A HEXIM1 and 7SK Cooperate to Inhibit PTEFb

The activities of CDKs involved in cell cycle regulation are negatively regulated by proteins that associate with either CDKs or CDK/cyclin complexes to inhibit their kinase activities (Sherr and Roberts, 1999). Similarly, in the nucleus, not every CDK9/ CycTl heterodimer displays the P-TEFb kinase and transcriptional activities. In human HeLa cells, only about of half of cellular P-TEFb are in the active state and the regulation of P-TEFb activity is a dynamic and tightly controlled process involving the P-TEFb-associated factors. In an effort to isolate nuclear factors that can bind to and control the activity of human P-TEFb, we and others have identified the 7SK snRNA as a specific P-TEFb-associated factor (Nguyen et al., 2001; Yang et al., 2001). Transcribed by RNA Pol III, the human 7SK snRNA is an abundant (2 x 105 copies/cell) and evolutionarily conserved small nuclear RNA (snRNA) of 331 nucleotides (Murphy et al., 1987; Zieve et al., 1977). Despite the first description of 7SK in the 1970's, little is known about its function for the next quarter of century (Murphy et al., 1987; Zieve et al., 1977). Our data indicate that there exist two major forms of P-TEFb with distinct 7SK-binding abilities in human cells. The 7SK-free form, which accounts for about half of total P-TEFb in the cell, can function as both a general and HIV-specific transcription factor. In contrast, the 7SK-bound P-TEFb is inactive as its kinase and transcriptional activities are suppressed. Moreover, when associated with 7SK, P-TEFb cannot even be recruited to the HIV promoter in vivo and in vitro (Nguyen et al., 2001; Yang et al., 2001).

While investigating the functional significance of a reconstituted interaction between 7SK and P-TEFb, we found that the association with 7SK is necessary but not sufficient to inactivate P-TEFb, implicating the presence of another factor in the 7SK snRNP for P-TEFb inactivation (Yik et al., 2003). Indeed, through affinity-purification, a protein factor called HEXIM1 has been identified as the third protein component of the 7SK:P-TEFb snRNP formed in vivo (Michels et al., 2003; Yik et al., 2003). Importantly, HEXIM1 can potently and specifically inhibit the kinase and transcriptional activities of P-TEFb in a 7SK-dependent manner (Yik et al., 2003). HEXIM1 has previously been identified as a nuclear protein whose expression is induced in many transformed cell types treated with hexamethylene bisacetamide (HMBA) (Ouchida et al.,

2003), a potent inducer of cell differentiation. The 7SK-dependent inhibition of P-TEFb by HEXIM1 can be explained by the fact that 7SK plays a scaffolding role in mediating the interaction between HEXIM1 and P-TEFb, enabling HEXIM1 to exert its inhibitory effect on P-TEFb in vivo and in vitro (Yik et al., 2003).

B: A Similar Architectural Plan for Forming the Tat: TAR: P-TEFb and HEXIM1-.7SK:P-TEFb Complexes

In an effort to define the sequence requirements for HEXIM1 to interact with 7SK and inactivate P-TEFb, an arginine-rich motif that overlaps with the nuclear localization signal (NLS) near the center of HEXIM1 (Fig. 14.5) has been shown to mediate a direct and specific interaction of HEXIM1 with 7SK (Yik et al.,

2004). This motif, together with the HEXIM1 C-terminal domain that is required for the interaction with and inhibition of P-TEFb, allow HEXIM1 to inhibit Pol II transcription through the 7SK-mediated inactivation of P-TEFb. Interestingly, the 7SK-binding motif in HEXIM1 is highly homologous to and functionally interchangeable with the arginine-rich TAR-binding motif in HIV Tat (Fig. 14.5) (Yik et al., 2004), suggesting that a similar architectural plan may exist to form both the Tat:TAR:P-TEFb and the HEXIM1:7SK:P-TEFb ternary complexes. This hypothesis, while yet to be confirmed through further structural analysis, raises an intriguing possibility that the nuclear level of HEXIM1 can be therapeutically manipulated to effectively modulate the amount of P-TEFb in the Tat:TAR:P-TEFb complex, which in turn would affect HIV transcription.

C: Dynamic Exchange of Partners between Active and Inactive P-TEFb Complexes

In HeLa cell, about half of nuclear P-TEFb are sequestered in the inactive 7SK:HEXIMl:P-TEFb snRNP. Importantly, the amount of the HEXIM1/

7SK-bound P-TEFb does not remain static in the cell but rather undergoes dynamic changes in response to various stimuli. For example, treatment of cells with several stress-inducing agents, such as the global transcription inhibitors actinomycin D and DRB or the DNA-damaging agent UV-irradiation, rapidly dissociates 7SK and HEXIM1 from P-TEFb without affecting the expressions of CDK9 and CycTl or the CDK9/CycTl dimer formation (Fig. 14.6) (Nguyen et al., 2001; Yang et al., 2001). Although these agents generally cause a global inhibition of transcription, earlier observations have indicated that at least for actinomycin D and UV, a low dosage can induce the phosphorylation of the Pol II CTD and activation of HIV transcription (Casse et al., 1999; Valerie et al., 1988). Moreover, UV irradiation of human T-cells prior to HIV infection also significantly shortens the viral growth cycle (Valerie et al., 1988). The ability of these agents to dissociate HEXIM1 and 7SK and activate P-TEFb provides a mechanistic explanation for their effects on HIV transcription and replication. In contrast to the stress treatment that causes the disruption of the 7SK:HEXIMl:P-TEFb snRNP, HEXIM1 expression is known to be elevated in cells treated with HMBA (Ouchida et al., 2003). The induced HEXIM1 expression could potentially sequester more P-TEFb into the inactive 7SK snRNP (Fig. 14.6), although this notion is yet to be tested experimentally.

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