Volume 60, Issue 3, Pages 422-434 (November 2015) Nucleosome Stability Distinguishes Two Different Promoter Types at All Protein-Coding Genes in Yeast  Slawomir Kubik, Maria Jessica Bruzzone, Philippe Jacquet, Jean-Luc Falcone, Jacques Rougemont, David Shore  Molecular Cell  Volume 60, Issue 3, Pages 422-434 (November 2015) DOI: 10.1016/j.molcel.2015.10.002 Copyright © 2015 Elsevier Inc. Terms and Conditions

Molecular Cell 2015 60, 422-434DOI: (10.1016/j.molcel.2015.10.002) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 1 Chromatin MNase Sensitivity Profiles Define 2 Distinct Promoter Classes (A) Scatterplot representing dependency between MNase sensitivity and WW dinucleotide count within the central 100 bp of all genomic nucleosomes (left) or −1 nucleosomes (right). Color scale represents density of nucleosome particles; dashed line indicates the threshold used to determine the subset of FNs. (B) Map showing nucleosome occupancy of genes with well-determined TSS (van Bakel et al., 2013), (top panel) genes were aligned to the center of their −1 nucleosome, in the least (left) and most (right) digested samples, genes were clustered into 2 classes depending on the stability of their −1 nucleosome and then sorted by the occupancy of this nucleosome; (bottom panel) regions were aligned to the center of +1 nucleosome and sorted by the distance to the center of the first non-FN upstream from +1. (C) Plots representing distance from the center of the +1 nucleosome to the first non-fragile upstream nucleosome for all genes categorized based on the fragility of their −1 nucleosome. Box, 25th–75th percentile; whiskers 10th–90th percentile; dots, 5th and 95th percentile. (D) V-plots (Henikoff et al., 2011) showing size distribution of sequenced DNA fragments obtained by digestion at low MNase concentration (y axis) as a function of distance from the center of either stable −1 nucleosomes (top panel), fragile −1 nucleosomes (center panel), or NFRs (bottom panel) (x axis). See also Figure S1 for further details. Molecular Cell 2015 60, 422-434DOI: (10.1016/j.molcel.2015.10.002) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 2 Nucleosome Fragility Is Associated with Underlying DNA Sequence Features (A) Nucleosome occupancy at the REX2/FRS1 divergent promoter region taken from genome-wide MNase-seq maps at low and high MNase concentration (top panel). qPCR analysis of chromatin derived from either a wild-type strain or strains where the DNA sequence of the stable −1 nucleosome at REX2/FRS1 was replaced with the sequence of −1 FNs from the CHA1 (green) or RPL22B (orange) promoters. Chromatin was digested with 0.2 U (middle, upper panel) or 2 U (middle, lower panel) of MNase; (bottom). FRS1 and REX2 mRNA level measurements for the indicated constructs. Signals were normalized to ACT1 mRNA in each sample and then to levels in the wild-type promoter strain. The error bars in mRNA level plots represent the SD of at least three independent measurements. (B) Nucleosome occupancy for RPS11A (left) or RPL22B (right) promoters and mRNA levels measurements, as in (A), in a wild-type strain or strains where the DNA sequence of the fragile −1 nucleosome of each promoter was substituted with the sequence of the REX2/FRS1 stable −1 nucleosome. See also Figure S2 and Table S1 for further details. Molecular Cell 2015 60, 422-434DOI: (10.1016/j.molcel.2015.10.002) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 3 Specific Sequence Motifs Shape Promoter Architecture through the Action of the RSC Chromatin Remodeler (A) Motifs overrepresented in −1 FNs and their distribution in promoters, superimposed on nucleosome positions determined at high MNase concentration (gray) and aligned to the +1 nucleosome (as shown in Figure 1B, bottom right). (B) Motif frequency, for the two indicated sequences, as a function of distance from the midpoints of −1 FNs, NFRs, and −1 SNs smoothed by a running average with a 5-bp-wide window. (C) Maps of promoters containing −1 SNs, whose NFR width decreased by >10 bp and to a +1 to −1 distance of <190 bp following Sth1 depletion (two panels top left), or where little of no NFR shrinkage was observed (two panels bottom left). Maps are aligned to the centers of NFRs. Panels on the right show the distribution of polyA and G/C-rich motifs, as indicated, for the corresponding genes to the left. (D) Same as in (C) but aligned to centers of −1 FNs whose occupancy significantly changed (top) or did not change (bottom). See also Figure S3 for further details. Molecular Cell 2015 60, 422-434DOI: (10.1016/j.molcel.2015.10.002) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 4 PolyA and G/C-Rich Motifs Link Promoter DNA Sequence to Nucleosome Positioning and Transcription (A) Nucleosome occupancy, measured by MNase-qPCR at either low (top) or high (bottom) MNase concentration, across the GFD2 promoter in wild-type and strains where the polyA tract was progressively shortened by point mutant substitutions (mut1 to mut3). (B and C) Nucleosome occupancy measurements, as in (A), across the SSH1 promoter of wild-type and the indicated point mutants. (D and E) Nucleosome occupancy measurements, as in (A), for strains where a C/G-rich motif was inserted in the promoters of NDE2 and FRS1, respectively. See also Figure S4 for further details. Molecular Cell 2015 60, 422-434DOI: (10.1016/j.molcel.2015.10.002) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 5 Rap1 Mediates the Fragility of a Subset of FNs (A) Average binding signals (as Z-scores) of Abf1, Reb1 (Kasinathan et al., 2014), Cbf1 (Zhou and O’Shea, 2011), Tbf1 (Preti et al., 2010), and Rap1 (Knight et al., 2014) over the centers of fragile (left) and stable (right) −1 nucleosomes. (B) Plots of average sequenced DNA fragment size and a function of distance of fragment center from FN center for affected (left) and not affected (right) Rap1-proximal FNs, in control (blue) and Rap1-depleted (red) cells. MNase-seq data were derived from mildly digested samples. (C) Tracks showing Rap1 ChIP-seq signal (top) and nucleosome occupancy in samples where Rap1 was present or depleted using the anchor-away technique (as indicated), in promoters differently affected by the depletion: NFR shrinkage (GUP1, HAT2), 1 FN stabilized (RPL31A/RXT3, RPS17B/ADA2), one of two FNs stabilized (RPL27A/YHR009, SER1/RPS30B), two FNs stabilized (RPL17B/ATG27, RPS0A). FNs affected by the depletion are highlighted in red. (D) Average nucleosome occupancy plots for the four cases illustrated above in (C), in the presence (top) and absence (middle) of Rap1 (n indicates the number of cases). A schematic representation of the observed effects is shown for each case, with FNs indicated in red (bottom). See also Figure S5 for further details. Molecular Cell 2015 60, 422-434DOI: (10.1016/j.molcel.2015.10.002) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 6 Abf1 and Reb1 Have Diverse Effects on Chromatin Surrounding Their Binding Sites (A) Average nucleosome occupancy profiles in cells in which Abf1 was present (blue) or depleted using the anchor away technique (red), centered on NFRs that decrease in size (left), FNs that disappear (center), and FNs that become stabilized (right) upon Abf1 depletion. Vertical dashed lines indicate average nucleosome center positions in the presence (blue) or absence (red) of Abf1, with the center-to-center distance marked above in bp. Schematic representations (as in Figure 5) are shown below for the three different cases, both with and without Abf1. (B) Effects of Reb1 depletion presented as in (A). See also Figure S6 for further details. Molecular Cell 2015 60, 422-434DOI: (10.1016/j.molcel.2015.10.002) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 7 Schematic Representation of Elements Determining Promoter Nucleosome Architecture Illustrations at the top represent hypothetical situations in the absence of both GRF binding and RSC remodeling, as inferred from anchor-away experiments. Small NFR formation (left column) is critically dependent on RSC working through the G/C-rich and polyA motifs (left). Large NFRs (second column from left) contain, in addition, one or more GRF binding sites and are formed through the concerted action of the GRF(s) and RSC. Their size never exceeds 150 bp, and the average +1 to −1 SN distance is 270 bp. At FN-containing promoters (middle right column), we identify two basal conditions, one in which the GRF binding site is present in linker DNA (left) and one in which it is embedded in a SN when the GRF is not present (right). These promoters possess more abundant and broadly distributed G/C-rich and polyA motifs than do NFRs. GRF binding at these promoters, in the absence of RSC, increases the stable +1 to −1 distance and in some cases displaces the SN near the GRF binding site (FN column, middle). The additional action of RSC at these promoters increases the +1 to −1 SN distance, permitting the formation of a −1 FN (red) that is destabilized by GRF action (FN column, bottom). Promoters containing two FNs (right-most column) are often bound by Rap1 and contain a large destabilized nucleosome region that forms through an unknown mechanism, potentially involving additional TFs and/or chromatin remodelers. In all four columns the right-most nucleosome in every illustration represents the +1 nucleosome, and the dashed gray and black arrows represent the TSS. The order of events presented in these schemes is hypothetical. We imagine that the actual in vivo situation is highly dynamic and may involve configurations of nucleosomes, GRFs, and remodelers not indicated here. See also Figure S7 for NFR size distributions and Tables S3, S4, and S5 for further details. Molecular Cell 2015 60, 422-434DOI: (10.1016/j.molcel.2015.10.002) Copyright © 2015 Elsevier Inc. Terms and Conditions