Recruitment of active P-TEFb to chromatin

Subsequently to the identification of P-TEFb as a general transcription elongation factor, many studies have investigated the mechanisms by which P-TEFb is recruited to chromatin. Although free active P-TEFb complex is potentially able to activate transcription, its targeting to gene promoters or enhancers is necessary for efficient gene expression (Taube et al., 2002 ; Lin et al., 2002c). To date, a variety of transcriptional activators have been shown to interact with P-TEFb, allowing its recruitment to specific targets. A summary of these cofactors is provided in table 1. The first P-TEFb recruiting factor to be identified was CIITA, a class II transactivator which regulates the expression of MHC (major histocompatibility complex) genes (Kanazawa et al., 2000). CIITA binds CyclinT1 through the same domain as Tat. Consequently, both cofactors compete for P-TEFb recruitment to their respective target promoters in lymphoid tissues (Kanazawa et al., 2000). Subsequently, multiple transcription factors were found to interact with and to recruit P-TEFb to selective targets, including the nuclear factor-kappa B (NF-kB), the androgen receptor, c-Myc, the Aryl hydrocarbon receptor, the autoimmune regulator AIRE and MyoD (Table 1). In particular, P-TEFb was shown to be an essential factor for c-Myc-mediated transactivation. Myc interacts with the cyclin box of CyclinT1, and recruits P-TEFb to various E box-containing promoters (Eberhardy and Farnham, 2002 ; Kanazawa et al., 2003). Importantly, specific P-TEFb inhibition eventually impaired the Myc-induced proliferation and apoptosis in vivo (Kanazawa et al., 2003 ; Gargano et al., 2007). Because inactivation of P-TEFb does not prevent its recruitment by Myc, it is likely that RNAPII phosphorylation at Ser-2 represents an important limiting step for transcription of Myc-target genes (Gargano et al., 2007).

While the majority of P-TEFb recruiting factors listed above were shown to interact with CyclinT1, the transcription factor MyoD specifically associates with CyclinT2a-containing complex (Simone et al., 2002a ; Giacinti et al., 2006). During myogenesis, MyoD promotes cell differentiation by its ability to arrest the cell cycle and to activate muscle-specific transcription (Puri and Sartorelli, 2000). MyoD interacts with and is phosphorylated

Figure 8. Regulation of P-TEFb activity through interactions with 7SK/HEXIM and Brd4.

The P-TEFb inhibitory complex 7SK snRNP comprise the 7SK snRNA, a HEXIM dimer and two P-TEFb molecules. The 5’ end of 7SK is protected by the capping enzyme MEPCE while its 3’ extremity associates with LARP7/PIP7S. The complex assembly involves the sequential interaction of HEXIM with the 5’ stem-loop of 7SK followed by P-TEFb binding to HEXIM and the 3’ stem loop of 7SK. P-TEFb interaction with HEXIM and 7SK required auto-phosphorylation of Cdk9 at its activating Thr-186 residue, and possibly deacetylation of Cdk9 by N-CoR/HDAC3. The release of P-TEFb from 7SK snRNP eventually involve dephosphorylation of Cdk9 by PP2B/PP1α phosphatases and Cdk9 acetylation. Free P-TEFb can then be recruited to active promoters through interaction with Brd4 and/or gene-specific cofactors. P-TEFb-free 7SK RNA can be converted into others 7SK snRNP complexes by association with several hnRNP proteins such as hnRNP A1, A2/B1, Q, R and RHA (RNA helicase A).

by Cdk9/CyclinT2a resulting in myogenic differentiation, while inhibition of Cdk9 kinase activity prevents the activation of the myogenic program (Simone et al., 2002a). Additionally, Cdk9/CyclinT2 can also phosphorylate pRb, an essential cofactor for MyoD-dependent transcription (Simone et al., 2002b). Recent data indicate that concomitantly with Cdk9/CyclinT2, MyoD also recruits the histone acetyltransferases p300 and PCAF as well as the chromatin remodeling complex SWI/SNF to myogenic promoters (Giacinti et al., 2006).

Therefore, MyoD likely stimulates muscle-specific transcription by facilitating nucleosome acetylation, chromatin remodeling and RNAPII phosphorylation by P-TEFb.

All the data presented above support a model in which distinct P-TEFb complexes are recruited to selective promoters by specific transcription factors or cofactors. In other hand, two recent studies provided evidence that a large fraction of active P-TEFb associates with the bromodomain-containing protein Brd4 to activate transcription in a more general manner (Jang et al., 2005 ; Yang et al., 2005). Originally named MCAP (mitotic chromosome-associated protein), Brd4 is a ubiquitously expressed protein which binds acetylated nucleosomes, via its bromodomains, at actively transcribed genes (Dey et al., 2003). The bromodomains of Brd4 were also shown to mediate interaction with P-TEFb in vivo, presumably with either Cdk9 or CyclinT1 subunit (Jang et al., 2005 ; Yang et al., 2005). In HeLa cells, roughly half of nuclear P-TEFb is associated with Brd4, and Brd4 depletion causes a decrease of RNAPII transcription of many genes (Jang et al., 2005 ; Mochizuki et al., 2008). In particular, Brd4 knockdown was recently shown to impaired G1 genes transcription in a P-TEFb-dependent manner, thus preventing cell cycle progression toward S phase (Yang et al., 2008 ; Mochizuki et al., 2008).

Interestingly, Brd4 also interacts with a subset of Mediator complexes (Houzelstein et al., 2002 ; Wu et al., 2003b ; Yang et al., 2005). The current data suggest a model where Brd4 binding to acetylated chromatin facilitates recruitment of Mediator to the promoterregion.

Following PIC assembly and transcription initiation, Brd4-associated Mediator would then recruit P-TEFb to promote transcription elongation (Yang et al., 2005 ; Wu and Chiang, 2007). Although Brd4 appears to be an important P-TEFb-recruiting factor in mammals, it is unlikely that Brd4 tracks along with P-TEFb during transcription elongation. Instead, when overexpressed, Brd4 eventually prevents activation of the HIV-1 promoter due to the competition between Tat and Brd4 for P-TEFb binding (Yang et al., 2005 ; Urano et al., 2008). A recent report has indeed demonstrated that Brd4 and P-TEFb interact only transiently at the HIV-1 promoter, and the release of Brd4 following transcription initiation

coincides with a change in the phosphorylation status of Cdk9 resulting in full P-TEFb kinase activity (Zhou et al., 2009).