High-Resolution Structural Analysis of Chromatin at Specific Loci: Saccharomyces cerevisiae Silent Mating Type Locus HMLα

Abstract

Genetic studies have suggested that chromatin structure is involved in repression of the silent mating type loci in Saccharomyces cerevisiae. Chromatin mapping at nucleotide resolution of the transcriptionally silent HML␣ and the active MAT␣ shows that unique organized chromatin structure characterizes the silent state of HML␣. Precisely positioned nucleosomes abutting the silencers extend over the ␣1 and ␣2 coding regions. The HO endonuclease recognition site, nuclease hypersensitive at MAT␣, is protected at HML␣. Although two precisely positioned nucleosomes incorporate transcription start sites at HML␣, the promoter region of the ␣1 and ␣2 genes is nucleosome free and more nuclease sensitive in the repressed than in the transcribed locus. Mutations in genes essential for HML silencing disrupt the nucleosome array near HML-I but not in the vicinity of HML-E, which is closer to the telomere of chromosome III. At the promoter and the HO site, the structure of HML␣ in Sir protein and histone H4 N-terminal deletion mutants is identical to that of the transcriptionally active MAT␣. The discontinuous chromatin structure of HML␣ contrasts with the continuous array of nucleosomes found at repressed a-cell-specific genes and the recombination enhancer. Punctuation at HML␣ may be necessary for higher-order structure or karyoskeleton interactions. The unique chromatin architecture of HML␣ may relate to the combined requirements of transcriptional repression and recombinational competence.Transcriptional repression of the silent-mating-type loci is fundamental for the haploid yeast life cycle. The a or ␣ mating type is determined by expression of master regulatory genes of the active MAT locus near the centromere of chromosome III. Identical genes present at the HM (haploid mating) loci near the telomeres of the same chromosome, HML carrying ␣ information and HMR bearing a information, are not transcribed, thus preserving the unique mating type. The HM loci serve as donors during the gene interconversion event that allows a homothallic haploid cell to switch mating type, ensuring a diploid population in the wild. In addition to transcriptional repression, the DNA of the silenced HM loci is protected from HO endonuclease, which makes a double-strand break at the MAT HO site to initiate mating-type switching (40,46,55,75).Silencing of the HM-mating-type loci in Saccharomyces cerevisiae is remarkably similar to long-term, epigenetic inactivation of specific genomic domains in complex eukaryotes. Xchromosome inactivation (33) and gene imprinting (82) in mammalian cells, telomeric silencing (22) in yeast, and position effect variegation (reviewed by Henikoff [28]) in Drosophila melanogaster are examples of such parallel situations. Epigenetic states are thought to be achieved by chromosomal condensation into heterochromatin. The molecular events leading to such position-dependent, gene-independent transcriptional repression of a chromosomal region are not well understood. Silencing mechanisms in yeast are likely to involve a r...

Asymmetric Positioning of Nucleosomes and Directional Establishment of Transcriptionally Silent Chromatin by Saccharomyces cerevisiae Silencers

“…On the Abf1p side, three consecutive nucleosomes could be inferred (Fig. 4A, right, filled ellipses labeled 1 to 3), which is consistent with data of chromatin mapping on the Abf1p side of native HML-I obtained by Weiss and Simpson (39). On the other hand, no stable nucleosomes were located immediately adjacent to ACS (Fig.…”

Dual Chromatin Remodeling Roles for RSC during DNA Double Strand Break Induction and Repair at the Yeast MAT Locus

“…The structure we determined for the MAT␣ locus prior to HO induction is entirely consistent with previous work comparing regions of the locus (such as the ␣1/␣2 divergent promoter) with its silent counterpart HML, which showed Sir protein-dependent structures involved in transcriptional repression and occlusion of the HO site (27,28). Upon induction of the HO endonuclease, we observed a highly localized chromatin remodeling event concomitantly with break formation.…”

Mating-Type Genes andMATSwitching inSaccharomyces cerevisiae

“…Among the trans-acting proteins that play critical roles in this process are four Silent Information Regulator (Sir) proteins, a set of silencer binding proteins, histone proteins, the multipurpose Rap1 protein, as well as several chromatin modifiers. Together, these gene products and cis-acting sequences create short regions (3 kb) of heterochromatin, in which the DNA sequences of HML and HMR are found as a highly ordered nucleosome structure (Nasmyth 1982;Weiss and Simpson 1998;Ravindra et al 1999) (Figure 4B). These heterochromatic regions are transcriptionally silent for both PolII-and PolIII-transcribed genes (Brand et al 1985;Schnell and Rine 1986) and resistant to cleavage by several endogenously expressed endonucleases, including the HO endonuclease (Connolly et al 1988;Loo and Rine 1994).…”