Figure 1.
Loss of H2Bub1 globally alters distribution of RNAPII in gene coding regions.
(A) Average distribution of H2Bub1 at 540 S. pombe genes, as determined by ChIP-chip. Genes were grouped according to total levels of RNAPII enrichment (see key at top). The grey box in the “average gene” representation at bottom denotes the gene coding region; 5′ and 3′ untranslated regions are denoted by thin black lines. The arrow denotes the transcription start site. (B) Average distribution of RNAPII at 540 S. pombe genes, as determined by ChIP-chip in a wild-type strain (JTB62-1). Genes were grouped according to total levels of RNAPII enrichment. (C) As in (B), determined in an htb1-K119R mutant strain (JTB67-1). Gene groupings were created using wild-type RNAPII enrichment values. (D) Average distributions of differences between mutant and wild-type RNAPII enrichment grouped according to RNAPII enrichment in wild-type cells. The key below the graph illustrates the statistical significance of the differences for each group at 50 positions along the average gene. The rows of the key are color-coded according to the graph. Open squares denote p>0.01; light shading denotes 0.01>p>10exp-5; dark shading denotes p<10exp-5 (one-sample t-tests; μ0 = 0). Note that there is only light shading for the last row (corresponding to the blue curve).
Figure 2.
H2Bub1 depends on Cdk9 activity and Spt5 phosphorylation.
(A) Immunoblots of whole-cell extracts from wild-type (wt) (JS78) or AS mutant strains (LV7, LV77, LV42), as indicated, grown in the absence (−) or presence (+) of 20 µM 3-MB-PP1, added 20 min prior to harvest. Antibodies are indicated at right. (B) Immunoblots of extracts from indicated strains probed for total histone H2B and H2Bub1, as indicated at right. “T212A” and “T212E” denote strains cdk9-T212A (HD7-24) and cdk9-T212E (HG127). (C) Immunoblots of extracts from wild-type (JS78) or indicated spt5 mutant strains.
Figure 3.
H2Bub1 independently stimulates Cdk9-mediated Spt5 phosphorylation and Set1-dependent H3K4 methylation.
(A) Immunoblots of extracts from strains carrying spt5-myc with or without an as kinase allele as indicated (CS111, CS112, CS155, CS159, LV125 and LV167, respectively). Cultures were grown in the absence (−) or presence (+) of 20 µM 3-MB-PP1, added 20 min prior to harvest. Antibody reactivities are indicated at right. (B) Immunoblots of extracts from indicated strains (JS78, JTB67-1, JTB62-1, JTB331, HD7-24 and JTB297, respectively), probed for phosphorylated (Spt5-P) or total Spt5. (C) Fluorescent images of DAPI/calcofluor-stained cells from indicated strains (spt5-WT, JTB350, spt5-T1A, JTB352, spt5-T1E, JTB354, respectively). (D) Quantification of abnormal septation in strains of indicated genotypes (JTB67-1, spt5-WT, JTB350, spt5-T1A, JTB352, spt5-T1E, JTB354, JTB418 and JTB428, respectively). Error bars represent standard deviations from 2 independent experiments; at least 200 cells were counted in each. (E) Immunoblots of extracts from indicated strains (JTB204, JTB80-2, and JTB331, respectively), probed for phosphorylated (Spt5-P) or total Spt5. (F) Immunoblots of extracts from indicated strains (JTB204, JTB80-2, JTB67-1, spt5-WT, spt5-T1A and spt5-T1E, respectively), probed for H3K4me3 or total H3.
Figure 4.
H2Bub1 enhances Cdk9 recruitment and Spt5 phosphorylation at transcribed genes.
(A,B) Spt5 phosphorylation was measured by ChIP using anti-Spt5-P in spt5-myc (MS265; black bars) and spt5-myc htb1-K119R (LV239; gray bars) strains and quantified at the indicated genes by qPCR. Enrichment is plotted as a percentage of input signal for each primer pair. Positions of PCR primer pairs within coding regions are indicated schematically at top. (C,D) Spt5-myc occupancy was measured by ChIP as in A and B. (E,F) Spt5-P enrichment normalized to total Spt5-myc occupancy. (G,H) Cdk9-myc occupancy was measured by ChIP in cdk9-myc (MS264; black bars) and cdk9-myc htb1-K119R (KL259; gray bars) strains. (I,J) H2Bub1 enrichment was measured by ChIP and normalized to H2B-FLAG occupancy. Error bars denote standard deviations from 3 independent experiments. Asterisks denote a significant difference between wild-type and mutant (“*” p<0.04, “**” p<0.02; unpaired t-test).
Figure 5.
Mutual phenotypic suppression in cdk9 htb1-K119R double mutants.
(A) For indicated strains (JS78, LV7, JTB62-1, JTB67-1, MS249, LV193), growth in increasing [3-MB-PP1] is plotted as a percentage of growth in the absence of 3-MB-PP1. Error bars denote standard deviations from 3 independent experiments. (B) Images of DAPI- and calcofluor-stained htb1-K119R (JTB67-1) and cdk9as htb1-K119R (LV193) cells grown in the absence (top) or presence (middle) of 10 µM 3-MB-PP1 for 7 hr, or after inhibitor washout and return to growth (bottom). (C) Fluorescent images of DAPI/calcofluor-stained wild-type (JS78), cdk9-T212A (HD7-24), brl2Δ (JTB331), brl2Δ cdk9-T212A (JTB335), ubp8Δ (JTB297), ubp8Δ cdk9-T212A (JTB336), htb1-K119R (JTB67-1), cdk9-T212A htb1-K119R (LV252) cdk9-T212E htb1-K119R (LV256), and mcs6-S165A htb1-K119R (LV254) cells. (D) Quantification of abnormal septation patterns in strains of indicated genotypes (JTB62-1, JTB67-1, JTB325, JTB326, JTB331, JTB335, JTB377, JTB333 respectively). Error bars represent standard deviations from 2 independent experiments; at least 200 cells were counted in each. (E) Flocculation of indicated htb1-FLAG strains (JTB62-1, JTB67-1, JTB325, JTB326) was quantified as described in Materials and Methods. Error bars denote standard deviations from 2 independent experiments.
Figure 6.
Cdk9 activity towards multiple substrates is required for abnormal morphologies of H2Bub1-deficient cells.
(A) Quantification of abnormal septation patterns in the indicated htb1-FLAG strains (JTB378, JTB351, JTB379, JTB353, JTB380, JTB355). Error bars represent standard deviations from 2 independent experiments; at least 200 cells were counted in each. (B) As in (A) for the indicated cdk9as strains (KL289, KL291, KL293). Cells were grown in the presence of either DMSO or 3-MB-PP1 as described in Figure 5B. (C) As in (A) for the indicated htb1-FLAG strains (MS260, MS256, MS261, MS257, MS272, MS259). (D) Flocculation of indicated strains (JS78, JTB67-1, JTB351, JTB353, KL291, KL293, MS259) was quantified as described in Materials and Methods. (E) Growth rates in liquid rich medium (YES) were measured for strains analyzed in (D).
Figure 7.
Opposing effects of H2Bub1 and Cdk9 activity on RNAPII distribution revealed by ChIP–chip.
(A) Average distribution of RNAPII at 540 S. pombe genes, as determined by ChIP-chip in a cdk9-T212A strain (JTB325). Genes were grouped according to total levels of RNAPII enrichment. (B) As in (A) for cdk9-T212A htb1-K119R (JTB326). (C) Average distributions of differences between cdk9-T212A (JTB325) and wild-type (JTB62-1) RNAPII enrichment grouped according to RNAPII enrichment in wild-type cells. (D) As in (C) for differences between cdk9-T212A htb1-K119R and wild-type RNAPII enrichment. (E) As in (C) for differences between cdk9-T212A htb1-K119R and cdk9-T212A RNAPII enrichment. (F) As in (C) for differences between cdk9-T212A htb1-K119R and htb1-K119R RNAPII enrichment. The keys below C-F illustrate the statistical significance of the differences for each group at 50 positions along the average gene. The rows of the key are color-coded according to the graph. Open squares denote p>0.01; light shading denotes 0.01>p>10exp-5; dark shading denotes p<10exp-5 (one-sample t-tests; μ0 = 0). Note that there is only light shading for the last row (corresponding to the blue curve).
Figure 8.
Model depicting positive and negative interactions between P-TEFb and H2Bub1 during transcript elongation (see text for details).