CDK9 activity is essential for maintaining global levels of H2Bub1. (A) H1299 cells were treated with the specific CDK9 inhibitors KM, DRB, FP or CDK9 inhibitor II (CDK9iII) at the indicated concentrations for 4 h and analysed by Western blot analysis. (B,C) H1299 cells were transfected with (B) control or CDK9 siRNAs or (C) expression plasmids and analysed by Western blot with the indicated antibodies. (D) The CTD and Ser 2 are necessary for maintaining H2Bub1 levels. HEK293 cells were transfected with α-amanitin-resistant RNAPII large subunit expression constructs containing wild-type (WT), deletion or mutant CTD. (E) Regulation of H2Bub1 by SUPT5H, NELF-E, RNF20/40 and PAF1. H1299 cells were transfected and analysed by Western blot using the indicated siRNAs and antibodies. H2B and HSC 70 are shown as loading controls. CDK9, cyclin-dependent kinase 9; CTD, carboxyl-terminal domain; DRB, 5,6-dichloro-β-D-ribofuranosyl benzimidazole; FP, flavopiridol; H2Bub1, histone H2B monoubiquitination; HEK, human embryonic kidney; HSC70, heat shock cognate protein 70; KM, KM05283; NELF-E, negative elongation factor E; PAF, polymerase II associated factor; RNAPII, RNA polymerase II; RNF, ring finger protein; siRNA, small interfering RNA; SUPT5H, suppressor of Ty 5 homologue.

CDK9 activity recruits proteins involved in H2B ubiquitination and histone messenger RNA 3′-end formation, and decreases RNAPII read-through. ChIP analysis of the HIST1H2BD (or HIST1H2AC in (E)) gene in HEK293 cells after transfection with control (grey bars) or CDK9 (black bars) siRNAs using the indicated antibodies. (A) TBP is specifically enriched at the TSS and is not affected by CDK9 knockdown, whereas CDK9 is significantly enriched both at the TSS and at 0.5 kb and these levels decrease on knockdown. (B) CDK9 knockdown similarly decreases RNAPII and P-Ser 5-RNAPII levels at the TSS without affecting elongation to the 3′-end cleavage site. By contrast, P-Ser 2 is markedly decreased to near-background levels both at the TSS and at 0.5 kb. (C) The H2B-ubiquitinating complex (PAF1, RNF20 and RNF40) is enriched at 0.5 kb of the HIST1H2BD gene together with H2Bub1, and their levels decrease following CDK9 knockdown. (D) ChIP analysis of SLBP and the U7 snRNP component LSM11 shows decreased recruitment to the 3′-cleavage site. (E) CDK9 knockdown increases RNAPII read-through past the normal 3′-end cleavage site of the HIST1H2BD and HIST1H2AC genes. ChIP analysis of total RNAPII levels at the polyadenylation site (13 kb for HIST1H2BD or 15 kb for HIST1H2AC) were compared with the amount of total initiating RNAPII (at the TSS) and plotted as the percentage read-through of RNAPII. ChIP experiments are shown as percentage input as in Fig 1; mean values+s.d., n=3. Statistically significant differences are indicated: ^*P⩽0.05; ^**P⩽0.01; ^***P⩽0.001. The experimental background is shown in each graph as a dotted line. CDK9, cyclin-dependent kinase 9; ChIP, chromatin immunoprecipitation; H2Bub1, histone H2B monoubiquitination; HEK, human embryonic kidney; LSM11, Sm-like protein 11; PAF, RNA polymerase II associated factor; RNAPII, RNA polymerase II; RNF, ring finger protein; SLBP, stem–loop (histone) binding protein; siRNAs, small interfering RNAs; snRNP, small nuclear ribonucleoprotein; TBP, TATA-binding protein; TSS, transcriptional start site, 0.5 kb is equal to 3′ end cleavage site, 0.9 kb is equal to 400 bp 3′ to the cleavage site, 13 kb or 15 kb is equal to polyadenylation site of the read-through transcripts for HIST1H2BD and HIST1H2AC, respectively.

H2Bub1 depends on CDK9 activity rather than transcriptional activation per se. (A) H1299 cells were treated with actinomycin D for the indicated times to inhibit transcription, and protein lysates were analysed by Western blot for H2Bub1 and H2B protein levels. (B) Nutlin 3 and DRB both increase levels of p21 mRNA. U-2 OS cells were treated with 8 μM nutlin 3 or 50 μM DRB for the indicated times before RT–PCR analyses. Transcript levels were normalized to mitochondrial 16S ribosomal RNA and expressed relative to the control treatment; mean values+s.d., n=3. (C–E) P-Ser 2, rather than elongation by RNAPII, is essential for maintaining gene-associated H2Bub1. U-2 OS cells were treated with nutlin 3 or DRB for 8 h and analysed by ChIP using antibodies against (C) total RNAPII, (D) P-Ser 2 or (E) H2Bub1. qChIP values were normalized to their respective DNA inputs and expressed as DNA recovery (percentage input); mean values+s.d., n=3. The experimental background is shown as a dotted line. Act.D, actinomycin D; CDK9, cyclin-dependent kinase 9; ChIP, chromatin immunoprecipitation; DRB, 5,6-dichloro-β-D ribofuranosyl benzimidazole; H2Bub1, histone H2B monoubiquitination; mRNA, messenger RNA; qChIP, quantitative chromatin immunoprecipitation; RNAPII, RNA polymerase II; RT–PCR, reverse transcription PCR; TR, transcribed region; TSS, transcriptional start site.

CDK9 and H2Bub1 direct replication-dependent histone messenger RNA 3′ end processing but not transcription. (A) Schematic representation of the human HIST1H2BD and HIST1H2AC genes showing both normally processed and polyadenylated transcripts resulting from the splicing of a longer transcript. (B) Total HIST1H2BD and HIST1H2AC expression is not significantly affected by CDK9, RNF20 or RNF40 knockdown. Random-primed cDNAs were analysed for total HIST1H2BD or HIST1H2AC expression after transfection of HEK293 cells with control, CDK9, RNF20 or RNF40 siRNAs. The expression of total histone transcripts was normalized to a control gene, 36B4, and is represented as relative mRNA expression; mean values+s.d., n=3. (C) CDK9 knockdown increases the formation of read-through HIST1H2BD transcripts. Total RNA from HEK293 cells transfected with control or CDK9 siRNA was analysed by random-primed qRT–PCR using specific primers for total and read-through HIST1H2BD transcripts. The expression of processed (left panel) and read-through transcripts (right panel) was normalized to 36B4 and is shown as the percentage total transcript; mean values+s.d., n=3. (D,E) CDK9 and RNF40/20 knockdown increase the formation of (D) polyadenylated HIST1H2BD and (E) HIST1H2AC transcripts. Total mRNA from cells transfected with control, CDK9, RNF40 or RNF20 siRNA was analysed by oligo-dT-primed qRT–PCR. Expression levels were normalized and expressed as in (A); mean values+s.d., n=3. CDK9, cyclin-dependent kinase 9; cDNA, complementary DNA; ChIP, chromatin immunoprecipitation; H2Bub1, histone H2B monoubiquitination; HEK, human embryonic kidney; mRNA, messenger RNA; RNF, ring finger protein; siRNAs, small interfering RNAs, qRT–PCR, quantitative reverse transcription PCR; RNF, ring finger protein; siRNAs, small interfering RNAs.