Set2 methyltransferase facilitates cell cycle progression by maintaining transcriptional fidelity

Abstract

Methylation of histone H3 lysine 36 (H3K36me) by yeast Set2 is critical for the maintenance of chromatin structure and transcriptional fidelity. However, we do not know the full range of Set2/H3K36me functions or the scope of mechanisms that regulate Set2-dependent H3K36 methylation. Here, we show that the APC/CCDC20 complex regulates Set2 protein abundance during the cell cycle. Significantly, absence of Set2-mediated H3K36me causes a loss of cell cycle control and pronounced defects in the transcriptional fidelity of cell cycle regulatory genes, a class of genes that are generally long, hence highly dependent on Set2/H3K36me for their transcriptional fidelity. Because APC/C also controls human SETD2, and SETD2 likewise regulates cell cycle progression, our data imply an evolutionarily conserved cell cycle function for Set2/SETD2 that may explain why recurrent mutations of SETD2 contribute to human disease.

Figure 1.

Long genes prone to cryptic transcripts are associated with cell cycle related functions. (A) Network analysis, following pathway enrichment analysis, of genes that give rise to cryptic transcript from (32) showing the ontology relationships between the pathways. Genes that give rise to cryptic transcripts (429 genes from Lickwar et al. (32)) in set2Δ cells reveal an enrichment of these genes with the cell cycle, mitosis, DNA replication and cell polarity. The direction of the arrows shows the ontogeny of related functional categories. (B) A Venn diagram comparing genes that are periodic (top 800 from Cyclebase) to those that give rise to cryptic transcripts in set2Δ cells reveal a significant overlap (p = 0.00548). (C) Venn diagram showing that genes which give rise to cryptic transcription in set2Δ cells also tend to be long (P < 1 × 10⁻²⁰). (D) Plot showing the relationship between long genes (800 random genes) and periodic genes (top 800 from Cyclebase) reveal periodic genes tend to be longer than expected by chance (P< 2.531 × 10⁻⁵ Kolmogorov–Smirnov test). (E) and (F) RT-qPCR analysis of sense and antisense transcripts originating from the SWE1 (SUT201) and CDC28 genes (SUT_FS0074), demonstrating the reduced sense transcript levels of SWE1 and CDC28 in set2Δ cells.

Figure 2.

The transcriptional fidelity of cell cycle-regulated genes is maintained by Set2. (A) Diagrammatic representation of the FAR1 locus and its corresponding antisense stable un-annotated transcript (SUT204). (B) qRT-PCR showing the absence of SET2 results in mis-regulation of the levels of both sense and antisense FAR1 transcripts. (C) FAR1 antisense transcript, SUT204, is increased in set2Δ cells at all points across the cell cycle, as assessed by qRT-PCR. (D) Schematic representation of the genomic locus of SWE1 and its corresponding antisense transcript, SUT201. (E) Loss of SET2 results in the mis-regulation in the levels of SWE1 sense transcript (compare the levels of SWE1 transcript at 30 and 45 min between WT and set2Δ). (F) qRT-PCR of SWE1 antisense transcript SUT201, shows altered expression kinetics in set2Δ cells. (G) Schematic representation of the FAR1 locus with an insertion of a 2 kb KANMX cassette to displace the SUT204 from the FAR1 3′ end (H) qRT-PCR to detect the levels of FAR1 transcript in a strain that lacks the 2 kb KANMX cassette and showing a similar down-regulation of FAR1 as in Figure 2B. (I) levels of SUT204 in a strain containing the 2 kb KANMX cassette showing partial abrogation of the transcript. The data in all the above experiments are represented as the standard deviations of three biological replicates with three technical replicates in individual experiments. The statistical significance was calculated using paired t test (* represents P value <0.01).

Figure 3.

Set2 is required to repress cryptic unstable transcripts at the cell cycle-regulated DBF2 locus. (A) Schematics of DBF2 gene organization alongside its neighboring gene (DRN1) and the cognate antisense transcripts CUT613 and CUT614, respectively. (B) Genome browser shot of the sense and antisense transcripts originating from the WT and set2Δ cells. (C) and (D) WT or set2Δ cells were arrested with α-factor, then released into fresh medium, after which samples were collected at indicated time points for strand-specific qRT-PCR. Results show that as DBF2 levels increase in G2/M, its corresponding antisense CUT613 is also detectable. Absence of Set2 results in significant increase in CUT613 production that correlates with sense decrease, suggesting that CUT613 antisense transcripts can interfere with sense transcription. The data in are represented as the standard deviations of three biological replicates with three technical replicates in individual experiments. Statistical significance was calculated using paired t test (* represents P value <0.01).

Figure 4.

Loss of Set2 and H3K36 methylation abrogates yeast cell cycle progression. (A) Representative flow cytometry profiles of WT and set2Δ cells after α-factor arrest and release, which reveal defects in the ability of set2Δ cells to properly progress through the cell cycle. Comparison of the profiles at 30, 45 and 60 min after release in G1 show set2Δ cells have a delayed entry into S-phase, which then progresses faster than that observed for WT cells. (B–D) Quantification of multiple flow cytometry profiles of WT and set2Δ cells (n > 3) in which G1, S and G2/M cells from WT and set2Δ cells are counted.

Figure 5.

Yeast and human Set2 are cell cycle regulated proteins. (A) Set2 protein is cell cycle regulated. WT and set2Δ cells were arrested in the G1 phase using α-factor and released into fresh medium. Whole cell extracts were prepared at indicated time points and probed for Set2, H3, H3K36me3, Clb2 (marker for cell cycle progression) and G6PDH (total protein loading). (B) Set2 protein levels are down-regulated during progression through mitosis and mitotic exit. Whole cell extracts were prepared at designated time points after release from nocodazole. WT cells were released into fresh medium and immunoblotted for Set2, Clb2 (a marker for cell cycle progression), Pds1 (a marker for metaphase to anaphase transition) and G6PDH (total protein loading control). Analogous to Set2, H3K36me3 levels similarly change across the cell cycle, as analyzed by immunoblotting for H3K36me2/me3, and anti-H3 (loading control). (C) Human SETD2 is a cell cycle regulated protein. U2OS cells were synchronized using nocodazole, collected at indicated time points, and immunoblotted for SETD2. CycA, CycE and H3S10P serve as markers for different cell cycle stages, whereas RAN was used as a loading control.

Figure 6.

The Anaphase Promoting Complex (APC) promotes Set2 protein turnover across the cell cycle. (A) Compromised APC/C function stabilizes Set2 protein levels. Cycloheximide chase experiments were performed using a WT or APC mutant allele (cdc23–1) after shift to restrictive temperature (37°C), and cell extracts were immunoblotted for Set2. G6PDH was used as a loading control. (B) Set2 is not regulated by APC/C^Cdh1. Cycloheximide chase experiments were performed as described in panel (A) in WT and CHD1 deleted cells (cdh1Δ) to detect the rate of Set2 loss. (C) Set2 is regulated by the APC/C^Cdc20. A GAL-CDC20 strain was grown in dextrose for 2 h to shut off expression of CDC20, and protein extracts were immunoblotted to probe for Set2, Clb2 and G6PDH. (D) An immunoblot showing the decrease in the levels of Set2 and H3K36me3 in a strain that contains a kinase-dead allele of BUB1, a checkpoint kinase that negatively regulates CDC20. (E) Representation of Set2 protein showing the domains and two putative D-boxes in the protein sequence. For comparison, a canonical D-box sequence is shown. (F) Mutation of the conserved D-box residues in Set2 (RXXL → AXXA) enhances Set2 stability. Cycloheximide chase experiments were performed at the indicated time points on WT and the D-box mutant strains and probed for Set2 and G6PDH. (G) Set2 protein levels become uncoupled from cell cycle regulation upon D-box mutation. Lysates were prepared from WT and D-Box mutants of Set2 post G1 arrest and released at indicated time points. Lysates were probed for Set2, Clb2 and G6PDH. (H) Appropriate turnover of Set2 protein is required for proper response to mitotic poison, benomyl. Five-fold serial dilutions of indicated strains were spotted on either SC-Ura or SC-Ura with benomyl (30 μg/mL).

Figure 7.

Model for the function of Set2 in regulating the cell cycle by maintaining transcriptional fidelity. In WT cells, Set2-dependent H3K36 methylation is required for the recruitment/activation of the Rpd3S histone deacetylase complex, which maintains a repressed chromatin state that is refractory to pervasive cryptic transcription. Our results show that Set2 protein and H3K36me levels are cell cycle regulated, with lowest levels in the G1- and S-phases and highest in the G2/M-phase. In addition, we found that Set2 and H3K36 methylation is required for the proper expression of cell cycle regulated genes that tend to be long and highly dependent on Set2. Because Set2/H3K36me levels fluctuate across the cell cycle, we speculate that these changing levels fine-tune the levels of cryptic transcription across the cell cycle to allow for optimal expression of genes required for transition through the different cell cycle phases.