1. Volume 59, Issue 5, Pages 732-743 (September 2015)
Global Promoter Targeting of a Conserved Lysine Deacetylase for Transcriptional Shutoff during Quiescence Entry 
Jeffrey N. McKnight, Joseph W. Boerma, Linda L. Breeden, Toshio Tsukiyama 
Molecular Cell 
Volume 59, Issue 5, Pages (September 2015)
DOI: /j.molcel
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2. Molecular Cell 2015 59, 732-743DOI: (10.1016/j.molcel.2015.07.014)
Copyright © 2015 Elsevier Inc. Terms and Conditions

3. Figure 1 Global Transcriptional Shutoff Accompanies Quiescence Entry
(A) Boxplot of transcript abundance for all genes (left) or ribosomal protein genes (right) during logarithmic growth in rich media (log), 2 hr after glucose exhaustion (DS), or purified quiescent cells from a 7-day culture (Q).
(B) Browser shot of strand-specific, normalized transcript abundance across chromosome III. Blue indicates Watson strand and red indicates Crick strand.
(C–E) Scatter plots comparing normalized transcript abundance between log and DS cells (C), DS and Q cells (D), or log and Q cells (E). Yellow dots indicate ribosomal protein transcripts. Red diagonal line indicates y = x axis to represent where expected transcript distribution should occur in the absence of global transcription changes. Normalized FPKM denotes fragments per kilobase of exon per million fragments mapped determined by Cufflinks and scaled to normalize for standard-corrected RNA per cell.
See also Figure S1 and Table S1.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
Global Changes in Chromatin Reflect Transcriptional Repression in Q Cells
(A) Nucleosome dyad signal aligned at 4,550 transcription start sites (TSSs) (Nagalakshmi et al., 2008) for matched MNase digests from Q and log cells.
(B and C) Nucleosome dyad signal for two different digestion levels in log cells (B) or Q cells (C) at TSS. Corresponding MNase digestion ladders are shown in Figure S2A.
(D) Individual nucleosome dyad signal aligned at TSS and sorted by increase and decrease in dyad signal at nucleosome-depleted regions (NDR) as cells transition from log growth (left) to quiescence (right).
(E) Western blot using antibodies against acetylated histone tails as indicated in log, DS, or Q cells. Total H3 was used as a loading control.
See also Figure S2.
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5. Figure 3
Tight Correlations between Chromatin Transition and Transcriptional Shutoff
(A) Nucleosome dyad signal (left), histone H3 density (center), and hyperacetylated H4 signals (right) in log or Q cells at TSSs for top 500 genes most repressed in Q cells. Dyad signal is from 80% mononucleosome dataset, H3 signal is first Z score normalized, and H4 hyperacetylation signal is corrected for histone density. H3K23 acetylation profiles are show in Figures S3A and S3B.
(B) Same as (A) but for the 500 least-repressed (or induced) transcripts in Q cells.
(C) The changes in signals between most- and least-repressed genes upon Q entry.
(D) Correlation between changes in H3 occupancy (from ChIP-seq) and changes in transcription between Q and log cells (left). Heatmap of changed H3 signals (Q/log, normalized to input), sorted by changes in transcription between Q and log cells (right).
(E) Same as (D) but for changes in H3-normalized, hyperacetylated H4ac levels.
(F–H) Changes in MNase dyad signals (F, from 50% mononucleosome digest), H3 occupancy (G), or H4 hyperacetylation (H) at 166 transcription factor motifs from the JASPAR database (Mathelier et al., 2014) (see Supplemental Experimental Procedures) sorted by difference in signals. Green bars indicate motifs within indicated cluster corresponding to transcription factor with known interactions with Rpd3 (from BioGRID database: Transcription factor identity and expanded Rpd3 interaction motifs are shown in Figures S3E–S3G.
See also Figure S3.
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6. Figure 4 Rpd3 Is Required for Quiescence Entry and Survival
(A) Representative image of WT (left) and Δrpd3 (right) stationary-phase cultures after 7 days of growth prior to Q cell isolation, depicting distinct culture morphologies.
(B) Growth analysis of WT and Δrpd3 cells through 48 hr in YPD. Arrows indicate time of glucose exhaustion.
(C) Viability assay showing cell survival as cells enter the Q state in WT and Δrpd3 cells.
(D) Representative image of Q cells purified from 7-day stationary-phase cultures indicating similar gross morphology.
(E) Western blot of indicated acetylated histones in WT and Δrpd3 cells. H3 serves as a loading control.
(F) Survival curve for purified Q cells, measured by colony-forming units, after incubation in pure water for indicated time at 30°C. Data are presented as mean ± SEM.
See also Figure S4.
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7. Figure 5 Rpd3 Plays the Key Role for Transcriptional Quiescence
(A) Histone H3 density (top) or hyperacetylated H4ac signal (bottom) for 500 most- and 500 least-repressed genes in Q state for indicated strains. Orange lines highlight the points of similarity between WT DS shift cells and Δrpd3 Q cells.
(B) Scatterplot showing correlation of H4ac signal at TSSs between indicated strains.
(C) Boxplot showing normalized transcript abundance globally (left) or for ribosomal protein transcripts (right). Orange lines highlight similarities between WT DS shift and Δrpd3 Q.
(D) Browser shot comparing normalized transcription levels in WT DS, WT Q, and Δrpd3 Q cells.
(E and F) Scatterplots showing correlation and global differences between the transcriptomes of wild-type Q and Δrpd3 Q cells (E) or wild-type DS and Δrpd3 Q cells (F). Red diagonal line indicates y = x axis, and yellow dots correspond to ribosomal protein genes.
See also Figure S5.
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8. Figure 6 Global Relocalization of Rpd3 upon Quiescent Entry
(A) Browser shot of Rpd3 binding in log cells (light blue) and Q cells (dark blue) along the length of chromosome IX (top) or a zoomed in (translucent red rectangle) segment of chromosome IX (bottom).
(B) Global distribution of Rpd3 localization at TSSs in aggregate (left) or individual genes ranked by Rpd3-Myc signal (right).
(C) Browser shot of Rpd3-Myc ChIP-seq signals in WT (black), Δstb3 (blue), and Δxbp1 (orange) strains showing TF dependence of Rpd3 binding. Gene names indicate promoters where Rpd3 binding is dictated by the associated TF.
(D) Global comparison of Rpd3 promoter binding in Δstb3 and Δxbp1 strains. Rpd3 ChIP-seq signal ratio was calculated at 4,550 TSSs. Blue indicates promoters where deletion of STB3 reduces Rpd3 binding, while orange indicates promoters where XBP1 deletion reduces Rpd3 binding.
(E) Scatterplot showing mutual exclusivity of Rpd3 binding and H4 hyperacetylation in Q cells at gene promoters.
(F) Histone acetylation levels, relative transcriptional repression, and change in Pol II binding during the log-to-Q transition at annotated TSS ranked by Rpd3 binding in Q cells. Transcriptional repression is represented as log2 transcription ratio centered at median repression value. Acetylation difference is the log2 ratio normalized to H3, Pol II change is log2 ratio of Rpb3 (Pol II subunit) binding between log and Q cells.
(G) Difference in acetylation and transcription between WT Q and Δrpd3 Q cells ranked by Rpd3 binding in Q cells.
See also Figure S6.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7 Model for Rpd3-Mediated Quiescence
(A) Cartoon representation of chromatin state in log and Q cells. See text for details.
(B) Characteristic locus demonstrating Rpd3-mediated repression. The TKL1 gene is actively transcribed in log cells, where Rpd3 is absent (top). In Q cells, Xbp1 binds to a specific motif (green rectangle) and recruits Rpd3 upstream of the TKL1 promoter (red arrow), histones are deacetylated into the coding region (yellow shaded region), nucleosomes are stabilized (green shaded region), and transcription is extensively repressed.
Molecular Cell  , DOI: ( /j.molcel )
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