Distinct histone methylation and transcription profiles are established during the development of cellular quiescence in yeast

Abstract

Background: Quiescent cells have a low level of gene activity compared to growing cells. Using a yeast model for cellular quiescence, we defined the genome-wide profiles of three species of histone methylation associated with active transcription between growing and quiescent cells, and correlated these profiles with the presence of RNA polymerase II and transcripts.

Results: Quiescent cells retained histone methylations normally associated with transcriptionally active chromatin and had many transcripts in common with growing cells. Quiescent cells also contained significant levels of RNA polymerase II, but only low levels of the canonical initiating and elongating forms of the polymerase. The RNA polymerase II associated with genes in quiescent cells displayed a distinct occupancy profile compared to its pattern of occupancy across genes in actively growing cells. Although transcription is generally repressed in quiescent cells, analysis of individual genes identified a period of active transcription during the development of quiescence.

Conclusions: The data suggest that the transcript profile and histone methylation marks in quiescent cells were established both in growing cells and during the development of quiescence and then retained in these cells. Together, this might ensure that quiescent cells can rapidly adapt to a changing environment to resume growth.

Keywords: Cellular quiescence; Histone methylation; Transcription.

Fig. 1

Histone modifications in growing and quiescent cells. a. TCA lysates were prepared from cells in log phase (Log), at the diauxic shift (DS), and in purified populations of quiescent (Q) and nonquiescent (NQ) cells isolated 7 and 14 days after culture inoculation. Western blots were probed with antibodies against histones and methylated histones. An empty lane between the DS samples and day-7 Q samples was removed from the image. b Western blots were probed with antibodies against H3K79me1, H3K79me2, and H3K79me3 in lysates from log cells and purified populations of Q and NQ cells isolated 3, 5, and 7 days after culture inoculation. c-e Lysates were prepared from log cells and purified Q cells isolated at 3, 5, and 7 days after culture inoculation and western blots were probed with (c) anti-Myc antibodies to detect Set1, Set2, and Dot1; (d) anti-Flag antibodies to detect H2B and monoubiquitinated H2B (H2Bub1); and (e) antibodies that recognize unmodified RNA polymerase II (Rpb3-Myc) and the serine 5 (CTD-Ser5P) and serine 2 (CTD-Ser2P) phosphorylated forms of the RNAP II CTD. Actin served as a loading control in all blots, and the images represent the results from a single time-course experiment. A representative Actin blot is shown in panel a

Fig. 2

Genome-wide distribution of RNAP II and H3 methylations in growing and quiescent cells. a-e Scatter plots showing correlation of RNAP II (a), H3K4me3 (b), H3K36me3 (c), H3K79me3 (d), and H3 (e) signals across the genome of log and 7-day Q cells. f Box plots showing the average enrichment of RNAP II, H3K4me3, H3K36me3, H3K79me3 and H3 on genes in log and Q cells. g Venn diagrams identifying genes marked with RNAP II and the three H3 PTMS only in log cells, only in Q cells, and in both log and Q cells

Fig. 3

Distribution of H3 methylations and RNAP II on genes in growing and quiescent cells. a The CHROMATRA tool was used to visualize the enrichment of H3K4me3, H3K36me3, and H3K79me3 in log and 7-day Q cells across all transcripts sorted by their length (bp). TSS represents transcription start site, and positions upstream (−500 bp) and downstream (5000 bp) of the TSS are indicated. b-e Averaged gene analysis of the top 25% binding sites on genes for H3K4me3 (b); H3K36me3 (c); H3K79me3 (d); and RNAP II (e), in Log and 7-day Q cells relative to the TSS and transcription termination sites (TES), with positions upstream (−250 bp) and downstream (+250 bp) of these sites indicated

Fig. 4

RNA content of growing and quiescent cells. a Scatter plot comparing transcript abundance between log and 7-day Q cells. b Box plots of transcript abundance for all genes, genes common to log and Q cells, and log-only and Q-only genes in log and Q cells. c Venn analysis showing the number of ORF transcripts that are unique and common in each cell type. d-g Venn analysis showing the number of ORF RNAs associated with RNAs classified as sequestered RNAs in Q cells (d) and log cells (e), and the number of ORFs associated with RNAP II and transcripts in log cells (f) and Q cells (g)

Fig. 5

Correlation of RNAP II and H3 methylation signals on genes between log and quiescent cells. a Spearman correlations were derived from scatter plots comparing RNAP II, H3K4me3, H3K36me3, H3K79me3, and H3 signals across the genome of log and 7-day Q cells. b Number of genes in log and 7-day Q cells co-enriched for RNAP II, H3K4me3, H3K36me3, and H3K79me3 were identified from 4-way Venn analysis. P-values represent Fisher’s exact test

Fig. 6

Transcript, RNAP II, and H3 methylation profiles of individual genes during the development of quiescence. Log and Q cells isolated at 3, 5, and 7 days after culture inoculation were analyzed for transcript levels and Rpb3-Myc, H3K4me3, H3K36me3, and H3K79me3 occupancy at (a) PMA1, a log cell expressed gene; (b) XBP1, a Q cell expressed gene; and (c) BAP2, a gene expressed in both cell types. Transcript levels represent the change in RNA abundance relative to levels in log cells. Rpb3-Myc and H3 modification occupancies were determined at the TATA and 5’ or 3’ ORF regions of each gene. Relative occupancy represents the IP/Input at each position relative to IP/Input at TELV (Rpb3-Myc) or to H3 IP/Input (H3K4me3, H3K36me3, H3K79me3). The data represent the average with STD of 2 biological replicates

Fig. 7

Histone modifications differentially affect the reproductive capacity of quiescent cells. a. Wild type cells (WT) and cells with mutations that abolish H2B ubiquitylation (htb-K123R), H3K4 methylation (hht-K4A), H3K36 methylation (hht-K36A), or H3K79 methylation (hht-K79A) were cultured in glucose-containing rich medium (YPD). Samples were removed from each culture at the diauxic shift (day 0) and at various times after glucose exhaustion for up to 30 days before spreading aliquots in triplicate onto YPD plates. The results are representative of a single experiment, and show the percentage of colony forming units relative to those at day 0, which was set as 100%. b Separation of wild type and hht-K79A cells on a Percoll gradient 7 days after inoculation into rich medium. c Relative proportions of quiescent and nonquiescent cells in 7-day cultures of wild type and hht-K79A strains. d Survival of wild type and hht-K79A quiescent cells incubated in water. The data represent the average with STD from two independent experiments. NQ, non-quiescent cell population; Q, quiescent cell population