Nan Zhou, Ying Zhu, Yuhua Xue, Haohong Luo, Huan Li, Qiang Zhou, Min Liu, Wanichaya N. Ramey, Nanping Ai, Ruichuan Chen, and Nanhai He
In eukaryotes, the transcription of protein-coding genes is performed by RNA polymerase (Pol) II in a cyclic process consisting of several tightly regulated stages (Sims et al. 2004). During the elongation stage, the C-terminal domain of the largest subunit of Pol II is phosphorylated by the positive transcription elongation factor b (P-TEFb) (Peterlin and Price 2006; Zhou and Yik 2006). This modification is crucial for Pol II to change from abortive to productive elongation and produce full-length RNA transcripts. Consisting of Cdk9 and cyclin T1 (or the minor forms T2 and K), P-TEFb is considered a general transcription factor required for the expression of a vast array of protein-coding genes (Chao and Price 2001; Shim et al. 2002). Not only is P-TEFb critical for cellular gene transcription, it is also a specific host cofactor for the HIV-1 Tat protein. Tat recruits P-TEFb to the TAR RNA element located at the 5′ end of nascent viral transcripts, allowing P-TEFb to phosphorylate stalled Pol II and enhance HIV-1 elongation (Peterlin and Price 2006). However, not every P-TEFb in the nucleus is in a transcriptionally active state. A major reservoir of P-TEFb (∼50% of total P-TEFb in HeLa cells) actually exists in an inactive complex termed 7SK snRNP that also contains the 7SK snRNA (Nguyen et al. 2001; Yang et al. 2001) and three nuclear proteins, HEXIM1 (Michels et al. 2003; Yik et al. 2003), BCDIN3 (Jeronimo et al. 2007), and PIP7S/LARP7 (He et al. 2008; Krueger et al. 2008). Within this complex, HEXIM1 inhibits the Cdk9 kinase in a 7SK-dependent manner (Yik et al. 2003). Underscoring the importance of 7SK as a molecular scaffold to maintain the integrity of 7SK snRNP, this RNA is protected from both 5′–3′ and 3′–5′ exonucleases by the respective actions of BCDIN3, a methylphosphate capping enzyme specific for 7SK (Jeronimo et al. 2007), and PIP7S/LARP7, a La-related protein bound to the 3′ UUU-OH sequence of 7SK (He et al. 2008). Besides P-TEFb sequestered in 7SK snRNP, a separate population of P-TEFb exists in a complex together with the bromodomain protein Brd4, which recruits P-TEFb to chromatin templates through interacting with acetylated histones and the mediator complex (Jang et al. 2005; Yang et al. 2005). Recent evidence shows that this recruitment occurs mostly at late mitosis and is essential to promote G1 gene expression and cell cycle progression (Yang et al. 2008). Importantly, the two populations of P-TEFb are kept in a functional equilibrium that can be perturbed by conditions that impact cell growth. For example, treating cells with global transcription inhibitors DRB and actinomycin D or the DNA-damaging agent UV disrupts 7SK snRNP and converts P-TEFb into the Brd4-bound form for stress-induced gene expression (Nguyen et al. 2001; Yang et al. 2001, 2005). Similarly, in cardiac myocytes, hypertrophic signals release P-TEFb from 7SK snRNP, leading to an overall increase in cellular protein and RNA contents, enlarged cells, and hypertrophic growth (Sano et al. 2002). Finally, RNAi-mediated depletion of PIP7S/LARP7 compromises the integrity of 7SK snRNP, resulting in P-TEFb-dependent transformation of mammary epithelial cells (He et al. 2008). Recent data indicate that the P-TEFb functional equilibrium can also be affected by HMBA (hexamethylene bisacetamide), which is known to inhibit growth and induce differentiation of many cell types (Marks et al. 1994). Interestingly, the response to HMBA displays a biphasic nature (He et al. 2006; Contreras et al. 2007). Shortly after the treatment begins, a disruption of 7SK snRNP and enhanced formation of the Brd4–P-TEFb complex occur. However, when the P-TEFb-dependent HEXIM1 expression markedly increases as the treatment continues, the elevated HEXIM1 levels eventually push the P-TEFb equilibrium back toward the 7SK snRNP side to accommodate an overall reduced transcriptional demand in terminally differentiated cells (He et al. 2006). Accumulating evidence indicates that 7SK snRNP represents a major reservoir of activity where P-TEFb can be recruited to activate transcription (Zhou and Yik 2006). However, the signaling pathway(s) that controls this process is mostly unknown. Of note, in the course of our studies, it was reported that the activity of PI3K/Akt is required for HMBA to release P-TEFb from 7SK snRNP (Contreras et al. 2007). However, since neither the treatment with various pharmacological activators of the PI3K/Akt pathway nor the expression of a CA form of Akt induces 7SK snRNP disruption (Supplemental Fig. S1), it is unlikely that PI3K/Akt alone is sufficient to accomplish this task. Here, through analyzing the disruption of 7SK snRNP by UV or short-term HMBA treatment, we show that both agents cause calcium ion (Ca2+) influx and activation of a Ca2+–calmodulin–PP2B (protein phosphatase 2B, also known as calcineurin) signaling pathway that is necessary although insufficient to cause the disruption. To dissociate HEXIM1 from P-TEFb, PP2B must act sequentially and cooperatively with PP1α (protein phosphatase 1α). Facilitated by a PP2B-induced conformational change in 7SK snRNP, PP1α releases P-TEFb from 7SK snRNP through dephosphorylating phospho-Thr186 located in the Cdk9 T-loop. This event is also necessary for the subsequent recruitment of P-TEFb by Brd4 to the preinitiation complex (PIC), where Cdk9 has been reported to remain unphosphorylated and inactive until after the synthesis of a short RNA transcript (Zhou et al. 2001). Together, our data are consistent with a model that PP2B and PP1α act cooperatively and in response to Ca2+ signaling to dephosphorylate Cdk9 T-loop, disrupt 7SK snRNP, and generate a pool of P-TEFb that can be recruited to the PIC.