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. 2009 Sep 11;35(5):626-41.
doi: 10.1016/j.molcel.2009.07.017. Epub 2009 Aug 13.

Linking cell cycle to histone modifications: SBF and H2B monoubiquitination machinery and cell-cycle regulation of H3K79 dimethylation

Julia M Schulze  1 , Jessica Jackson, Shima Nakanishi, Jennifer M Gardner, Thomas Hentrich, Jeff Haug, Mark Johnston, Sue L Jaspersen, Michael S Kobor, Ali Shilatifard
Affiliations

Linking cell cycle to histone modifications: SBF and H2B monoubiquitination machinery and cell-cycle regulation of H3K79 dimethylation

Julia M Schulze et al. Mol Cell. .

Abstract

To identify regulators involved in determining the differential pattern of H3K79 methylation by Dot1, we screened the entire yeast gene deletion collection by GPS for genes required for normal levels of H3K79 di- but not trimethylation. We identified the cell cycle-regulated SBF protein complex required for H3K79 dimethylation. We also found that H3K79 di- and trimethylation are mutually exclusive, with M/G1 cell cycle-regulated genes significantly enriched for H3K79 dimethylation. Since H3K79 trimethylation requires prior monoubiquitination of H2B, we performed genome-wide profiling of H2BK123 monoubiquitination and showed that H2BK123 monoubiquitination is not detected on cell cycle-regulated genes and sites containing H3K79me2, but is found on H3K79me3-containing regions. A screen for genes responsible for the establishment/removal of H3K79 dimethylation resulted in identification of NRM1 and WHI3, both of which impact the transcription by the SBF and MBF protein complexes, further linking the regulation of methylation status of H3K79 to the cell cycle.

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Figures

Figure 1
Figure 1. GPS reveals that SWI4 and SWI6 are required for H3K79 di- but not trimethylation
(A - F) Cell extracts from each of the nonessential yeast gene deletion mutants were subjected to SDS-PAGE, Western blotted, and probed with antibodies specific for (A and B) H3K79 dimethylation or (C and D) trimethylation. (E and F) H3 K4 dimethylation was used as a control. Red arrows represent (A, C, E) swi4Δ and (B, D, F) swi6Δ. Blue arrows indicate empty wells that serve as plate markers. (G) Protein extracts from two different mating strains of swi4Δ and swi6Δ were analyzed as in A-F. Anti-acetyl H3 was probed for a loading control. (H) The swi4::kanMX mutant from the deletion collection was mated with a wild type strain and spores of the resulting tetrads were dissected and scored for the KanR and H3K79 methylation pattern.
Figure 2
Figure 2. Role of SWI4 and SWI6 in H3K79 methylation
(A ,B) Cell extracts were subjected to SDS-PAGE blotted to a membrane, and probed with H3 Lys79 di- or trimethyl specific antibodies. Anti-acetyl H3 was probed as a loading control. H3 Lys79 dimethylation could be rescued by a plasmid carrying either (A) SWI4 or (B) SWI6 under control of the GAL1 promoter. (C) RT-PCR was performed using cDNA made from RNA extracted from SWI4, as well as, SWI6 deletion strains via reverse transcriptase (RT). Primers specific for DOT1 were used to detect its mRNA. (D) The effect of the loss of SWI4 on histone H2B monoubiquitination levels. Highly purified acid extracted histones from wild type strains (strains containing FLAG-tagged H2B as the only source of histone H2B) or strains deleted for SWI4 in the same background, were analyzed by Western blotting using (upper panel) antibodies against the FLAG epitope or (mid and lower panels) polyclonal antibodies raised against trimethylated H3K79. As indicated by the red arrows, monoubiquitinated H2B is the slower migrating, and the ubiquitinated species of histone H2B is the faster migrating form. rad6Δ cells lack the E2 ubiquitin-conjugating enzyme required for H2B ubiquitination.
Figure 3
Figure 3. Cell cycle regulation of H3K79 dimethylation
(A) Cells lacking DOT1 accumulate in G1. Wild-type and dot1Δ cells were grown to mid-log phase at 30°C in YPD and DNA content was analyzed by flow cytometry. The percentage of cells in G1 with 1N DNA content, S phase with intermediate DNA content and G2/M with 2N DNA content was quantitated using 3 independent samples. Error bars represent standard deviation of the mean. (B-C) H3K79 dimethylation increases during S phase. Wild-type cells (SLJ001) were arrested in G1 with α-factor at 30°C for 3 hr. They were released into fresh YPD at 30°C, and α-factor was added back when small buds appeared, to prevent cells from entering the next cell cycle. Samples were taken at the indicated times following release from α-factor; asynchronous cells (A) were also collected. (B) DNA content was analyzed by flow cytometry to estimate cell cycle position. Entry into S phase begins around 15-30 min. based on the shift of the 1N DNA peak, while cells have entered G2/M approximately 45-60 min. after α-factor release. By 90 min., cells have exited M phase and re-arrested in G1. (C) Cell cycle-dependent modification of H3 was analyzed by immunoblotting with anti-acetyl-H3 and anti-H3K79me2 and anti-H3K79me3 antibodies. Equal protein concentrations were loaded in all lanes as judged by total H3 levels (data not shown). (D-F) DNA replication is not required for cell cycle oscillations in H3K79 dimethylation. Wild-type and GAL-CDC6 cells were grown in YEP containing 2% raffinose and 4% galactose at 30°C then arrested in G1 with α-factor (αf1). Cells were released from G1 into YEP containing 2% raffinose and 4% galactose to allow CDC6 expression to initiate DNA replication. After 20 min., cells were transferred into YPD to repress GAL-CDC6 and α-factor was added at 45 min. to arrest cells in G1 (αf2). Cells were then released from G1 in glucose-containing media to analyze cell cycle progression in cells lacking Cdc6 protein. α-factor was added back when small buds appeared, to prevent cells from entering the next cell cycle. Samples were taken at the indicated times following release from the second α-factor release; asynchronous cells (A) were also collected. (D) DNA content was analyzed by flow cytometry. While wild-type cells replicate DNA (30 min.), progress through mitosis and rearrest in G1, similar to our results in (B), cells lacking Cdc6 protein do not undergo DNA replication. Peak drift in this sample is likely due to mitochondrial DNA since cells continue to increase in size. Cell cycle-dependent modification of H3 was analyzed by immunoblotting with anti-H3K79me2 and anti-acetyl-H3 antibodies in wild-type (E) and GAL-CDC6 (F) cells. Equal protein concentrations were loaded in all lanes as judged by total H3 levels (data not shown).
Figure 4
Figure 4. High-resolution profile of H3K79me2 and H3K79me3 across the yeast genome with global occupancy analysis
(A) H3K79me2 and H3K79me3 profiles. Sample genomic positions for chromosome 4 and 8 were plotted along the x-axis against the relative occupancy of H3K79me2 and H3K79me3 on the y-axis. ORFs are indicated as rectangles, above the axis for Watson genes and below the axis for Crick genes. Green boxes represent HMM-predicted well-positioned and fuzzy nucleosome positions derived from Lee et al. (Lee et al., 2007) (B) Venn diagram comparing the number of H3K79me2 and -me3-enriched ORFs. (C) Average lengths of H3K79me3 and -me2-enriched genes. Boxplots showing the lengths of 6576 ORFs in yeast, as well as, the lengths of H3K79me3 and H3K79me2-enriched ORFs. (D-E) Average profile of H3K79me3 (D) and H3K79me2 (E)-enriched ORFs. A gene was considered to be enriched if at least 50% of its ORF was covered by the modification. ORFs were aligned according to their translational start and stop sites similar to an approach by the Young lab (Pokholok et al., 2005). Each ORF was divided into 40 bins of equal length, probes were assigned accordingly, and average enrichment values were calculated for each bin. Probes in promoter regions (500 base pairs upstream of transcriptional start site) and 3′UTR (500 base pairs downstream of stop site) were assigned to 20 bins, respectively. The average enrichment value for each bin was plotted.
Figure 5
Figure 5. Functional characterization of H3K79 dimethylated genes
(A-B) Average profiles of H3K79me3 (A) and H3K79me2 (B)-enriched ORFs according to transcriptional activity. All genes for which information was available (Holstege et al., 1998) (Transcriptome 2005) were divided into five classes according to their transcriptional rate. Average gene profiles were computed and plotted as described in Figure 4D. (C) Percent enrichment of H3K79 di- and trimethylated ORFs in different transcriptional classes. As before genes were divided into five classes according to their transcriptional activity (Holstege et al., 1998) and the percent overlap with H3K79 di- and trimethylated ORFs was plotted. (D-E) Overlap of H3K79me2 and H3K79me3-enriched ORFs with transcriptionally regulated genes for each cell cycle stage (Spellman et al., 1998). Numbers below the x-axis represent total number of genes with periodic transcription. Numbers above x-axis represent the overlap of these genes with those enriched for H3K79me2 and H3K79me3. The percentage of the overlap was plotted on the y-axis. (D) H3K79me2-enriched ORFs in asynchronous cells. Expected by chance are 28% (1866 H3K79me2-enriched ORFs out of 6576 total). (E) H3K79me3-enriched ORFs in asynchronous cells. Expected by chance are 36% (2350 H3K79me3-enriched ORFs out of 6576 total). The p-values were calculated using the hypergeometric test.
Figure 6
Figure 6. Genome-wide characteristics of H3K79me2-enriched genes in G2/M
(A-B) Overlap of H3K79me2-enriched ORFs in G2/M-arrested cells with transcriptionally regulated genes for each cell cycle stage (Spellman et al., 1998). (A) H3K79me2-enriched ORFs in G2/M-arrested cells. Expected by chance are 38% (2444 H3K79me2-enriched ORFs out of 6576 total). (B) H3K79me2-enriched promoters in G2/M. Expected by chance are 23% (1483 H3K79me2-enriched promoters out of 6576 total). (C) Average profile of H3K79me2-enriched ORFs in G2/M-arrested cells. The profile for the average enriched ORF was determined as explained in Figure 4D. (D) Average profile of H3K79me2-enriched ORFs in G2/M-arrested cells according to their transcriptional activity. The profile was determined as described in Figure 5A. (E) Percent enrichment of H3K79 dimethylated ORFs in different transcriptional classes. As before genes were divided into five classes according to their transcriptional activity (Holstege et al., 1998) and the percent overlap with H3K79 dimethylated ORFs in G2/M- arrested cells was plotted. (F) Venn diagram showing overlap of the 137 Swi4-bound genes (Iyer et al., 2001) with H3K79me2 and H3K79me3-enriched ORFs and promoters, respectively. H3K79me2-enriched promoters in G2/M-arrested cells showed significant overlap with Swi4-bound genes. H3K79me3-enriched ORFs and promoters show significant under-representation of Swi4-bound genes. Promoters were called enriched when 450bp upstream of the ORF were covered by the methyl mark.
Figure 7
Figure 7. Genome-wide profile of H2B monoubiqitination demonstrates an association with H3K79 trim but not dimethylation
(A) Development of polyclonal antibodies specific to monoubiquitinated H2B. H2B specific antibodies generated in rabbit were affinity purified and used for testing extracts from strains either carrying either Flag::H2B (lane 1) Flag::H2BK123R (lane 2), or wild type H2B in strains deleted for RAD6 (lane 3) or BRE1 (lane4). (B) Overlay of H2BK123ub, H3K79me2, and H3K79me3 profiles. Sample genomic positions for chromosome 8, and 10 were plotted along the x-axis against the relative occupancy of the indicated histone modifications on the y-axis. ORFs are indicated as rectangles, above the axis for Watson and below the axis for Crick strand. (C) Diagram summarizing the percentage of genome-wide occupancy of H3K79me2 and H3K79me3 and their overlap with H2BK123ub. (D) Diagram illustrating the overlap of H3K79me2 and H3K79me3 enriched with H2BK123ub enriched genes. (E) Overlap of H2BK123 ubiquitinated ORFs with transcriptionally regulated genes for each cell cycle stage (Spellman et al., 1998). Numbers below and above x-axis represent total number of genes in each cell cycle class and their overlap with H2B ubiquitinated genes, respectively. The percentage of overlap is plotted on the y-axis with a dashed line indicating the percentage expected by chance. The p-values were calculated using the hypergeometric test. (F) Yeast cell arrest arrested in the G1 and S phase of the cell cycle. To better understand the role of factors responsible for the implementation/removal of H3K79 dimethylation during G1/S stages of cell cycle, we performed a biochemical screen with the entire collection of viable yeast gene deletion mutants arrested with HU (Supplementary Figure 6). (G) This screen resulted in the identification of Nrm1 and Whi3, which is required for exit from the G1 phase of the cell cycle, as a factor required for the removal of dimethylated H3K79, further linking H3K79 methylation status to cell cycle.
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