Latent HIV reservoirs are the primary hurdle to eradication of infection. Identification of agents, pathways and molecular mechanisms that activate latent provirus may, in the presence of highly active antiretroviral therapy, permit clearance of infected cells by the immune system. Promoter-proximal pausing of RNA polymerase (Pol) II is a major rate-limiting step in HIV gene expression. The viral Tat protein recruits human Super Elongation Complex (SEC) to paused Pol II to overcome this limitation. Here, we identify the bromodomain protein Brd4 and its inhibition of Tat-transactivation as a major impediment to latency reactivation. Brd4 competitively blocks the Tat–SEC interaction on HIV promoter. The BET bromodomain inhibitor JQ1 dissociates Brd4 from the HIV promoter to allow Tat recruitment of SEC to stimulate HIV elongation. JQ1 synergizes with another latency activator prostratin, which promotes Pol II loading onto the viral promoter. Because JQ1 activates viral latency without inducing global T cell activation, this and other closely related compounds and their antagonization of Brd4 to promote Tat–SEC interaction merit further investigations as effective agents/strategies for eliminating latent HIV.

Super Elongation Complexes (SECs) contain two different transcription elongation factors, P-TEFb and ELL1/2, linked by the scaffolding protein AFF4 or AFF1. They stimulate the expression of both normal and disease-related genes, especially those of HIV or involved in leukemogenesis. Among all SECs subunits, ELL2 is stoichiometrically limiting and uniquely regulated at the level of protein stability. Here we identify the RING domain protein Siah1, but not the homologous Siah2, as the E3 ubiquitin ligase for ELL2 polyubiquitination and proteasomal degradation. Siah1 cannot access and ubiquitinate ELL2 bound to AFF4, although at high concentrations, it also degrades AFF4/1 to destroy SECs. Prostratin and HMBA, two well-studied activators of HIV transcription and latency, enhance ELL2 accumulation and SECs formation largely through decreasing Siah1 expression and ELL2 polyubiquitination. Given its importance in formation of SECs, the Siah1 ubiquitination pathway provides a fresh avenue for developing strategies to control disease-related transcription.

The positive transcription elongation factor b (P-TEFb) stimulates RNA polymerase elongation by inducing the transition of promoter proximally paused polymerase II into a productively elongating state. P-TEFb itself is regulated by reversible association with various transcription factors/cofactors to form several multisubunit complexes [e.g., the 7SK small nuclear ribonucleoprotein particle (7SK snRNP), the super elongation complexes (SECs), and the bromodomain protein 4 (Brd4)-P-TEFb complex] that constitute a P-TEFb network controlling cellular and HIV transcription. These complexes have been thought to share no components other than the core P-TEFb subunits cyclin-dependent kinase 9 (CDK9) and cyclin T (CycT, T1, T2a, and T2b). Here we show that the AF4/FMR2 family member 1 (AFF1) is bound to CDK9-CycT and is present in all major P-TEFb complexes and that the tripartite CDK9-CycT-AFF1 complex is transferred as a single unit within the P-TEFb network. By increasing the affinity of the HIV-encoded transactivating (Tat) protein for CycT1, AFF1 facilitates Tat's extraction of P-TEFb from 7SK snRNP and the formation of Tat-SECs for HIV transcription. Our data identify AFF1 as a ubiquitous P-TEFb partner and demonstrate that full Tat transactivation requires the complete SEC.T ranscription by RNA polymerase II (Pol II) is a dynamic process consisting of several distinct but interconnected stages. Over the past three decades, much attention has been focused on the initiation and preinitiation stages of the transcription cycle, because they had been thought to be the principal points at which transcription is controlled (1, 2). Since 2007, however, accumulating evidence has revealed that promoter-proximal pausing of Pol II during early elongation is much more prevalent than previously thought, suggesting that intricate control of gene expression can occur frequently at this stage also (3, 4). Indeed, the importance of controlled pause and release of Pol II is illustrated by the observations that this process plays a prominent role in regulating cell growth, renewal, and differentiation (5, 6).Transcription of the integrated HIV-1 proviral genome is hypersensitive to elongation defects, thus making it an ideal model for elucidating the mechanism and factors that control elongation. It has long been known that in the absence of the HIVencoded transactivating protein (Tat), Pol II can initiate transcription from the viral promoter efficiently but pauses soon after the synthesis of a short RNA segment that folds into a stem-loop structure termed the "transactivation-response" (TAR) element. Tat overcomes Pol II pausing by recruiting the host positive transcription elongation factor b (P-TEFb) to the newly formed TAR RNA to stimulate the production of full-length HIV transcripts (7,8). Containing cyclin-dependent kinase 9 (CDK9) and cyclin T1 (CycT1), P-TEFb triggers the release of paused Pol II by phosphorylating and thereby antagonizing the inhibitory actions of two negative elongation factors, DSIF and NELF (5, 6). P-...

The AF4/FMR2 proteins AFF1 and AFF4 act as a scaffold to assemble the Super Elongation Complex (SEC) that strongly activates transcriptional elongation of HIV-1 and cellular genes. Although they can dimerize, it is unclear whether the dimers exist and function within a SEC in vivo. Furthermore, it is unknown whether AFF1 and AFF4 function similarly in mediating SEC-dependent activation of diverse genes. Providing answers to these questions, our current study shows that AFF1 and AFF4 reside in separate SECs that display largely distinct gene target specificities. While the AFF1-SEC is more potent in supporting HIV-1 transactivation by the viral Tat protein, the AFF4-SEC is more important for HSP70 induction upon heat shock. The functional difference between AFF1 and AFF4 in Tat-transactivation has been traced to a single amino acid variation between the two proteins, which causes them to enhance the affinity of Tat for P-TEFb, a key SEC component, with different efficiency. Finally, genome-wide analysis confirms that the genes regulated by AFF1-SEC and AFF4-SEC are largely non-overlapping and perform distinct functions. Thus, the SEC represents a family of related complexes that exist to increase the regulatory diversity and gene control options during transactivation of diverse cellular and viral genes.