13 research outputs found
Rif1 controls DNA replication by directing Protein Phosphatase 1 to reverse Cdc7-mediated phosphorylation of the MCM complex
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Analysis of Chromosome III Replicators Reveals an Unusual Structure for the ARS318 Silencer Origin and a Conserved WTW Sequence within the Origin Recognition Complex Binding Site▿ †
Saccharomyces cerevisiae chromosome III encodes 11 autonomously replicating sequence (ARS) elements that function as chromosomal replicators. The essential 11-bp ARS consensus sequence (ACS) that binds the origin recognition complex (ORC) has been experimentally defined for most of these replicators but not for ARS318 (HMR-I), which is one of the HMR silencers. In this study, we performed a comprehensive linker scan analysis of ARS318. Unexpectedly, this replicator depends on a 9/11-bp match to the ACS that positions the ORC binding site only 6 bp away from an Abf1p binding site. Although a largely inactive replicator on the chromosome, ARS318 becomes active if the nearby HMR-E silencer is deleted. We also performed a multiple sequence alignment of confirmed replicators on chromosomes III, VI, and VII. This analysis revealed a highly conserved WTW motif 17 to 19 bp from the ACS that is functionally important and is apparent in the 228 phylogenetically conserved ARS elements among the six sensu stricto Saccharomyces species
Yeast heterochromatin regulators Sir2 and Sir3 act directly at euchromatic DNA replication origins
<div><p>Most active DNA replication origins are found within euchromatin, while origins within heterochromatin are often inactive or inhibited. In yeast, origin activity within heterochromatin is negatively controlled by the histone H4K16 deacetylase, Sir2, and at some heterochromatic loci also by the nucleosome binding protein, Sir3. The prevailing view has been that direct functions of Sir2 and Sir3 are confined to heterochromatin. However, growth defects in yeast mutants compromised for loading the MCM helicase, such as <i>cdc6-4</i>, are suppressed by deletion of either <i>SIR2</i> or <i>SIR3</i>. While these and other observations indicate that <i>SIR2</i>,<i>3</i> can have a negative impact on at least some euchromatic origins, the genomic scale of this effect was unknown. It was also unknown whether this suppression resulted from direct functions of Sir2,3 within euchromatin, or was an indirect effect of their previously established roles within heterochromatin. Using MCM ChIP-Seq, we show that a <i>SIR2</i> deletion rescued MCM complex loading at ~80% of euchromatic origins in <i>cdc6-4</i> cells. Therefore, Sir2 exhibited a pervasive effect at the majority of euchromatic origins. Using MNase-H4K16ac ChIP-Seq, we show that origin-adjacent nucleosomes were depleted for H4K16 acetylation in a <i>SIR2</i>-dependent manner in wild type (i.e. <i>CDC6</i>) cells. In addition, we present evidence that both Sir2 and Sir3 bound to nucleosomes adjacent to euchromatic origins. The relative levels of each of these molecular hallmarks of yeast heterochromatin–<i>SIR2</i>-dependent H4K16 hypoacetylation, Sir2, and Sir3 –correlated with how strongly a <i>SIR2</i> deletion suppressed the MCM loading defect in <i>cdc6-4</i> cells. Finally, a screen for histone H3 and H4 mutants that could suppress the <i>cdc6-4</i> growth defect identified amino acids that map to a surface of the nucleosome important for Sir3 binding. We conclude that heterochromatin proteins directly modify the local chromatin environment of euchromatic DNA replication origins.</p></div
Depletion of H4K16ac from origin-adjacent nucleosomes required <i>SIR2</i>.
<p><b>A.</b> H4K16 acetylation status of nucleosomes was assessed by MNase ChIP-Seq in <i>SIR2</i> and <i>sir2Δ</i> cells. These cells were the same as those analyzed for MCM binding in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007418#pgen.1007418.g001" target="_blank">Fig 1</a>. <b>B.</b> The H4K16 acetylation status was plotted for the low, medium and high <i>SIR2</i>-responsive quintiles as defined in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007418#pgen.1007418.g003" target="_blank">Fig 3</a>.</p
Sir2 and Sir3 were physically associated with nucleosomes adjacent to origins.
<p><b>A.</b> Sir2 or (<b>B)</b> Sir3 binding to origin or non-origin adjacent nucleosomes was assessed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007418#pgen.1007418.g003" target="_blank">Fig 3</a> using data from [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007418#pgen.1007418.ref033" target="_blank">33</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007418#pgen.1007418.ref034" target="_blank">34</a>]. <b>C.</b> The normalized Sir3 ChIP-Seq signal (log<sub>2</sub> Sir3 ChIP signal, y-axis) for each nucleotide was plotted over the 1201 bp origin (blue) or non-origin (green) loci (coordinates on x-axis). The Sir3 ChIP-Seq signal represented total reads for each nucleotide normalized to the depth (total reads) and breadth (number of nucleotides with reads) of the sequencing reactions [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007418#pgen.1007418.ref049" target="_blank">49</a>]. Genomic regions previously established as <i>SIR</i>-heterochromatic domains were excluded from the normalization but see <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007418#pgen.1007418.s008" target="_blank">S8 Fig</a></b> for an analysis that includes these nucleotides in establishing the baseline. <b>D. and E.</b> The Sir3 ChIP-Signals were plotted as in ‘<b>C</b>’ or ‘<b>B</b>’, respectively, for the three different <i>SIR2</i>-responsive groups defined in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007418#pgen.1007418.g003" target="_blank">Fig 3</a>. <b>F.</b> The <i>SIR2</i>-responsive origins comprising the three groups assessed in ‘<b>D’</b> were randomized into three different sets and the Sir3 ChIP-Seq signals determined and plotted as in ‘<b>D</b>’.</p
<i>SIR2</i> inhibited MCM association at the majority of euchromatic origins in <i>cdc6-4</i> mutant cells.
<p><b>A.</b> Experimental outline: MCM binding to chromosomal DNA was examined by ChIP-Seq from congenic <i>CDC6 SIR2</i> (wild type), <i>CDC6 sir2Δ</i> (<i>sir2Δ</i>), <i>cdc6-4 SIR2</i> (<i>cdc6-4</i>), and <i>cdc6-4 sir2Δ</i> cells. Cells were released from a G2/M nocodazole-arrest into G1 phase at 37°C prior to formaldehyde crosslinking. <b>B.</b> MCM ChIP-Seq signals across Chromosome III. The x-axis indicates the chromosomal coordinates, the y-axis the normalized MCM read counts (MCM signal). The scale on the y-axis is the same for each of the four strains. Confirmed origins on Chromosome III are indicated by boxes and numbers. Black boxes indicate origins that were defined as <i>SIR2</i>-responsive because they provided for plasmid replication in <i>cdc6-4 sir2Δ</i> cells at the non-permissive temperature for <i>cdc6-4</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007418#pgen.1007418.ref023" target="_blank">23</a>]. The arrowhead and the line-with-circle indicate <i>ARS305</i> and <i>ARS307</i>, respectively that produced similar association with MCM in wild type cells but different degrees of MCM association in <i>cdc6-4 sir2Δ</i> cells. These origins are highlighted to illustrate that the wild type pattern of MCM origin distribution was not rescued fully in <i>cdc6-4 sir2Δ</i> cells. The starred origins (<i>ARS301</i>, <i>ARS302</i>, <i>ARS317</i> and <i>ARS318</i>) are associated with transcriptional silencers that direct the assembly of <i>SIR</i>-heterochromatin at the <i>HML</i> and <i>HMR</i> loci on chromosome III [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007418#pgen.1007418.ref015" target="_blank">15</a>]. They were excluded from further analyses in this study for this reason, as discussed in text. <b>C.</b> MCM ChIP-Seq signals across Chromosome VI. The arrowhead and the line-with-circle indicate <i>ARS606</i> and <i>ARS609</i>, respectively, which behaved analogously to <i>ARS305</i> and <i>ARS307</i>, respectively, as described in B. <b>D.</b> Origins’ <i>SIR2</i>-responsiveness was defined as the ratio of the MCM signal at the origin in <i>cdc6-4 sir2Δ</i> cells relative to <i>sir2Δ</i> cells. We confined analyses to confirmed origins that produced strong signals in both WT and <i>sir2Δ</i> cells as defined by inclusion among the top 400 chromosomal coordinates of enrichment (<b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007418#pgen.1007418.s002" target="_blank">S2 Fig</a></b>). The dotted line indicates the cut-off that captured the euchromatic origins (<i>ARS305</i>, <i>ARS315</i>, <i>ARS603 and</i> ARS606) that had previously been defined as <i>SIR2</i>-responsive origins based on a systematic plasmid-based screen of these chromosomal origins [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007418#pgen.1007418.ref023" target="_blank">23</a>].</p
Reduction in rDNA copy number did not account for the suppression of <i>cdc6-4</i> by <i>sir2Δ</i>.
<p><b>A.</b> Diagram of a single rDNA repeat is shown with the location of the primer pairs used to assess rDNA copy number by qPCR indicated by colored histogram. The rDNA origin is indicated as the open circle. NTS are the non-transcribed regions in the rDNA locus. RFB is the replication fork block element that binds Fob1. <b>B.</b> Enrichment values are equal to 2<sup>- ΔCt</sup> where ΔCt = Ct (rDNA locus (purple or teal) or <i>ERV46(</i>aqua<i>)</i>)—Ct (single copy control locus, <i>RIM15</i>) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007418#pgen.1007418.ref072" target="_blank">72</a>] and equal the predicted average copy number of the target locus. For each strain, three independent colonies were assessed (biological replicates), each represented by a bar with the standard error. Because we observed substantial variation in rDNA copy number between colonies from the same strains (purple and teal bars represent different primer pairs that amplify the rDNA locus), we also used qPCR to assess the copy number of a single copy gene (<i>ERV46</i>) for the same DNA preparations (aqua). Note the different scale for the single-copy experiment as this value should equal only 1.0. <b>C.</b> <i>SIR2</i> effect on the average enrichment values of the rDNA locus for each of the strains assessed in C. <b>D.</b> Growth of 10-fold dilutions of the indicated strains was assessed on solid YPD media at the indicated temperatures. <b>E.</b> <i>SIR3</i> effect on the average enrichment values of the rDNA locus for each of the indicated strains.</p
Acetylated H4K16 is depleted from origin-adjacent nucleosomes.
<p><b>A.</b> Two groups of loci were analyzed, origins, defined as experimentally-confirmed origins with a confirmed or high-confidence ORC site, n = 259, and intergenic non-origin loci with ORC site matches but for which no origin activity has been observed, n = 179 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007418#pgen.1007418.ref043" target="_blank">43</a>]. These two different groups of loci were compared to determine whether chromatin-states at origins were specific for origin function as opposed to being the result of underlying AT-rich sequence elements present in origins. “ORC in vitro” refers to loci that were bound by purified ORC in a genomic electrophoretic mobility assay [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007418#pgen.1007418.ref073" target="_blank">73</a>]; “ORC in vivo” and “MCM in vivo” refer to the fraction of sites in these two groups that were detected by ORC- or MCM-ChIP, respectively. <b>B.</b> The WebLogo consensus for the ORC sites (or matches) in origins and non-origins, respectively, are shown above the diagram of the fragments used in the analyses of adjacent nucleosomes. Each fragment analyzed was oriented with the T-rich strand of the ORC site 5’ to 3’ on the top strand, and the first nucleotide of the ORC site was designated as position “0”. The fragments were 1201 bp such that six proximal nucleosomes, shown as black ovals, three on each side of the ORC site, were assessed. <b>C.</b> Nucleosome occupancy surrounding the origin and non-origin nucleosome depleted regions are shown using the MNase-ChIP-Seq nucleosome occupancy data from [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007418#pgen.1007418.ref032" target="_blank">32</a>]. <b>D.</b> Normalized H4K16ac and H3K9ac for each of the six nucleosomes for the two different groups of loci examined. P-values are derived from Student’s T-test comparing the mean of acetylation status between each nucleosome to the mean acetylation status of nucleosomes from the 239 intergenic control loci. <b>E.</b> <i>SIR2</i>-responsiveness was defined as the ratio of the MCM ChIP-Seq signal in <i>cdc6-4 sir2Δ</i> to <i>sir2Δ</i> cells. The origins were ranked based on <i>SIR2</i>-responsiveness and then divided into quintiles, with the high quintile containing the most <i>SIR2</i>-responsive origins. <b>F.</b> H4K16ac status for the three quintiles of <i>SIR2</i>-responsive origins indicated in ‘<b>E</b>’ was determined as in ‘<b>D</b>’.</p