Nucleosomes disrupted in remodeler ATPase mutants are in more intrinsically preferred positions.

<p>A) WT (top row) and Chd1, Ino80 and Isw1 deletion mutant (middle row) occupancy profiles at genic nucleosome positions are shown in comparison to occupancy profiles (<i>in vitro</i> occupancy) based on intrinsic sequence preferences obtained from Kaplan et al. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003479#pgen.1003479-Kaplan2" target="_blank">[27]</a> (bottom row). Each panel corresponds to a genic nucleosome position, numbered relative to the TSS, and plots the <i>in vivo</i> or predicted occupancy for genes with a positive (red), negative (green), or no shift (blue) in nucleosomes at that position. Solid lines reflect the mean occupancy in a smoothing window with a bandwidth of 25 bp, with the corresponding 95% confidence interval indicated by shaded areas of the same color. B) Overview of the effects of nucleosome shifts at each +1 to +4 position on neighboring nucleosomes in the same genic array. Each panel shows the average shift profile (solid lines) and 95% confidence interval (shaded area) for all nucleosomes in the same array for genes with a positive (red), negative (green) or no shifts (blue) at the indicated genic nucleosome position (black triangle).</p

A Compendium of Nucleosome and Transcript Profiles Reveals Determinants of Chromatin Architecture and Transcription

<div><p>Nucleosomes in all eukaryotes examined to date adopt a characteristic architecture within genes and play fundamental roles in regulating transcription, yet the identity and precise roles of many of the trans-acting factors responsible for the establishment and maintenance of this organization remain to be identified. We profiled a compendium of 50 yeast strains carrying conditional alleles or complete deletions of genes involved in transcriptional regulation, histone biology, and chromatin remodeling, as well as compounds that target transcription and histone deacetylases, to assess their respective roles in nucleosome positioning and transcription. We find that nucleosome patterning in genes is affected by many factors, including the CAF-1 complex, Spt10, and Spt21, in addition to previously reported remodeler ATPases and histone chaperones. Disruption of these factors or reductions in histone levels led genic nucleosomes to assume positions more consistent with their intrinsic sequence preferences, with pronounced and specific shifts of the +1 nucleosome relative to the transcription start site. These shifts of +1 nucleosomes appear to have functional consequences, as several affected genes in Ino80 mutants exhibited altered expression responses. Our parallel expression profiling compendium revealed extensive transcription changes in intergenic and antisense regions, most of which occur in regions with altered nucleosome occupancy and positioning. We show that the nucleosome-excluding transcription factors Reb1, Abf1, Tbf1, and Rsc3 suppress cryptic transcripts at their target promoters, while a combined analysis of nucleosome and expression profiles identified 36 novel transcripts that are normally repressed by Tup1/Cyc8. Our data confirm and extend the roles of chromatin remodelers and chaperones as major determinants of genic nucleosome positioning, and these data provide a valuable resource for future studies.</p></div

Profiles of genic nucleosome shifts.

<p>Box plots of nucleosome shifts for 20 conditions that show a median deviation of at least 4 base pairs for at least one genic nucleosome position, organized in 7 functional groups: A) remodeler ATPases, B) histone depletion, C) CAF-1 complex subunits, D) Elongation factors and chaperones, E) histone gene regulators, F) transcription disruption and G) all other factors. Boxes correspond to the spread between the upper and lower quartile, with medians indicated by a solid horizontal line, and whiskers extend up to 1.5 times the inter-quartile range. Nucleosome positions are relative to the transcription start site. Positive and negative numbers correspond to shifts away or towards the TSS, respectively.</p

Novel transcripts identified in <i>tup1-Δ</i> and <i>cyc8-Δ</i> strains.

<p>A) Hierarchical clustering of 36 novel Tup1/Cyc8-repressed transcripts (TCR) showing changes in nucleosome occupancy directly upstream the transcription start site (blue/yellow; left) and expression changes across each transcript and 500 bp intergenic flanking region (red/green; right). The degree of change is indicated by the color bars. B) Example of de-repressed novel transcripts (<i>TCR4</i> and <i>TCR5</i>) in the <i>tup1-Δ</i> strain. C) Hierarchical clustering of average expression changes at the 36 TCR loci across all 55 compendium conditions. D) Results of phenotype analysis of 15 <i>tcr</i> deletion mutants in a <i>tup1-</i>Δ background across a panel of 14 different stress conditions. Growth rates were measured by spot assay and quantified using ImageQuant. Differences in growth rate between <i>tup-Δ</i> and <i>tup-Δ/tcr-Δ</i> strains are indicated by the color bar.</p

Loss of Ino80 or Isw1 leads to +1 nucleosome shifts at a subset of genes.

<p>A) Number of +1 to +4 nucleosomes with significant shifts in strains bearing deletions of the remodeler ATPases Chd1, Ino80 or Isw1, compared to 36 WT conditions. B) Hierarchical clustering of shifts at +1 nucleosomes found as significant in at least one of the remodeler ATPase mutants. Two biological replicates, each grown on different days, are shown for each mutant. The direction and degree of shift relative to the transcription start site is indicated by the color bar. Positions for which a shift could not be determined because of absent nucleosome calls in WT or mutant strains are colored grey. C) Box plot of the change in expression for genes with positive (red) or negative (green) +1 nucleosome shifts in <i>ino80-Δ</i> or <i>isw1-Δ</i> strains, compared to genes without shifts (blue). Box and whisker definitions follow those in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003479#pgen-1003479-g002" target="_blank">Figure 2</a>. D) qPCR analysis of <i>FRE1</i> and <i>FRE7</i> expression changes after 30 min treatment with 100 µM 1,10-phenantroline in a WT or <i>ino80-Δ</i> background. E) qPCR analysis of <i>MIG1</i> and <i>RGT1</i> expression changes upon shifts from YPG to YPD (left) or YPD to YPG (right) in a WT or <i>ino80-Δ</i> background.</p

Cryptic antisense transcripts in CAF-1 complex mutants.

<p>Hierarchical clustering of average expression changes in 3,578 antisense (AS) regions. The set of AS transcripts was compiled by first identifying all AS transcripts with a more than 2-fold change in expression and a p-value<0.05 over at least 80 bp (10 probes) in at least one compendium condition, and then combining these transcripts in a non-redundant union set. Four strains (<i>rpa190-tet</i>, <i>rpb2-tet</i>, <i>rrn3-tet</i>, <i>bdp1-tet</i>) were excluded from the analysis as the large decrease in gene expression and/or global changes in total RNA composition in these strains made them unsuitable to our tiling array normalization procedure. Profiles corresponding to deletions of subunits of the CAF-1 complex are indicated in bold.</p

Disruption of nucleosome-excluding general transcription factors results in cryptic promoter transcripts.

<p>A) Correlation between changes in promoter nucleosome occupancy (left panels, yellow/blue) and transcription changes across the gene body and 1 kb intergenic flanking regions (right panels, red/green). In each panel, genes are ranked according to the average change in NDR nucleosome occupancy. NDRs were defined as the 200 bp region directly upstream of curated transcription start sites. B) Representative examples of promoter transcripts in each TF mutant (arrows). Positions of NDRs are highlighted in grey. C) Fraction of diverging and tandem genes among promoters that show significant changes in nucleosome occupancy and expression of cryptic transcripts. The number of promoter regions considered is indicated above each bar. D) Top: nucleosome occupancy relative to transcript starts for promoter transcripts in the <i>tbf1-ts</i>, <i>rap1-tet</i>, <i>abf1-ts</i> and <i>rsc3-ts</i> mutants. Bottom: changes in nucleosome occupancy compared to wild-type strains.</p

Global nucleosome occupancy profiles.

<p>A) Schematic overview of compendium mutants and conditions. B) Average nucleosome occupancy relative to the curated transcription start sites of 5,043 <i>S. cerevisiae</i> genes, expressed as the log2 ratio of probe intensities of MNase-treated nucleosomal DNA samples over un-crosslinked DNA samples. 52 mutants and conditions with a canonical nucleosome distribution are shown in the top panel and differentially colored according to average NDR occupancy. Mutants with disrupted global nucleosome occupancy profiles are shown below.</p

Control MNase-seq data aligned to Haloferax genome (BAI file)

Control MNase-seq data aligned to Haloferax genome (BAI file

Control MNase-seq data aligned to Haloferax genome (BAM file PART1)

naked DNA control (MNase-seq