of yeast silent SIR-nucleosome pre-heterochromatin by stabilizing the intermolecular interaction between Sir3 N- and C-terminal regions.
Recruitment and allosteric stimulation of a histone-deubiquitinating enzyme during heterochromatin assembly
2018, Journal of Biological Chemistry
Citation Excerpt :
Three general steps in establishing heterochromatin involve the following: 1) recruitment of the silencing complex to the target locus; 2) nucleation via histone-modifying activities; and 3) spreading of the silencing complexes due to iterative cycles of recruitment and nucleation. In budding yeast, silent information regulator (SIR) complex–dependent heterochromatin is a well-studied and simplified silencing model (13–15). The SIR complex is composed of three proteins, Sir2, Sir3, and Sir4.
Heterochromatin formation in budding yeast is regulated by the silent information regulator (SIR) complex. The SIR complex comprises the NAD-dependent deacetylase Sir2, the scaffolding protein Sir4, and the nucleosome-binding protein Sir3. Transcriptionally active regions present a challenge to SIR complex–mediated de novo heterochromatic silencing due to the presence of antagonistic histone post-translational modifications, including acetylation and methylation. Methylation of histone H3K4 and H3K79 is dependent on monoubiquitination of histone H2B (H2B-Ub). The SIR complex cannot erase H2B-Ub or histone methylation on its own. The deubiquitinase (DUB) Ubp10 is thought to promote heterochromatic silencing by maintaining low H2B-Ub at sub-telomeres. Here, we biochemically characterized the interactions between Ubp10 and the SIR complex machinery. We demonstrate that a direct interaction between Ubp10 and the Sir2/4 sub-complex facilitates Ubp10 recruitment to chromatin via a co-assembly mechanism. Using hydrolyzable H2B-Ub analogs, we show that Ubp10 activity is lower on nucleosomes compared with H2B-Ub in solution. We find that Sir2/4 stimulates Ubp10 DUB activity on nucleosomes, likely through a combination of targeting and allosteric regulation. This coupling mechanism between the silencing machinery and its DUB partner allows erasure of active PTMs and the de novo transition of a transcriptionally active DNA region to a silent chromatin state.
Human sirtuins: Structures and flexibility
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Citation Excerpt :
In 1987, Rine et al. identified four proteins responsible for the silencing of the Hidden MAT loci of Saccharomyces cerevisiae, implicated in the mating process of the yeast (Rine and Herskowitz, 1987). These four proteins, called silent information regulatory (Sir) proteins, have been found to bind to the nucleosome through interactions with histone proteins H3 and H4, and to repress gene expression (Johnson et al., 1990; Murphy et al., 2013; Cubizolles et al., 2006; Ehrentraut et al., 2011; Oppikofer et al., 2013). Sir proteins have aroused great hope when it was found that they may participate in extending the yeast life span (Lin et al., 2004).
In recent years, sirtuins (SIRTs), members of histone deacetylases (HDACs) class III, have been found to modulate cellular processes related to the development of human aging-related pathologies (i.e. cancer, neurodegeneration, metabolic disorders). Several crystallographic structures and computational studies have shed light into their catalytic mechanism of action, identifying also the structural elements for the design of selective drug candidates. In this review, we first aim at summarizing the structural features characterizing human SIRTs. We then describe the observed mass and one-off movements related to conformational changes upon SIRT-mediated recognition events. Such information will be useful not only for rationalizing the design of new SIRT modulators, but also for improving the comprehension of SIRT-related biological roles.
The Dynamics of Histone Modifications During Aging
2016, Epigenomics in Health and Disease
Aging is a biologic process characterized by a progressive loss of functionality leading to increased sensitivity to pathology. As more knowledge is gained through the study of the epigenome, we are quickly realizing that dramatic changes in the chromatin fiber accompany the aging process. Since most studies correlate changes in histone density or modifications with the aging process, distinguishing between driver and passenger events remains challenging. In this chapter, we will discuss how changes in histone density, histone variants, and histone modifications either drive or accompany the aging process. We will also discuss how the DNA damage response and cellular senescence link histone modifications to aging phenotypes. Finally, we will discuss the contribution of histone modifications in progeria. Overall, we hypothesize that through their direct involvement in age-related phenotypes, histone modifications may represent an intriguing therapeutic target for the treatment of premature and normal aging.
Stabilization of mini-chromosome segregation during mitotic growth by overexpression of YCR041W and its application to chromosome engineering inSaccharomyces cerevisiae
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Citation Excerpt :
Because the stabilizing effect was not observed in the absence of Sir4, however, it seems likely that segregation processes related to telomere function are involved in the stabilization of mini-chromosomes by YCR041W and ZDS1. In the budding yeast, the Sir complex has two physiological roles at the telomere: (i) formation of heterochromatin and silencing (29), and (ii) anchoring telomeres to the nuclear envelope and telomere clustering, thereby stabilizing the chromosome (30). Among the Sir proteins, Sir2 is involved only in heterochromatin formation, whereas Sir3 and Sir4 are involved in both heterochromatin formation and telomere anchoring and clustering (30–32).
Chromosome engineering enables large-scale genome manipulation and can be used as a novel technology for breeding of yeasts. PCR-mediated chromosome splitting (PCS) offers a powerful tool for chromosome engineering by enabling a yeast chromosome to be split at any desired site. By applying PCS, a huge variety of chromosome combinations can be created and the best strain under specific conditions can be selected—a technology that we have called genome reorganization. Once the optimal strain is obtained, chromosome constitutions need to be maintained stably; however, mini-chromosomes of less than 50 kb are at relatively high frequency lost during cultivation. To overcome this problem, in this study we screened for multicopy suppressors of the high loss of mini-chromosomes by using a multicopy genomic library of Saccharomyces cerevisiae. We identified a novel gene, YCR041W, that stabilizes mini-chromosomes. The translational product of YCR041W was suggested to play an important role in increasing stability for mini-chromosome maintenance, probably by decreasing the rate of loss during mitotic cell division. The stabilization of mini-chromosomes conferred by YCR041W overexpression was completely dependent on the silencing protein Sir4, suggesting that a process related to telomere function might be involved in mini-chromosome stabilization. Overexpression of YCR041W stabilized not only a yeast artificial chromosome vector, but also a mini-chromosome derived from a natural chromosome. Taking these results together, we propose that YCR041W overexpression can be used as a novel chromosome engineering tool for controlling mini-chromosome maintenance and loss.

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¹: Current address: Department of Early Discovery Biochemistry, Genentech Research and Early Development, 1 DNA Way, South San Francisco, CA 94080, USA.

²: Current address: F. Hoffmann-La Roche Ltd. Pharmaceuticals Division, Grenzacherstrasse 124, 4070 Basel, Switzerland.

View full text

ReviewSIR–nucleosome interactions: Structure–function relationships in yeast silent chromatin

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Structural aspects of the chromatin fiber

Chromatin regulates gene expression

Silent chromatin in S. cerevisiae

Outlook

Conflict of interest

Acknowledgments

Mol. Cell

Mol. Cell

Cell

Electron Microsc. Rev.

Cell

Cell

Cell

J. Biol. Chem.

Curr. Biol.

FEBS Lett.

Structure

Cell

Genome Res.

Mol. Cell

Cell

Mol. Cell

J. Mol. Biol.

Micron

J. Mol. Biol.

J. Mol. Biol.

Curr. Biol.

Trends Biochem. Sci.

Cell

Gene

Trends Genet.

J. Mol. Biol.

Cell

Cell

Exp. Cell Res.

Cell

J. Biol. Chem.

Reciprocal regulation of anaerobic and aerobic cell wall mannoprotein gene expression in Saccharomyces cerevisiae

J. Bacteriol.

Acetylation and methylation of histones and their possible role in the regulation of rna synthesis

Proc. Natl. Acad. Sci. U. S. A.

Nucleosome structure(s) and stability: variations on a theme

Annu. Rev. Biophys.

Perinuclear localization of chromatin facilitates transcriptional silencing

Nature

Esc1, a nuclear periphery protein required for Sir4-based plasmid anchoring and partitioning

Mol. Cell. Biol.

Structural basis of silencing: Sir3 BAH domain in complex with a nucleosome at 3.0 A resolution

Science

Regulation of chromatin by histone modifications

Cell Res.

Silencing chromatin: comparing modes and mechanisms

Nat. Rev. Genet.

Yeast origin recognition complex functions in transcription silencing and DNA replication

Science

Determinants and dynamics of genome accessibility

Nat. Rev. Genet.

Visualization of G1 chromosomes: a folded, twisted, supercoiled chromonema model of interphase chromatid structure

J. Cell Biol.

Components of the Ku-dependent non-homologous end-joining pathway are involved in telomeric length maintenance and telomeric silencing

EMBO J.

Transcriptional silencing in yeast is associated with reduced nucleosome acetylation

Genes Dev.

A map of nucleosome positions in yeast at base-pair resolution

Nature

Rapid accessibility of nucleosomal DNA in yeast on a second time scale

EMBO J.

Sir3–nucleosome interactions in spreading of silent chromatin in Saccharomyces cerevisiae

Mol. Cell. Biol.

Two DNA-binding factors recognize specific sequences at silencers, upstream activating sequences, autonomously replicating sequences, and telomeres in Saccharomyces cerevisiae

Mol. Cell. Biol.

Action of a RAP1 carboxy-terminal silencing domain reveals an underlying competition between HMR and telomeres in yeast

Genes Dev.

In silicio identification of glycosyl-phosphatidylinositol-anchored plasma-membrane and cell wall proteins of Saccharomyces cerevisiae

Review
SIR–nucleosome interactions: Structure–function relationships in yeast silent chromatin