1. Volume 18, Issue 2, Pages 171-183 (April 2005)
Genome-Wide Analyses Reveal RNA Polymerase II Located Upstream of Genes Poised for Rapid Response upon S. cerevisiae Stationary Phase Exit 
Marijana Radonjic, Jean-Christophe Andrau, Philip Lijnzaad, Patrick Kemmeren, Thessa T.J.P. Kockelkorn, Dik van Leenen, Nynke L. van Berkum, Frank C.P. Holstege 
Molecular Cell 
Volume 18, Issue 2, Pages (April 2005)
DOI: /j.molcel
Copyright © 2005 Elsevier Inc. Terms and Conditions

2. Figure 1 Global Changes during Quiescence Exit and Entry
(A) Schematic representation of a glucose starvation experiment, with circles representing the 39 samples analyzed. The single filled circle in the rapid exponential growth phase represents the common reference sample used in the microarray experiments. The open circle, left of the horizontal axis, represents the aliquot of the SP culture (day 9) that was used to start the experiment. See Experimental Procedures for a complete list of time points.
(B) Cell count per OD600 throughout the culture (left) and for the first 40 hr (right). The colored bar represents the various culture periods, with abbreviations according to (A).
(C) Number of cells/ml throughout the culture (left) and for the first 40 hr (right).
(D) Total RNA per 108 cells.
(E) Average poly(A) dot blot 32P radioactive signal per 108 cells as determined by analysis of two dot-blot experiments, one of which is depicted in (G).
(F) poly(A) signal per 108 cells (open circles) and total RNA per 108 cells (filled circles) for the first 3 hr.
(G) poly(A) dot blot for determination of mRNA levels throughout the experiment. The first dot (left upper corner) represents the 9-day SP culture from which the experiment was started. The 20th and 21st spot (counting from left to right) are duplicates of the 6.5 hr time point.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
Genome-Wide Changes in Transcript Levels during Quiescence Exit and Entry
(A) Standard, all genes normalized scatter-plot of average background-subtracted microarray gene spot intensities, comparing ML samples with SP samples (9 days). The three lines represent 2-fold up, no change, and 2-fold down boundaries, from top to bottom, respectively. Colored in red and green are genes that were determined to show statistically significant changes in mRNA expression in the analysis of the eight SP versus ML replicate measurements.
(B) External control normalized result of the experiment shown in (A), with the same genes colored red and green as in (A).
(C) External control-normalized result of a same versus same, ML replicate microarray hybridization.
(D) Standard, all genes normalized, hierarchical clustering diagram for the entire time course (left to right) of all 6357 yeast genes represented on the microarrays. The colored bar on top represents the various culture periods, with abbreviations according to Figure 1A. Clustering was by average linkage analysis (UPGMA) with a standard (cosine) correlation. Table S1 contains a fully annotated version of the cluster diagram.
(E) Hierarchical clustering diagram of the external control-normalized microarray data.
(F) Expression ratios of 123 ribosomal protein genes throughout the time course when the data are normalized by the standard, all genes approach. The collective expression ratio of all genes is artificially set at 1 (no change) in each individual time point by this approach. The RPGs appear only to fluctuate once during the time course, at 20 hr. This actually represents the time point when these transcripts deviate most from the behavior of all the genes, with which the data are normalized here.
(G) Expression ratios of 123 ribosomal protein genes when data are normalized with external controls. This results in ratios for each individual transcript, relative to the level of that transcript in the reference sample. The reference sample was ML, which is why all transcripts show a ratio of 1 at this time point.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

4. Figure 3
Many Distinct Patterns of Gene Expression during Quiescence Exit and Entry
(A) The average expression ratio of all genes across the time course.
(B) Genes that most closely follow the average transcript behavior.
(C) Rapid SP exit genes, transiently induced prior to both periods of proliferation.
(D) Rapid SP exit genes, transiently induced only prior to proliferation on glucose.
(E) Rapid SP exit genes, induced prior to proliferation and maintained at high levels during exponential growth.
(F) Transcripts that fluctuate several times during the time course. These are mainly genes with uncharacterized molecular function and/or biological processes.
(G) A group of uncharacterized/hypothetical ORFs induced at the end of lag phase.
(H) Genes that escape the general DS repression and that are induced very early in DS, at 10–11 hr.
(I) Similar to (H), but with less prominent induction.
(J) Transcripts that peak at the end of rapid exponential growth and late in the DS.
(K) Transposon TyB Gag-Pol genes. The tight coregulation is likely due to their overall identity.
(L) Transposon TyA Gag genes.
(M) Subtelomeric Y′ helicases and homologs.
(N) A cluster of various functionally related genes induced late in lag-phase and including negative regulators of the Swe1p kinase, SPT6 and SPT16, genes involved in bud site selection and replication.
(O) Transcripts that show two transient peaks, late in lag phase and very early during DS. Half of these genes are involved in mating.
(P) SP enriched transcripts, induced late in DS and remaining high thereafter. This includes many of the stress and aerobic respiratory genes enriched in SP.
(Q) SP enriched transcripts, more transiently induced late in DS, but still remaining elevated in SP compared to ML. This also includes many of the stress and aerobic respiratory genes enriched in SP.
(R) SP enriched transcripts, as in (Q), but with an additional induction early in lag phase, although not immediately upon exit. This suggests that these genes may be required for transitions to any new carbon source.
(S) SP enriched transcripts as in (Q) and (R) but that show a second induction upon SP entry after the postdiauxic growth phase.
(T) SP enriched transcripts that start to rise late in lag phase and continue to do so into SP. This group includes previously characterized SP genes such as SNO1, SNZ1, and SNZ2.
(U) SP enriched transcripts showing induction upon the transition between slow postdiauxic growth and SP.
Groups (B)–(O) were obtained by clustering across the entire time course. Groups (P)–(U) were clustered based on their behavior only in the latter part of the time course. The genes depicted in each cluster are listed in Table S2. More genes with similar behavior can be found in Table S1.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

5. Figure 4
The General Transcription Machinery Is Maintained during Quiescence
(A) Rapid and broad transcription activation upon exit from stationary phase (SP). The expression ratios throughout the time course for the 769 genes induced more than 2-fold within the first 3 min of SP exit are depicted. The genes are listed in Table S3.
(B) Western blot analysis of mid-log (ML) and stationary phase (SP) protein extracts for RNA polymerase II subunits Rpb1p, its C-terminal domain (CTD), and Rpb3p. Ser2-P and Ser5-P refer to specific phosphorylation sites within the heptapeptide repeat of the CTD. TBP refers to TATA binding protein and Srb4p is a subunit of the Mediator complex. Extracts from equivalent amounts of cells were loaded and this was verified using an antibody specific for the invariant part of Histone H3 (H3).
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 Genome-Wide Location Analysis of RNA Polymerase II
Histograms of Pol II binding ratios of all genomic sequences for ML (A) and SP (B). Each histogram is the result of a single microarray hybridization. The vertical axis represents the normalized binding ratios. In red are genomic features selected as significantly enriched in each collection of four hybridizations. Conventional ChIP analysis of PUT4 (C) and SUT1 (D), analyzed by PCR in the linear range of amplification, loaded on a 7% polyacrylamide gel and quantified using a phosphoimager as described in Experimental Procedures. Relative positions of the PCR products are indicated below the graphs. The asterisk represents the position of the internal normalization control for PCR. The binding ratios depicted on the graphs are from the microarray (dashed lines) and conventional ChIP (solid lines), for ML (blue) and SP (red), respectively.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions