Saccharomyces cerevisiae telomeres have been a paradigm for studying telomere position effects on gene expression. Telomere position effect was first described in yeast by its effect on the expression of reporter genes inserted adjacent to truncated telomeres. The reporter genes showed variable silencing that depended on the Sir2/3/4 complex. Later studies examining subtelomeric reporter genes inserted at natural telomeres hinted that telomere position effects were less pervasive than previously thought. Additionally, more recent data using the sensitive technology of chromatin immunoprecipitation and massively parallel sequencing (ChIP-Seq) revealed a discrete and noncontinuous pattern of coenrichment for all three Sir proteins at a few telomeres, calling the generality of these conclusions into question. Here we combined the ChIP-Seq of the Sir proteins with RNA sequencing (RNA-Seq) of messenger RNAs (mRNAs) in wild-type and in SIR2, SIR3, and SIR4 deletion mutants to characterize the chromatin and transcriptional landscape of all native S. cerevisiae telomeres at the highest achievable resolution. Most S. cerevisiae chromosomes had subtelomeric genes that were expressed, with only 6% of subtelomeric genes silenced in a SIR-dependent manner. In addition, we uncovered 29 genes with previously unknown cell-type-specific patterns of expression. These detailed data provided a comprehensive assessment of the chromatin and transcriptional landscape of the subtelomeric domains of a eukaryotic genome.KEYWORDS Sir complex; telomeres; ChIP-Seq; RNA-Seq; mating-type regulation T ELOMERES are specialized structures at the ends of eukaryotic chromosomes that are critical for various biological functions. Telomeres bypass the problem of replicating the ends of linear DNA, protect chromosome ends from exonucleases and nonhomologous end joining, prevent the linear DNA ends from activating a DNA-damage checkpoint, and exhibit suppressed recombination [reviewed in Wellinger and Zakian (2012)]. In Saccharomyces cerevisiae, telomeres are composed of three sequence features: telomeric repeats, which consist of 300 6 75 bp of (TG 1-3 ) n repeated units produced by telomerase; X elements; and Y9 elements, which contain an ORF for a putative helicase gene. The X elements are subdivided into a core X [consisting of an autonomously replicating sequence (ARS) consensus sequence and an Abf1-binding site] and subtelomeric repeats that have variable numbers of repeated units containing a binding site for Tbf1 (Louis 1995). All telomeres contain telomeric repeats plus an X element, and about half of S. cerevisiae's 32 telomeres also contain a Y9 element (X-Y9 telomeres). X-only telomeres contain an X element but not a Y9 element. Unlike the Y9 elements, the telomeric repeats and X elements are bound by proteins that are critical for maintenance of telomeres. Rap1 binds the TG 1-3 telomeric repeats and recruits the Sir2/3/4 protein complex, the trio of heterochromatin structural proteins critical for repression of the silent mating ...

Yta7 is a highly conserved bromodomain-containing protein with AAA-ATPase homology originally implicated in heterochromatin boundary function in Saccharomyces cerevisiae. Although increased activity of the human ortholog has been implicated in malignant breast tumors, Yta7's precise mode of action is unknown. Transcriptional analysis in yeast cells revealed a role for Yta7 and its ATPase function in gene induction, including galactose-and sporulation-induced transcription. This requirement was direct and activating, because Yta7 associated with the GAL gene cluster only upon transcriptional induction. Suggestive of a role in transcriptional elongation, Yta7 localized to the ORFs of highly transcribed genes. Intriguingly, the yta7Δ mutant's transcriptional defects were partially suppressed by decreased dosage of histones H3 and H4. Consistent with this suppression, cells lacking Yta7 exhibited both increased levels of chromatin-incorporated histone H3 and decreased nucleosome spacing. Importantly, this modulation of H3 levels occurred independently of changes in H3 transcript level. Because Yta7 binds histone H3 in vitro, these results suggested a direct role for Yta7 in H3 eviction or degradation. Further, local loss of Yta7 activity at a long inducible gene resulted in accumulation of H3 at the 3′ end upon transcriptional activation, implying Yta7 may regulate H3 cotranscriptionally.ANCCA | H2A.Z

We used the budding yeasts Saccharomyces cerevisiae and Torulaspora delbrueckii to examine the evolution of Sir-based silencing, focusing on Sir1, silencers, the molecular topography of silenced chromatin, and the roles of SIR and RNA interference (RNAi) genes in T. delbrueckii. Chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) analysis of Sir proteins in T. delbrueckii revealed a different topography of chromatin at the HML and HMR loci than was observed in S. cerevisiae. S. cerevisiae Sir1, enriched at the silencers of HML␣ and HMRa, was absent from telomeres and did not repress subtelomeric genes. In contrast to S. cerevisiae SIR1's partially dispensable role in silencing, the T. delbrueckii SIR1 paralog KOS3 was essential for silencing. KOS3 was also found at telomeres with T. delbrueckii Sir2 (Td-Sir2) and Td-Sir4 and repressed subtelomeric genes. Silencer mapping in T. delbrueckii revealed single silencers at HML and HMR, bound by Td-Kos3, Td-Sir2, and Td-Sir4. The KOS3 gene mapped near HMR, 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. delbrueckii RNAi genes AGO1 and DCR1 did not function in heterochromatin formation. These results highlighted the shifting role of silencing genes and the diverse chromatin architectures underlying heterochromatin.