Chromatin immunoprecipitation (ChIP) is the gold-standard technique for localizing nuclear proteins in the genome. We used ChIP, in combination with deep sequencing (Seq), to study the genomewide distribution of the Silent information regulator (Sir) complex in Saccharomyces cerevisiae. We analyzed ChIP-Seq peaks of the Sir2, Sir3, and Sir4 silencing proteins and discovered 238 unexpected euchromatic loci that exhibited enrichment of all three. Surprisingly, published ChIP-Seq datasets for the Ste12 transcription factor and the centromeric Cse4 protein indicated that these proteins were also enriched in the same euchromatic regions with the high Sir protein levels. The 238 loci, termed "hyper-ChIPable", were in highly expressed regions with strong polymerase II and polymerase III enrichment signals, and the correlation between transcription level and ChIP enrichment was not limited to these 238 loci but extended genome-wide. The apparent enrichment of various proteins at hyper-ChIPable loci was not a consequence of artifacts associated with deep sequencing methods, as confirmed by ChIP-quantitative PCR. The localization of unrelated proteins, including the entire silencing complex, to the most highly transcribed genes was highly suggestive of a technical issue with the immunoprecipitations. ChIP-Seq on chromatin immunoprecipitated with a nuclear-localized GFP reproduced the above enrichment in an expression-dependent manner: induction of the GAL genes resulted in an increased ChIP signal of the GFP protein at these loci, with presumably no biological relevance. Whereas ChIP is a broadly valuable technique, some published conclusions based upon ChIP procedures may merit reevaluation in light of these findings.ChIP-chip | HOT regions | yeast | tRNA C hromatin immunoprecipitation, followed either by microarrays (ChIP-chip) or deep sequencing (ChIP-Seq) is the standard method for in vivo genome-wide protein localization analysis (reviewed in refs. 1-3). Since the first applications of deep sequencing to ChIP in 2007, the ChIP-Seq technique has quickly become accepted as superior to ChIP-chip hybridization and is now the dominant and most preferred approach for studying DNA-and chromatin-interacting proteins (2, 4, 5). Because of known biases in chromatin preparation and sequencing, nearly all ChIP-Seq studies compare the mapped reads of the immunoprecipiated (IP) sample to an input control with chromatin that is cross-linked but not immunoprecipitated (2, 5, 6). We applied ChIP-Seq to study the distribution of the silencing protein complex, consisting of Sir2, Sir3, and Sir4, in Saccharomyces cerevisiae. Unexpectedly, the well-characterized biology of silencing enabled the resulting data to illuminate a technical artifact introduced by the ChIP technique.Silencing in S. cerevisiae is established by the Sir2, Sir3, and Sir4 protein complex that binds and deacetylates key positions on nucleosomes, forming a heterochromatic structure that inhibits transcription (reviewed in ref. 7). Prior work raised the possibility th...

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 ...

Heterochromatin imparts regional, promoter-independent repression of genes and is epigenetically heritable. Understanding how silencing achieves this regional repression is a fundamental problem in genetics and development. Current models of yeast silencing posit that Sir proteins, recruited by transcription factors bound to the silencers, spread throughout the silenced region. To test this model directly at high resolution, we probed the silenced chromatin architecture by chromatin immunoprecipitation (ChIP) followed by next-generation sequencing (ChIP-seq) of Sir proteins, histones, and a key histone modification, H4K16-acetyl. These analyses revealed that Sir proteins are strikingly concentrated at and immediately adjacent to the silencers, with lower levels of enrichment over the promoters at HML and HMR, the critical targets for transcriptional repression. The telomeres also showed discrete peaks of Sir enrichment yet a continuous domain of hypoacetylated histone H4K16. Surprisingly, ChIP-seq of cross-linked chromatin revealed a distribution of nucleosomes at silenced loci that was similar to Sir proteins, whereas native nucleosome maps showed a regular distribution throughout silenced loci, indicating that cross-linking captured a specialized chromatin organization imposed by Sir proteins. This specialized chromatin architecture observed in yeast informs the importance of a steric contribution to regional repression in other organisms.