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. 2009 Jul 3;10(8):894–900. doi: 10.1038/embor.2009.108

Figure 4.

Figure 4

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.