Distinguishing between recruitment and spread of silent chromatin structures in Saccharomyces cerevisiae

The formation of heterochromatin at HML, HMR, and telomeres in Saccharomyces cerevisiae involves two main steps: Recruitment of Sir proteins to silencers and their spread throughout the silenced domain. We developed a method to study these two processes at single base-pair resolution. Using a fusion protein between the heterochromatin protein Sir3 and the non-site-specific bacterial adenine methyltransferase M.EcoGII, we mapped sites of Sir3-chromatin interactions genome-wide using long-read Nanopore sequencing to detect adenines methylated by the fusion protein. A silencing-deficient mutant of Sir3 lacking its Bromo-Adjacent Homology (BAH) domain, sir3-bahΔ, was still recruited to HML, HMR, and telomeres. However, in the absence of the BAH domain, it was unable to spread away from those recruitment sites. Overexpression of Sir3 did not lead to further spreading at HML, HMR, and most telomeres. A few exceptional telomeres, like 6R, exhibited a small amount of Sir3 spreading, suggesting that boundaries at telomeres responded variably to Sir3 overexpression. Finally, by using a temperature-sensitive allele of SIR3 fused to M.ECOGII, we tracked the positions first methylated after induction and found that repression of genes at HML and HMR began before Sir3 occupied the entire locus.

SWI/SNF antagonizes SIR heterochromatin to promote transcription of genes expressed during mitotic exit in Saccharomyces cerevisiae

ABSTRACTHeterochromatin is a repressive, specialized chromatin structure that is central to eukaryotic transcriptional regulation and genome stability. In the budding yeast, Saccharomyces cerevisiae, heterochromatin formation requires Sir2p, Sir3p, and Sir4p, and these Sir proteins create specialized chromatin structures at telomeres and silent mating type loci. Previously, we reported that the SWI/SNF chromatin remodeling enzyme can evict Sir3 from chromatin fibers in vitro, though whether this activity contributes to the role of SWI/SNF as a transcriptional activator at euchromatic loci is unknown. Here, we characterize genetic interactions between the SIR genes (SIR2, SIR3, and SIR4) and genes encoding subunits of the chromatin remodelers SWI/SNF and INO80C, as well genes encoding the histone deacetylases Hst3 and Hst4. We find that loss of SIR genes partially rescues the growth defects of swi2, ino80, and hst3/hst4 mutants during replication stress conditions. Interestingly, partial suppression of swi2, ino80, and hst3 hst4 mutant phenotypes is due to the pseudo-diploid state of sir mutants, but a significant portion is due to more direct functional interactions. Consistent with this view, transcriptional profiling of strains lacking Swi2 or Sir3 identifies a set of genes whose expression in the M/G1 phase of the cell cycle requires SWI/SNF to antagonize the repressive impact of Sir3.

SIR proteins create compact heterochromatin fibers

Heterochromatin is a silenced chromatin region essential for maintaining genomic stability and driving developmental processes. The complicated structure and dynamics of heterochromatin have rendered it difficult to characterize. In budding yeast, heterochromatin assembly requires the SIR proteins—Sir3, believed to be the primary structural component of SIR heterochromatin, and the Sir2–4 complex, responsible for the targeted recruitment of SIR proteins and the deacetylation of lysine 16 of histone H4. Previously, we found that Sir3 binds but does not compact nucleosomal arrays. Here we reconstitute chromatin fibers with the complete complement of SIR proteins and use sedimentation velocity, molecular modeling, and atomic force microscopy to characterize the stoichiometry and conformation of SIR chromatin fibers. In contrast to fibers with Sir3 alone, our results demonstrate that SIR arrays are highly compact. Strikingly, the condensed structure of SIR heterochromatin fibers requires both the integrity of H4K16 and an interaction between Sir3 and Sir4. We propose a model in which a dimer of Sir3 bridges and stabilizes two adjacent nucleosomes, while a Sir2–4 heterotetramer interacts with Sir3 associated with a nucleosomal trimer, driving fiber compaction.

SIR proteins create compact heterochromatin fibers

SummaryHeterochromatin is a silenced chromatin region essential for maintaining genomic stability and driving developmental processes. The complicated structure and dynamics of heterochromatin have rendered it difficult to characterize. In budding yeast, heterochromatin assembly requires the SIR proteins -- Sir3, believed to be the primary structural component of SIR heterochromatin, and the Sir2/4 complex, responsible for the targeted recruitment of SIR proteins and the deacetylation of lysine 16 of histone H4. Previously, we found that Sir3 binds but does not compact nucleosomal arrays. Here we reconstitute chromatin fibers with the complete complement of SIR proteins and use sedimentation velocity, molecular modeling, and atomic force microscopy to characterize the stoichiometry and conformation of SIR chromatin fibers. In contrast to previous studies, our results demonstrate that SIR arrays are highly compact. Strikingly, the condensed structure of SIR heterochromatin fibers requires both the integrity of H4K16 and an interaction between Sir3 and Sir4. We propose a model in which two molecules of Sir3 bridge and stabilize two adjacent nucleosomes, while a single Sir2/4 heterodimer binds the intervening linker DNA, driving fiber compaction.

Modulations of SIR-nucleosome interactions of reconstructed yeast silent pre-heterochromatin by O-acetyl-ADP-ribose and magnesium

Yeast silent heterochromatin provides an excellent model with which to study epigenetic inheritance. Previously we developed an in vitro assembly system to demonstrate the formation of filament structures with requirements that mirror yeast epigenetic gene silencing in vivo. However, the properties of these filaments were not investigated in detail. Here we show that the assembly system requires Sir2, Sir3, Sir4, nucleosomes, and O-acetyl-ADP-ribose. We also demonstrate that all Sir proteins and nucleosomes are components of these filaments to prove that they are SIR-nucleosome filaments. Furthermore, we show that the individual localization patterns of Sir proteins on the SIR-nucleosome filament reflect those patterns on telomeres in vivo. In addition, we reveal that magnesium exists in the SIR-nucleosome filament, with a role similar to that for chromatin condensation. These results suggest that a small number of proteins and molecules are sufficient to mediate the formation of a minimal yeast silent pre-heterochromatin in vitro.

Heterochromatin assembly by interrupted Sir3 bridges across neighboring nucleosomes

Heterochromatin is a conserved feature of eukaryotic chromosomes with central roles in regulation of gene expression and maintenance of genome stability. Heterochromatin formation involves spreading of chromatin-modifying factors away from initiation points over large DNA domains by poorly understood mechanisms. In Saccharomyces cerevisiae, heterochromatin formation requires the SIR complex, which contains subunits with histone-modifying, histone-binding, and self-association activities. Here, we analyze binding of the Sir proteins to reconstituted mono-, di-, tri-, and tetra-nucleosomal chromatin templates and show that key Sir-Sir interactions bridge only sites on different nucleosomes but not sites on the same nucleosome, and are therefore 'interrupted' with respect to sites on the same nucleosome. We observe maximal binding affinity and cooperativity to unmodified di-nucleosomes and propose that nucleosome pairs bearing unmodified histone H4-lysine16 and H3-lysine79 form the fundamental units of Sir chromatin binding and that cooperative binding requiring two appropriately modified nucleosomes mediates selective Sir recruitment and spreading.

Evolution and Functional Trajectory of Sir1 in Gene Silencing

We used the budding yeastsSaccharomyces cerevisiaeandTorulaspora delbrueckiito examine the evolution of Sir-based silencing, focusing on Sir1, silencers, the molecular topography of silenced chromatin, and the roles ofSIRand RNA interference (RNAi) genes inT. delbrueckii. Chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) analysis of Sir proteins inT. delbrueckiirevealed a different topography of chromatin at theHMLandHMRloci than was observed inS. cerevisiae. S. cerevisiaeSir1, enriched at the silencers ofHMLα andHMRa, was absent from telomeres and did not repress subtelomeric genes. In contrast toS. cerevisiaeSIR1's partially dispensable role in silencing, theT. delbrueckiiSIR1paralogKOS3was essential for silencing.KOS3was also found at telomeres withT. delbrueckiiSir2 (Td-Sir2) and Td-Sir4 and repressed subtelomeric genes. Silencer mapping inT. delbrueckiirevealed single silencers atHMLandHMR, bound by Td-Kos3, Td-Sir2, and Td-Sir4. TheKOS3gene mapped nearHMR, and its expression was regulated by Sir-based silencing, providing feedback regulation of a silencing protein by silencing. In contrast to the prominent role of Sir proteins in silencing,T. delbrueckiiRNAi genesAGO1andDCR1did not function in heterochromatin formation. These results highlighted the shifting role of silencing genes and the diverse chromatin architectures underlying heterochromatin.

Heterochromatin formation via recruitment of DNA repair proteins

Heterochromatin formation and nuclear organization are important in gene regulation and genome fidelity. Proteins involved in gene silencing localize to sites of damage and some DNA repair proteins localize to heterochromatin, but the biological importance of these correlations remains unclear. In this study, we examined the role of double-strand-break repair proteins in gene silencing and nuclear organization. We find that the ATM kinase Tel1 and the proteins Mre11 and Esc2 can silence a reporter gene dependent on the Sir, as well as on other repair proteins. Furthermore, these proteins aid in the localization of silenced domains to specific compartments in the nucleus. We identify two distinct mechanisms for repair protein–mediated silencing—via direct and indirect interactions with Sir proteins, as well as by tethering loci to the nuclear periphery. This study reveals previously unknown interactions between repair proteins and silencing proteins and suggests insights into the mechanism underlying genome integrity.

Reinventing Heterochromatin in Budding Yeasts: Sir2 and the Origin Recognition Complex Take Center Stage

ABSTRACTThe transcriptional silencing of the cryptic mating-type loci inSaccharomyces cerevisiaeis one of the best-studied models of repressive heterochromatin. However, this type of heterochromatin, which is mediated by the Sir proteins, has a distinct molecular composition compared to the more ubiquitous type of heterochromatin found inSchizosaccharomyces pombe, other fungi, animals, and plants and characterized by the presence of HP1 (heterochromatin protein 1). This review discusses how the loss of important heterochromatin proteins, including HP1, in the budding yeast lineage presented an evolutionary opportunity for the development and diversification of alternative varieties of heterochromatin, in which the conserved deacetylase Sir2 and the replication protein Orc1 play key roles. In addition, we highlight how this diversification has been facilitated by gene duplications and has contributed to adaptations in lifestyle.