Reconstitution of Yeast Silent Chromatin: Multiple Contact Sites and O-AADPR Binding Load SIR Complexes onto Nucleosomes In Vitro

Abstract

At yeast telomeres and silent mating-type loci, chromatin assumes a higher-order structure that represses transcription by means of the histone deacetylase Sir2 and structural proteins Sir3 and Sir4. Here, we present a fully reconstituted system to analyze SIR holocomplex binding to nucleosomal arrays. Purified Sir2-3-4 heterotrimers bind chromatin, cooperatively yielding a stable complex of homogeneous molecular weight. Remarkably, Sir2-3-4 also binds naked DNA, reflecting the strong, albeit nonspecific, DNA-binding activity of Sir4. The binding of Sir3 to nucleosomes is sensitive to histone H4 N-terminal tail removal, while that of Sir2-4 is not. Dot1-mediated methylation of histone H3K79 reduces the binding of both Sir3 and Sir2-3-4. Additionally, a byproduct of Sir2-mediated NAD hydrolysis, O-acetyl-ADP-ribose, increases the efficiency with which Sir3 and Sir2-3-4 bind nucleosomes. Thus, in small cumulative steps, each Sir protein, unmodified histone domains, and contacts with DNA contribute to the stability of the silent chromatin complex.

Rpd3-dependent boundary formation at telomeres by removal of Sir2 substrate

“…We observed that mutations in the putative OAADPR binding site of Sir3 abrogate its silencing function and reduce its ability to interact with itself and Sir4 and spread on chromatin, thus providing experimental support for the notion that binding of OAADPR to Sir3 carries out an important function in heterochromatin formation. This notion is in agreement with a recent study that showed increased in vitro binding of the SIR complex to chromatin in the presence of OAADPR (39). Perhaps the metabolite is necessary for SIR-SIR complex interactions, which are important for propagation of heterochromatin along the chromatin fiber.…”

A Prototypic Lysine Methyltransferase 4 from Archaea with Degenerate Sequence Specificity Methylates Chromatin Proteins Sul7d and Cren7 in Different Patterns

“…Nevertheless, Dot1 participates in heterochromatin formation through competing with Sir3 for a binding site on histone H4 (13). In addition, H3K79 methylation could reduce the affinity of SIR complex for chromatin (4,14). Mammalian Dot1L has been shown to play crucial roles not only in transcription, cell cycle regulation, and DNA damage response, but also in embryogenesis, development, and differentiation (4).…”

Symmetry, asymmetry, and kinetics of silencing establishment in Saccharomyces cerevisiae revealed by single-cell optical assays

“…HML and HMR loci are constitutively repressed in wild-type cells. However, conditional alleles of the SIR genes have led to a greater understanding of heterochromatin establishment, loss, and maintenance (5)(6)(7)(8)(9). Investigating the dynamics of transitions between heterochromatin and euchromatin in mutants with altered chromatin structure should reveal how chromatin modifications impact the formation of these two states.…”

“…The apparent spreading of Sir proteins from silencers requires Sir2-dependent deacetylation of the critical histone residue H4 K16 and possibly other acetylated lysines as well (3,(11)(12)(13)(14). Because nucleosomes composed of unacetylated histones have a higher affinity for Sir protein complexes, histone deacetylation promotes the localization of Sir proteins throughout HML and HMR, leading to loss of transcription at those loci (9,15,16).…”

“…This particular lysine is located on the loss of rDNA silencing (LRS) face of H3, a surface whose electrostatic properties are important for association between the nucleosome and the BAH domain of Sir3 (32)(33)(34)(35)(36). H3 K79 methylation, therefore, interferes with the nucleosome's ability to adequately bind Sir3 (9). Dot1 can impact silencing formation in a second manner.…”