P-TEFb Kinase Complex Phosphorylates Histone H1 to Regulate Expression of Cellular and HIV-1 Genes

Abstract

Transcription of HIV-1 genes depends on the RNA polymerase II kinase and elongation factor positive transcription elongation factor b (P-TEFb), the complex of cyclin T1 and CDK9. Recent evidence suggests that regulation of transcription by P-TEFb involves chromatin binding and modifying factors. To determine how P-TEFb may connect chromatin remodeling to transcription, we investigated the relationship between P-TEFb and histone H1. We identify histone H1 as a substrate for P-TEFb involved in cellular and HIV-1 transcription. We show that P-TEFb interacts with H1 and that P-TEFb inhibition by RNAi, flavopiridol, or dominant negative CDK9 expression correlates with loss of phosphorylation and mobility of H1 in vivo. Importantly, P-TEFb directs H1 phosphorylation in response to wild-type HIV-1 infection, but not Tat-mutant HIV-1 infection. Our results show that P-TEFb phosphorylates histone H1 at a specific C-terminal phosphorylation site. Expression of a mutant H1.1 that cannot be phosphorylated by P-TEFb also disrupts Tat transactivation in an HIV reporter cell line as well as transcription of the c-fos and hsp70 genes in HeLa cells. We identify histone H1 as a novel P-TEFb substrate, and our results suggest new roles for P-TEFb in both cellular and HIV-1 transcription.The transcription of many viral and eukaryotic genes is controlled at the point of mRNA elongation by positive transcription elongation factor b (P-TEFb), 2 the complex of CDK9 and cyclin T1. This P-TEFb control has been shown in the HIV-1 genes encoding proviral peptides (1, 2), oncogenes such as c-fos (3), and inducible genes such as hsp70 (4). The transcription of these genes is initiated by RNA polymerase II (Pol II), but is inhibited shortly thereafter by Pol II pausing (1, 2). This pause is alleviated by P-TEFb, which increases Pol II processivity by hyperphosphorylating the Ser-2 sites of its C-terminal domain, thus promoting the transition from abortive to productive mRNA elongation (1,2). Importantly, HIV-1 transcription can be prevented by inhibiting P-TEFb phosphorylation of Pol II, such as by siRNA-mediated knockdown of CDK9 or by treating infected cells with the potent CDK9 inhibitor, flavopiridol (5, 6).Active P-TEFb also binds to multiple chromatin-binding and -modifying proteins such as the bromodomain-containing protein Brd4. Brd4 recruits P-TEFb to promoters by binding both acetylated histone proteins and components of the Mediator complex (7-9). P-TEFb not only interacts with Brd4 at cellular promoters, but also binds specific transcription factors such as NFB during cellular and HIV transcription (10, 11). Thus, P-TEFb may function in coupling chromatin modification to transcription.Histone H1 is a highly abundant linker histone protein that binds to nucleosomes and the DNA that connects them (12). H1 facilitates the compaction of chromatin, thus maintaining chromatin patterns during differentiation and development (13,14). In addition to its role in chromatin compaction, H1 exists in equilibrium between chromatin-bound and -fre...

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“…Besides the CTD, P-TEFb also phosphorylates the negative elongation factors DSIF and NELF to antagonize their inhibitory actions, triggering the release of Pol II from promoter-proximal pausing (Figure 1A). Recently, histone H1 was also found to be phosphorylated by P-TEFb, representing another process that promotes cellular and HIV transcription and is modulated by Tat (O’Brien et al, 2010). …”