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>’.