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. 2023 Oct 3;120(40):e2304343120.
doi: 10.1073/pnas.2304343120. Epub 2023 Sep 28.

Context-dependent function of the transcriptional regulator Rap1 in gene silencing and activation in Saccharomyces cerevisiae

Eliana R Bondra  1 , Jasper Rine  1
Affiliations

Context-dependent function of the transcriptional regulator Rap1 in gene silencing and activation in Saccharomyces cerevisiae

Eliana R Bondra et al. Proc Natl Acad Sci U S A. .

Abstract

In Saccharomyces cerevisiae, heterochromatin is formed through interactions between site-specific DNA-binding factors, including the transcriptional activator Repressor Activator Protein (Rap1), and Sir proteins. Despite an understanding of the establishment and maintenance of Sir-silenced chromatin, the mechanism of gene silencing by Sir proteins has remained a mystery. Utilizing high-resolution chromatin immunoprecipitation, we found that Rap1, the native activator of the bidirectional HMLα promoter, bound its recognition sequence in silenced chromatin, and its binding was enhanced by the presence of Sir proteins. In contrast to prior results, various components of transcription machinery were not able to access HMLα in the silenced state. These findings disproved the long-standing model of indiscriminate steric occlusion by Sir proteins and led to investigation of the role of the transcriptional activator Rap1 in Sir-silenced chromatin. Using a highly sensitive assay that monitors loss-of-silencing events, we identified a role for promoter-bound Rap1 in the maintenance of silent chromatin through interactions with the Sir complex. We also found that promoter-bound Rap1 activated HMLα when in an expressed state, and aided in the transition from transcription initiation to elongation. Highlighting the importance of epigenetic context in transcription factor function, these results point toward a model in which the duality of Rap1 function was mediated by local chromatin environment rather than binding-site availability.

Keywords: chromatin; epigenetics; gene silencing.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
Rap1 bound the promoter of HML in a silenced state but failed to recruit the preinitiation complex. For all ChIP-seq experiments, read counts were normalized to the nonheterochromatic genome-wide median. IP, input, and untagged control values are plotted on the same scale. Data shown are the average of two ChIP-seq experiments unless otherwise noted. (A) Schematic of HMLα and MATα on chromosome III. Rap1-binding sites at HML-EHML-I, and the promoter of both HML and MAT are noted. (B, Left) averaged normalized reads for ChIP-seq in two 3xV5-Rap1 samples at HML in SIR cells. Black bars represent 200 bp surrounding Rap1-binding sites at HML-E, HML-p, and HML-I, respectively. (Middle) same as left but showing MAT. (Right) same as (Left, Middle) but at RPS4A. (C). Same as B but for Taf1-3xFLAG-KanMX. (D) Same as B and C but for Rpb3-3xFLAG-KanMX.
Fig. 2.
Fig. 2.
Rap1 contributes to the maintenance of silent chromatin at the native HML promoter. Unless otherwise stated, ChIP-seq data represented averaged reads of two biological replicates over the locus, normalized as in Fig. 1. Black bars along X-axis represent 200 bp surrounding Rap1-binding sites at HML-E, HML-p, and HML-I, respectively. IP, input, and untagged control values are plotted on the same scale. (A) Normalized reads mapped to HML in two 3xV5-Rap1 ChIP-seq experiments for wild-type and mutant Rap1-binding motif at the promoter. (B) Representative CRASH colonies for SIR and sir1Δ cells with wild-type and mutant Rap1-binding site at HML-p. (C) Apparent silencing-loss rate for genotypes described in B ± SD. The following number of events was recorded for each sample: SIR wt promoter (n = 271,933); SIR rap1 bs mutant (n = 773,105); sir1Δ wt promoter (n = 151,846); sir1Δ rap1 bs mutant (n = 90,211). P-values (P < 2.2e-16) for both comparisons were calculated using a two-sided t-test. (D) Normalized ChIP-seq reads for Sir3-13xMyc mapped to HML for wild-type, mutant Rap1-binding motif at the promoter, and in sir4Δ cells. (E) Normalized ChIP-seq reads for 3xV5-Rap1 mapped to HML in sir4Δ and SIR cells.
Fig. 3.
Fig. 3.
Promoter-bound Rap1 was able to activate transcription of unsilenced α2 and aided the transition from initiation to elongation at unsilenced HML. For all ChIP-seq experiments, read counts were normalized to the nonheterochromatic genome-wide median. IP, input, and untagged control values are plotted on the same scale. Data shown are the average of two ChIP-seq experiments, unless otherwise noted. (A) RT-qPCR quantification of α2 expression at HML and MAT normalized to control locus ALG9. Each plot consists of an average of two biological replicates, each represented with individual dots. (B) Normalized reads for ChIP-seq of Sua7-3xFLAG at HML in sir4∆ cells. (C) Same as B but for Rpb3-3xFLAG-KanMX. (D) Same as B and C but for Elf1-3xFLAG-KanMX.
Fig. 4.
Fig. 4.
In vivo Rap1 residence time did not reflect differences in chromatin state. Decay of Rap1 occupancy at HML and MAT by Anchor Away. (A, Top), schematic of introduction of SNPs to enable unique mapping of HML and MAT in a strain that contains both. (Below) Rap1 enrichment by two, averaged ChIP-seq experiments at HML (Left) and MAT (Right) over time-course, plotted on the same Y-axes. (B) Fitted nonlinear regressions for residence times of HML-p and MAT-p. Each replicate is shown separately ± SE of average residence time. (C) Fitted nonlinear regressions for residence times of HML-E and HML-I as in B.
Fig. 5.
Fig. 5.
Genome-wide analysis of in vivo Rap1 apparent residence times supports and extends previous models that Rap1 dwell-time is correlated with transcriptional output. All figures comprise data obtained from the average of two biological replicates. Peak set (n = 377) was divided into quartiles based on residence time for analysis unless otherwise noted. For gene-level analyses, Rap1 peaks were assigned to ORFs for which a Rap1 peak summit was within 300 bp upstream of ORF start. (A) Average apparent Rap1 residence time in minutes of the 377 binding sites evaluated genome-wide. (B) Correlation between average Rap1 occupancy before depletion (enrichment at t = 0) and the average apparent residence times for all 377 Rap1-bound peaks (Pearson correlation r = 0.45, P-value < 2.2e-16). (C) Differences in apparent residence times between sites that are classified as regulating Ribosomal Protein genes (n = 104, dark green) and all other sites (n = 273, light green). The P-value was calculated using a Mann–Whitney U test (P < 2.2e-16). (D) Quantification, by mean apparent residence time quartile, of normalized Taf1 occupancy levels at the Rap1-binding sites. These levels are defined as the amount of Taf1 enrichment (in reads) covering the Rap1-bound loci. Significance was calculated using a one-way ANOVA followed by Tukey’s HSD test (P < 2.2e-16). (E) Mean profiles display NET-seq coverage (57) with 95% confidence intervals (CI, displayed as transparent filling) within neighboring transcript(s). Coverage was scaled according to transcript length (58). (F) Summary distribution plots of average H3 enrichment (59) centered on Rap1 peaks and spanning 500 bp+/−. Coverage was grouped by apparent-residence time quartiles. The CI are indicated as in Fig. 5E, with a transparent fill denoting 95% CI. (G) Same as F, but peaks grouped by ranked Rap1 occupancy at t = 0.
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References

    1. Rando O. J., Winston F., Chromatin and transcription in yeast. Genetics 190, 351–387 (2012). - PMC - PubMed
    1. Grunstein M., Gasser S. M., Epigenetics in saccharomyces cerevisiae. Cold Spring Harbor. Pers. Biol. 5, a017491–a017491 (2013). - PMC - PubMed
    1. Rine J., Strathern J. N., Hicks J. B., Herskowitz I., A suppressor of mating-type locus mutations in saccharomyces cerevisiae: Evidence for and identification of cryptic mating-type locI. Genetics 93, 877–901 (1979). - PMC - PubMed
    1. Rine J., Herskowitz I., Four genes responsible for a position effect on expression from HML and HMR in Saccharomyces cerevisiae. Genetics 116, 9–22 (1987). - PMC - PubMed
    1. Rusche L. N., Kirchmaier A. L., Rine J., The establishment, inheritance, and function of silenced chromatin in saccharomyces cerevisiae. Annu. Rev. Biochem. 72, 481–516 (2003). - PubMed

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