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. 2005 Jul;25(14):6123-39.
doi: 10.1128/MCB.25.14.6123-6139.2005.

Ubp10/Dot4p regulates the persistence of ubiquitinated histone H2B: distinct roles in telomeric silencing and general chromatin

Richard G Gardner  1 , Zara W Nelson, Daniel E Gottschling
Affiliations

Ubp10/Dot4p regulates the persistence of ubiquitinated histone H2B: distinct roles in telomeric silencing and general chromatin

Richard G Gardner et al. Mol Cell Biol. 2005 Jul.

Abstract

We previously discovered that the ubiquitin protease Ubp10/Dot4p is important for telomeric silencing through its interaction with Sir4p. However, the mechanism of Ubp10p action was unknown. We now provide evidence that Ubp10p removes ubiquitin from histone H2B; cells with UBP10 deleted have increased steady-state levels of H2B ubiquitination. As a consequence, ubp10delta cells also have increased steady-state levels of histone H3 Lys4 and Lys79 methylation. Consistent with its role in silencing, Ubp10p is preferentially localized to silent chromatin where its ubiquitin protease activity maintains low levels of H3 Lys4 and Lys79 methylation to allow optimal Sir protein binding to telomeres and global telomeric silencing. The ubiquitin protease Ubp8p has also been shown to remove ubiquitin from H2B, and ubp8delta cells have increased steady-state levels of H2B ubiquitination similar to those in ubp10delta cells. Unlike ubp10delta cells, however, ubp8delta cells do not have increased steady-state levels of H3 Lys4 and Lys79 methylation, nor is telomeric silencing affected. Despite their separate functions in silencing and SAGA-mediated transcription, respectively, deletion of both UBP10 and UBP8 results in a synergistic increase in the steady-state levels of H2B ubiquitination and in the number of genes with altered expression, indicating that Ubp10p and Ubp8p likely overlap in some of their _target chromatin regions. We propose that Ubp10p and Ubp8p are the only ubiquitin proteases that normally remove monoubiquitin from histone H2B and, while there are regions of the genome to which each is specifically _targeted, both combine to regulate the global balance of H2B ubiquitination.

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Figures

FIG. 1.
FIG. 1.
Ubp10p negatively regulates histone H2B ubiquitination and H3 Lys4 and Lys79 methylation levels. Global steady-state H2B ubiquitination levels from whole-cell lysates were assayed using anti-FLAG antibodies that recognize the FLAG epitope placed on the N terminus of H2B (76). Global steady-state H3 Lys4 and Lys79 methylation levels were also assayed using antibodies specific for mono-, di-, and trimethylated Lys4 forms of H3 as well as an antibody generated against the dimethylated Lys79 form of H3 (92). Whole-cell lysates were derived from strains UCC6369 (wild type), UCC6390 (ubp10Δ), UCC6392 (ubp8Δ), UCC6393 (ubp10Δ ubp8Δ), and UCC6163 (rad6Δ) that were harvested during log-phase growth. rad6Δ cells served as a negative control for H2B lacking ubiquitin. Changes in levels (n-fold) of H2B ubiquitination and H3 Lys4 and Lys79 methylation for each mutant strain relative to the wild-type strain are shown below each corresponding lane and are the average of three independent experiments. Quantitation was performed using National Institutes of Health ImageJ software.
FIG. 2.
FIG. 2.
Ubp10p is preferentially localized to silent chromatin. In vivo cross-linking analysis was performed using strains UCC6389 (untagged UBP10), UCC6390 (ubp10Δ), UCC6408 (UBP10-6Myc), UCC6406 (ubp10C371S-6Myc), UCC6407 (ubp10Δ94-250-6Myc), UCC6475 (UBP10-6Myc, sir2Δ), UCC6477 (UBP10-6Myc, sir3Δ), and UCC6479 (UBP10-6Myc, sir4Δ). A ChIP assay of Ubp10 proteins was performed using anti-Myc antibodies specific for the six-Myc tag fused to the C termini of the wild-type Ubp10, mutant Ubp10C371S, and mutant Ubp10Δ94-255 proteins. ChIP was also performed using untagged wild-type Ubp10p as a control. Multiplex PCR amplifications were performed to assess Ubp10p binding at the silent domains of telomere VIR and HMRa, the active SAN1 gene, and the repressed GAL1 gene. DNA of the total lysate was amplified as a control. (A) Representative example of Vistra Green-stained PCR amplifications. (B) Quantitative analysis of data in shown in panel A. Values represent the ratio of immunoprecipitate to total lysate for the query gene normalized to the ratio of immunoprecipitate to total lysate for SAN1. All values are the averages of at least two independent experiments. Error bars represent the standard deviation.
FIG. 3.
FIG. 3.
Loss of UBP10 primarily affects the expression of telomeric regions. Transcript array analysis was performed using strains UCC6389 (wild type) and UCC6390 (ubp10Δ) for panel A and strains UCC6432 (wild type), UCC6435 (ubp10Δ), UCC6406 (ubp10C371S), and UCC6407 (ubp10Δ94-250) for panel B. Cells were grown to log phase in rich medium (YEPD), and transcripts were isolated and analyzed. (A) Increased expression in ubp10Δ cells shows a telomeric bias. Number of genes increased in ubp10Δ cells plotted by position from the telomere. Histograms represent 20-kb increments from the telomeres. Red histograms represent genes with increased expression; green histograms represent genes with decreased expression. (B) ubp10Δ94-250 cells are specifically defective in telomeric silencing. Positional cluster analysis shows expression changes of genes based on distance from telomeres. Only genes expressed greater than or less than 1.5-fold in at least one of the strains are shown (total number of genes shown is 222). Genes located within 20 kbp of their respective telomeres are marked on the left as “telomeric regions.” See Table S1 in the supplemental material for the entire normalized data set.
FIG. 4.
FIG. 4.
Loss of Ubp10p activity results in increased histone H3 Lys4 and Lys79 methylation and decreased Sir3p binding at telomeres. In vivo cross-linking analysis was performed using strains UCC4825 (wild type), UCC4857 (ubp10Δ), UCC4870 (ubp10C371S), UCC4836 (ubp10Δ94-250), and UCC4836 (sir4Δ). ChIP assays of H3 Lys4 and Lys79 methylation and Sir3p binding were performed using antibodies specific for H3 Lys4 trimethylation, H3 Lys79 dimethylation, or Sir3p. Multiplex PCR amplifications were performed to assess the degree of H3 Lys4 methylation, H3 Lys79 methylation, and Sir3p binding at the silent domains of telomeres VIR and HMRa, the active SAN1 gene, and the repressed GAL1 gene. DNA of the total lysate was amplified as a control. (A) Representative example of Vistra Green-stained PCR amplifications. (B to D) Quantitative analysis of data in panel A. Values for H3 Lys4 and Lys79 methylation represent the ratio of immunoprecipitate to total lysate for the query gene normalized to the ratio of immunoprecipitate to total lysate for SAN1. Values for Sir3p binding represent the ratio of immunoprecipitate to total lysate for telomere VIR normalized to the ratio of immunoprecipitate to total lysate for HMRa. All values are the averages of at least two independent experiments. Error bars represent the standard deviation. (B) Relative fold change of H3 Lys4 tri-methylation at telomere VIR and GAL1. (C) Relative increase in H3 Lys79 methylation at telomere VIR and GAL1. (D) Relative decrease in Sir3p binding at telomere VIR.
FIG. 5.
FIG. 5.
Loss of UBP8 does not result in increased histone H3 Lys4 and Lys79 methylation and decreased Sir3p binding at telomeres. In vivo cross-linking analysis was performed as described in the legend of Fig. 4 using stains UCC6389 (wild type), UCC6392 (ubp8Δ), UCC6390 (ubp10Δ), UCC6393 (ubp10Δ ubp8Δ), and UCC6391 (sir4Δ). (A) Representative example of Vistra Green-stained PCR amplifications. (B to D) Quantitative analysis of data in shown in panel A was performed identically as described in the legend of Fig. 4B to D. (B) Relative increase in H3 Lys4 trimethylation at telomere VIR and GAL1. (C) Relative increase in H3 Lys79 methylation at telomere VIR and GAL1. (D) Relative decrease in Sir3p binding at telomere VIR. (E) Ubp8p does not function in telomeric silencing. Overnight, saturated cultures of yeast strains UCC6422 (wild type), UCC6423 (ubp10Δ), UCC6424 (sir4Δ), UCC6425 (ubp8Δ), and UCC6426 (ubp10Δ ubp8Δ), which all carry the URA3 gene located near telomere VIIL (85), were serially diluted and spotted onto YC plates, with or without uracil. Cells were grown at 30°C for 3 days.
FIG. 6.
FIG. 6.
Loss of silencing does not affect global H2B ubiquitination levels. (A) Loss of Ubp10p catalytic activity but not loss of Sir4p binding affects H2B ubiquitination and H3 Lys4 methylation. Global steady-state H2B ubiquitination and H3 Lys4 dimethylation levels from whole-cell lysates derived from strains UCC6195 (wild-type UBP10), UCC6199 (ubp10Δ), UCC6184 (ubp10C371S-6Myc), UCC6185 (ubp10Δ94-250-6Myc), and UCC6186 (UBP10-6Myc) were assayed using anti-FLAG antibodies (FLAG-tagged H2B) or antibodies that specifically recognized H3 Lys4 dimethylation. (B) Loss of silencing does not affect H2B ubiquitination or H3 Lys4 methylation. Global steady-state H2B ubiquitination and H3 Lys4 dimethylation levels were assayed as described in panel A using strains UCC6195 (UBP10), UCC6196 (sir2Δ), UCC6197 (sir3Δ), UCC6198 (sir4Δ), and UCC6199 (ubp10Δ).
FIG. 7.
FIG. 7.
Ubp10p and Ubp8p overlap in their _target chromatin regions. (A) Global steady-state levels of H2B ubiquitination and H3 Lys4 and Lys79 methylation from whole-cell lysates were assayed as described in the legend of Fig. 1. Whole-cell lysates were derived from strains UCC6369 (wild type), UCC6390 (ubp10Δ), UCC6392 (ubp8Δ), UCC6393 (ubp10Δ ubp8Δ), and UCC6163 (rad6Δ) that were harvested during log-phase growth, diauxie (overnight growth to saturation), and stationary phase (7-day saturation). (B) Some H2B ubiquitination persists in ubp10Δ and ubp8Δ cells in diauxie. H2B ubiquitination was assayed as described in the legend of Fig. 1. Arrow indicates where diubiquitinated form of H2B would run as expected in ubp10Δ ubp8Δ cells.
FIG. 8.
FIG. 8.
Loss of UBP10 and UBP8 has synergistic transcriptional effects primarily in active chromatin. Transcript array analysis using strains UCC6389 (wild type), UCC6390 (ubp10Δ), UCC6392 (ubp8Δ), and UCC6393 (ubp10Δ ubp8Δ) was performed as described in the legend of Fig. 2. (A) Venn diagrams show the degree of overlap in expression changes between ubp10Δ, ubp8Δ, and ubp10Δ ubp8Δ cells. (B) Expression changes in ubp10Δ ubp8Δ cells are primarily in nontelomeric regions. Positional cluster analysis shows expression changes of genes based on distance from telomeres. Only genes that were expressed greater than or less than 1.5-fold in at least one of the strains are shown (total genes shown is 368). Genes located in telomeric regions are marked on the left. See Table S1 in the supplemental material for the entire normalized data set.
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