Reversible phosphorylation of cyclin T1 promotes assembly and stability of P-TEFb.

The positive transcription elongation factor b (P-TEFb) is a critical coactivator for transcription of most cellular and viral genes, including those of HIV. While P-TEFb is regulated by 7SK snRNA in proliferating cells, P-TEFb is absent due to diminished levels of CycT1 in quiescent and terminally differentiated cells, which has remained unexplored. In these cells, we found that CycT1 not bound to CDK9 is rapidly degraded. Moreover, productive CycT1:CDK9 interactions are increased by PKC-mediated phosphorylation of CycT1 in human cells. Conversely, dephosphorylation of CycT1 by PP1 reverses this process. Thus, PKC inhibitors or removal of PKC by chronic activation results in P-TEFb disassembly and CycT1 degradation. This finding not only recapitulates P-TEFb depletion in resting CD4+ T cells but also in anergic T cells. Importantly, our studies reveal mechanisms of P-TEFb inactivation underlying T cell quiescence, anergy, and exhaustion as well as proviral latency and terminally differentiated cells.

Structural transitions in the RNA 7SK 5' hairpin and their effect on HEXIM binding.

7SK RNA, as part of the 7SK ribonucleoprotein complex, is crucial to the regulation of transcription by RNA-polymerase II, via its interaction with the positive transcription elongation factor P-TEFb. The interaction is induced by binding of the protein HEXIM to the 5' hairpin (HP1) of 7SK RNA. Four distinct structural models have been obtained experimentally for HP1. Here, we employ computational methods to investigate the relative stability of these structures, transitions between them, and the effects of mutations on the observed structural ensembles. We further analyse the results with respect to mutational binding assays, and hypothesize a mechanism for HEXIM binding. Our results indicate that the dominant structure in the wild type exhibits a triplet involving the unpaired nucleotide U40 and the base pair A43-U66 in the GAUC/GAUC repeat. This conformation leads to an open major groove with enough potential binding sites for peptide recognition. Sequence mutations of the RNA change the relative stability of the different structural ensembles. Binding affinity is consequently lost if these changes alter the dominant structure.

An ultra-high affinity ligand of HIV-1 TAR reveals the RNA structure recognized by P-TEFb.

The HIV-1 trans-activator protein Tat binds the trans-activation response element (TAR) to facilitate recruitment of the super elongation complex (SEC) to enhance transcription of the integrated pro-viral genome. The Tat-TAR interaction is critical for viral replication and the emergence of the virus from the latent state, therefore, inhibiting this interaction has long been pursued to discover new anti-viral or latency reversal agents. However, discovering active compounds that directly target RNA with high affinity and selectivity remains a significant challenge; limiting pre-clinical development. Here, we report the rational design of a macrocyclic peptide mimic of the arginine rich motif of Tat, which binds to TAR with low pM affinity and 100-fold selectivity against closely homologous RNAs. Despite these unprecedented binding properties, the new ligand (JB181) only moderately inhibits Tat-dependent reactivation in cells and recruitment of positive transcription elongation factor (P-TEFb) to TAR. The NMR structure of the JB181-TAR complex revealed that the ligand induces a structure in the TAR loop that closely mimics the P-TEFb/Tat1:57/AFF4/TAR complex. These results strongly suggest that high-affinity ligands which bind the UCU bulge are not likely to inhibit recruitment of the SEC and suggest that targeting of the TAR loop will be an essential feature of effective Tat inhibitors.

Schizosaccharomyces pombe Pol II transcription elongation factor ELL functions as part of a rudimentary super elongation complex.

ELL family transcription factors activate the overall rate of RNA polymerase II (Pol II) transcription elongation by binding directly to Pol II and suppressing its tendency to pause. In metazoa, ELL regulates Pol II transcription elongation as part of a large multisubunit complex referred to as the Super Elongation Complex (SEC), which includes P-TEFb and EAF, AF9 or ENL, and an AFF family protein. Although orthologs of ELL and EAF have been identified in lower eukaryotes including Schizosaccharomyces pombe, it has been unclear whether SEC-like complexes function in lower eukaryotes. In this report, we describe isolation from S. pombe of an ELL-containing complex with features of a rudimentary SEC. This complex includes S. pombe Ell1, Eaf1, and a previously uncharacterized protein we designate Ell1 binding protein 1 (Ebp1), which is distantly related to metazoan AFF family members. Like the metazoan SEC, this S. pombe ELL complex appears to function broadly in Pol II transcription. Interestingly, it appears to have a particularly important role in regulating genes involved in cell separation.

Substrate Specificity of the Kinase P-TEFb towards the RNA Polymerase II C-Terminal Domain.

The positive transcription elongation factor b (P-TEFb) promotes transcription elongation through phosphorylation of the RNA polymerase II C-terminal domain. This process is not well understood, partly due to difficulties in determining the specificity of P-TEFb toward the various heptad repeat motifs within the C-terminal domain. A simple assay using mass spectrometry was developed to identify the substrate specificity of the Drosophila melanogaster P-TEFb (DmP-TEFb) in vitro. This assay demonstrated that DmP-TEFb preferentially phosphorylates Ser5 and, surprisingly, that pre-phosphorylation or conserved amino acid variation at the 7-position in the heptad can alter DmP-TEFb specificity, leading to the creation of distinct double-phosphorylation marks.

Multiplex Substrate Profiling by Mass Spectrometry for Kinases as a Method for Revealing Quantitative Substrate Motifs.

The more than 500 protein kinases comprising the human kinome catalyze hundreds of thousands of phosphorylation events to regulate a diversity of cellular functions; however, the extended substrate specificity is still unknown for many of these kinases. We report here a method for quantitatively describing kinase substrate specificity using an unbiased peptide library-based approach with direct measurement of phosphorylation by tandem liquid chromatography-tandem mass spectrometry (LC-MS/MS) peptide sequencing (multiplex substrate profiling by mass spectrometry, MSP-MS). This method can be deployed with as low as 10 nM enzyme to determine activity against S/T/Y-containing peptides; additionally, label-free quantitation is used to ascertain catalytic efficiency values for individual peptide substrates in the multiplex assay. Using this approach we developed quantitative motifs for a selection of kinases from each branch of the kinome, with and without known substrates, highlighting the applicability of the method. The sensitivity of this approach is evidenced by its ability to detect phosphorylation events from nanogram quantities of immunoprecipitated material, which allows for wider applicability of this method. To increase the information content of the quantitative kinase motifs, a sublibrary approach was used to expand the testable sequence space within a peptide library of approximately 100 members for CDK1, CDK7, and CDK9. Kinetic analysis of the HIV-1 Tat (transactivator of transcription)-positive transcription elongation factor b (P-TEFb) interaction allowed for localization of the P-TEFb phosphorylation site as well as characterization of the stimulatory effect of Tat on P-TEFb catalytic efficiency.

The emerging picture of CDK9/P-TEFb: more than 20 years of advances since PITALRE.

CDK9 is a prominent member of the transcriptional CDKs subfamily, a group of kinases whose function is to control the primary steps of mRNA synthesis and processing by eukaryotic RNA polymerase II. As a cyclin-dependent kinase, CDK9 activation in vivo depends upon its association with T-type cyclins to assemble the positive transcription elongation factor (P-TEFb). Although CDK9/P-TEFb phosphorylates the C-terminal domain of RNAP II in the same positions targeted by CDK7 (TFIIH) and CDK8 (Mediator), the former does not participate in the transcription initiation, but rather plays a unique role by driving the polymerase to productive elongation. In addition to RNAP II CTD, the negative transcription elongation factors DSIF and NELF also represent major CDK9 substrates, whose phosphorylation is required to overcome the proximal pause of the polymerase. CDK9 is recruited to specific genes through proteins that interact with both P-TEFb and distinct elements in DNA, RNA or chromatin, where it modulates the activity of individual RNAP II transcription complexes. The regulation of CDK9 function is an intricate network that includes post-translational modifications (phosphorylation/dephosphorylation and acetylation/deacetylation of key residues) as well as the association of P-TEFb with various proteins that can stimulate or inhibit its kinase activity. Several cases of CDK9 deregulation have been linked to important human diseases, including various types of cancer and also AIDS (due to its essential role in HIV replication). Not only HIV, but also many other human viruses have been shown to depend strongly on CDK9 activity to be transcribed within host cells. This review summarizes the main advances made on CDK9/P-TEFb field in more than 20 years, introducing the structural, functional and genetic aspects that have been elucidated ever since.

An Overview on Small Molecule Inhibitors of BRD4.

BRD4, an epigenetic regulator that recognizes and binds the acetylated lysine residues in histone, has been reported as a potential therapeutic target for cancers. Since the first BRD4 inhibitor JQ1 developed in 2010, numerous BRD4 inhibitors have been discovered in past five years. In this review, we have systematically summarized a series of BRD4 binding compounds, which are divided into five categories based on the similarity of their chemical structures and respectively referred as JQ1 derivatives, tetrahydroquinoline derivatives, 3,5- dimethylisoxazole derivatives, 2-thiazolidinone derivatives and others. The binding affinities for each class of compounds are also discussed.

Development of a sensitive amplified luminescent proximity homogeneous assay to monitor the interactions between pTEFb and Tat.

The viral transactivator protein (Tat) plays an essential role in the replication of human immunodeficiency type 1 virus (HIV-1) by recruiting the host positive transcription elongation factor (pTEFb) to the RNA polymerase II transcription machinery to enable an efficient HIV-1 RNA elongation process. Blockade of the interaction between Tat and pTEFb represents a novel strategy for developing a new class of antiviral agents. In this study, we developed a homogeneous assay in AlphaLISA (amplified luminescent proximity homogeneous assay) format using His-tagged pTEFb and biotinylated Tat to monitor the interaction between Tat and pTEFb. On optimizing the assay conditions, the signal-to-background ratio was found to be greater than 10-fold. The assay was validated with untagged Tat and peptides known to compete with Tat for pTEFb binding. The Z' of the assay is greater than 0.5, indicating that the assay is robust and can be easily adapted to a high-throughput screening format. Furthermore, the affinity between Tat and pTEFb was determined to be approximately 20 pM, and only 7% of purified Tat was found to be active in forming tertiary complex with pTEFb. Development of this assay should facilitate the discovery of a new class of antiviral agents providing HIV-1 patients with broader treatment choices.

Acute hypoxia affects P-TEFb through HDAC3 and HEXIM1-dependent mechanism to promote gene-specific transcriptional repression.

Hypoxia is associated with a variety of physiological and pathological conditions and elicits specific transcriptional responses. The elongation competence of RNA Polymerase II is regulated by the positive transcription elongation factor b (P-TEFb)-dependent phosphorylation of Ser2 residues on its C-terminal domain. Here, we report that hypoxia inhibits transcription at the level of elongation. The mechanism involves enhanced formation of inactive complex of P-TEFb with its inhibitor HEXIM1 in an HDAC3-dependent manner. Microarray transcriptome profiling of hypoxia primary response genes identified ∼79% of these genes being HEXIM1-dependent. Hypoxic repression of P-TEFb was associated with reduced acetylation of its Cdk9 and Cyclin T1 subunits. Hypoxia caused nuclear translocation and co-localization of the Cdk9 and HDAC3/N-CoR repressor complex. We demonstrated that the described mechanism is involved in hypoxic repression of the monocyte chemoattractant protein-1 (MCP-1) gene. Thus, HEXIM1 and HDAC-dependent deacetylation of Cdk9 and Cyclin T1 in response to hypoxia signalling alters the P-TEFb functional equilibrium, resulting in repression of transcription.

Crystal structure of HIV-1 Tat complexed with human P-TEFb and AFF4.

Developing anti-viral therapies targeting HIV-1 transcription has been hampered by the limited structural knowledge of the proteins involved. HIV-1 hijacks the cellular machinery that controls RNA polymerase II elongation through an interaction of HIV-1 Tat with the positive transcription elongation factor P-TEFb, which interacts with an AF4 family member (AFF1/2/3/4) in the super elongation complex (SEC). Because inclusion of Tat•P-TEFb into the SEC is critical for HIV transcription, we have determined the crystal structure of the Tat•AFF4•P-TEFb complex containing HIV-1 Tat (residues 1-48), human Cyclin T1 (1-266), human Cdk9 (7-332), and human AFF4 (27-69). Tat binding to AFF4•P-TEFb causes concerted structural changes in AFF4 via a shift of helix H5' of Cyclin T1 and the α-3 10 helix of AFF4. The interaction between Tat and AFF4 provides structural constraints that explain tolerated Tat mutations. Analysis of the Tat-binding surface of AFF4 coupled with modeling of all other AF4 family members suggests that AFF1 and AFF4 would be preferred over AFF2 or AFF3 for interaction with Tat•P-TEFb. The structure establishes that the Tat-TAR recognition motif (TRM) in Cyclin T1 interacts with both Tat and AFF4, leading to the exposure of arginine side chains for binding to TAR RNA. Furthermore, modeling of Tat Lys28 acetylation suggests that the acetyl group would be in a favorable position for H-bond formation with Asn257 of TRM, thereby stabilizing the TRM in Cyclin T1, and provides a structural basis for the modulation of TAR RNA binding by acetylation of Tat Lys28.

AFF1 is a ubiquitous P-TEFb partner to enable Tat extraction of P-TEFb from 7SK snRNP and formation of SECs for HIV transactivation.

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.

Transcription factors mediate the enzymatic disassembly of promoter-bound 7SK snRNP to locally recruit P-TEFb for transcription elongation.

The transition from transcription initiation into elongation is controlled by transcription factors, which recruit positive transcription elongation factor b (P-TEFb) to promoters to phosphorylate RNA polymerase II. A fraction of P-TEFb is recruited as part of the inhibitory 7SK small nuclear ribonucleoprotein particle (snRNP), which inactivates the kinase and prevents elongation. However, it is unclear how P-TEFb is captured from the promoter-bound 7SK snRNP to activate elongation. Here, we describe a mechanism by which transcription factors mediate the enzymatic release of P-TEFb from the 7SK snRNP at promoters to trigger activation in a gene-specific manner. We demonstrate that Tat recruits PPM1G/PP2Cγ to locally disassemble P-TEFb from the 7SK snRNP at the HIV promoter via dephosphorylation of the kinase T loop. Similar to Tat, nuclear factor (NF)-κB recruits PPM1G in a stimulus-dependent manner to activate elongation at inflammatory-responsive genes. Recruitment of PPM1G to promoter-assembled 7SK snRNP provides a paradigm for rapid gene activation through transcriptional pause release.

The AFF4 scaffold binds human P-TEFb adjacent to HIV Tat.

Human positive transcription elongation factor b (P-TEFb) phosphorylates RNA polymerase II and regulatory proteins to trigger elongation of many gene transcripts. The HIV-1 Tat protein selectively recruits P-TEFb as part of a super elongation complex (SEC) organized on a flexible AFF1 or AFF4 scaffold. To understand this specificity and determine if scaffold binding alters P-TEFb conformation, we determined the structure of a tripartite complex containing the recognition regions of P-TEFb and AFF4. AFF4 meanders over the surface of the P-TEFb cyclin T1 (CycT1) subunit but makes no stable contacts with the CDK9 kinase subunit. Interface mutations reduced CycT1 binding and AFF4-dependent transcription. AFF4 is positioned to make unexpected direct contacts with HIV Tat, and Tat enhances P-TEFb affinity for AFF4. These studies define the mechanism of scaffold recognition by P-TEFb and reveal an unanticipated intersubunit pocket on the AFF4 SEC that potentially represents a target for therapeutic intervention against HIV/AIDS. DOI:http://dx.doi.org/10.7554/eLife.00327.001.

Transition step during assembly of HIV Tat:P-TEFb transcription complexes and transfer to TAR RNA.

Transcription factors regulate eukaryotic RNA polymerase II (Pol II) activity by assembling and remodeling complexes at multiple steps in the transcription cycle. In HIV, we previously proposed a two-step model where the viral Tat protein first preassembles at the promoter with an inactive P-TEFb:7SK snRNP complex and later transfers P-TEFb to TAR on the nascent transcript, displacing the inhibitory snRNP and resulting in Pol II phosphorylation and stimulation of elongation. It is unknown how the Tat:P-TEFb complex transitions to TAR to activate the P-TEFb kinase. Here, we show that P-TEFb artificially recruited to the nascent transcript is not competent for transcription but rather remains inactive due to its assembly with the 7SK snRNP. Tat supplied in trans is able to displace the kinase inhibitor Hexim1 from the snRNP and activate P-TEFb, thereby uncoupling Tat requirements for kinase activation and TAR binding. By combining comprehensive mutagenesis of Tat with multiple cell-based reporter assays that probe the activity of Tat in different arrangements, we genetically defined a transition step in which preassembled Tat:P-TEFb complexes switch to TAR. We propose that a conserved network of residues in Tat has evolved to control this transition and thereby switch the host elongation machinery to viral transcription.

The CDK9 tail determines the reaction pathway of positive transcription elongation factor b.

CDK9, the kinase of positive transcription elongation factor b (P-TEFb), stimulates transcription elongation by phosphorylating RNA polymerase II and transcription elongation factors. Using kinetic analysis of a human P-TEFb complex consisting of CDK9 and cyclin T, we show that the CDK9 C-terminal tail sequence is important for the catalytic mechanism and imposes an ordered binding of substrates and release of products. Crystallographic analysis of a CDK9/cyclin T complex in which the C-terminal tail partially blocks the ATP binding site reveals a possible reaction intermediate. Biochemical characterization of CDK9 mutants supports a model in which the CDK9 tail cycles through different conformational states. We propose that this mechanism is critical for the pattern of CTD Ser2 phosphorylation on actively transcribed genes.

Separate domains of fission yeast Cdk9 (P-TEFb) are required for capping enzyme recruitment and primed (Ser7-phosphorylated) Rpb1 carboxyl-terminal domain substrate recognition.

In fission yeast, discrete steps in mRNA maturation and synthesis depend on a complex containing the 5'-cap methyltransferase Pcm1 and Cdk9, which phosphorylates the RNA polymerase II (Pol II) carboxyl-terminal domain (CTD) and the processivity factor Spt5 to promote transcript elongation. Here we show that a Cdk9 carboxyl-terminal extension, distinct from the catalytic domain, mediates binding to both Pcm1 and the Pol II CTD. Removal of this segment diminishes Cdk9/Pcm1 chromatin recruitment and Spt5 phosphorylation in vivo and leads to slow growth and hypersensitivity to cold temperature, nutrient limitation, and the IMP dehydrogenase inhibitor mycophenolic acid (MPA). These phenotypes, and the Spt5 phosphorylation defect, are suppressed by Pcm1 overproduction, suggesting that normal transcript elongation and gene expression depend on physical linkage between Cdk9 and Pcm1. The extension is dispensable, however, for recognition of CTD substrates "primed" by Mcs6 (Cdk7). On defined peptide substrates in vitro, Cdk9 prefers CTD repeats phosphorylated at Ser7 over unmodified repeats. In vivo, Ser7 phosphorylation depends on Mcs6 activity, suggesting a conserved mechanism, independent of chromatin recruitment, to order transcriptional CDK functions. Therefore, fission yeast Cdk9 comprises a catalytic domain sufficient for primed substrate recognition and a multivalent recruitment module that couples transcription with capping.

Structure and dynamics of a stabilized coiled-coil domain in the P-TEFb regulator Hexim1.

The positive transcription elongation factor P-TEFb mediates the transition from transcription initiation to productive elongation by phosphorylation of the C-terminal domain of RNA polymerase II. P-TEFb is negatively regulated by the cellular protein Hexim1 (hexamethylene bisacetamide-inducible protein 1), which is highly conserved in higher eukaryotes. The C-terminal coiled-coil domain of Hexim1 recognizes the Cyclin T subunit of P-TEFb, whereas a central PYNT motif is required to inhibit the cyclin-dependent kinase Cdk9 by a yet unknown mechanism. Here, the crystal structure of the Cyclin T-binding domain (TBD) of human Hexim1 was determined at 2.1 Å resolution using a deletion mutant of three residues in its central stammer motif. The structure showed a continuous parallel coiled-coil domain of nine hepta-repeats with a preceding helix encompassing up to 15 residues. Two uncommon residues at heptad a positions in the N-terminal part of the coiled-coil structure, Lys284 and Tyr291, stabilize the preceding helix by a tight intermolecular hydrogen bond network with residues of the opposing chain. These interactions delineate a characteristic turn between both helices that is supposed to mediate binding to Cyclin T1. Stabilization of the coiled-coil domain by deletion of the stammer region was confirmed by NMR spectroscopic and backbone dynamic analyses analyzing wild-type TBD and three mutant variants. This study thus provides structural insights into the recognition of the regulator protein Hexim1 by P-TEFb and the modulation of coiled-coil dynamics by specific discontinuities.

The CDK9/cyclin T1 subunits of P-TEFb in mouse oocytes and preimplantation embryos: a possible role in embryonic genome activation.

BACKGROUND: Two stages of genome activation have been identified in the mouse embryo. Specifically, minor transcriptional activation is evident at the one-cell stage and a second major episode of activation occurs at the two-cell stage. Nuclear translocation of RNA polymerase II and phosphorylation of the C-terminal domain (CTD) of the largest enzyme subunit are major determinants of embryonic genome activation. P-TEFb, the Pol II CTD kinase, regulates transcriptional elongation via phosphorylation of the serine 2 residues of the CTD. RESULTS: Here, we show that the CDK9 and cyclin T1 subunits of P-TEFb are present in mouse oocytes and preimplantation embryos. Both proteins translocate to pronuclei at the late one-cell stage and are predominantly localized in nuclei at the two-cell stage. We additionally examine the effects of the CDK9-specific inhibitor, flavopiridol, on mouse preimplantation development. Our data show that treatment with the drug results in mislocalization of CDK9, cyclin T1, and phosphorylated Pol II, as well as developmental arrest at the two-cell stage. CONCLUSIONS: A change in CDK9 localization from the cytoplasm to the pronucleus occurs at the time of minor embryonic genome activation, and CDK9 accumulation at the two-cell stage is evident, concomitant with major transcriptional activation of the embryonic genome. Moreover, CDK9 inhibition triggers a developmental block at the two-cell stage. Our findings clearly indicate that CDK9 is essential for embryonic genome activation in the mouse.

Isolation and functional characterization of P-TEFb-associated factors that control general and HIV-1 transcriptional elongation.

Originally identified as a factor crucial for RNA polymerase (Pol) II transcriptional elongation of cellular genes, the P-TEFb kinase was subsequently shown to also serve as a specific host co-factor required for HIV-1 transcription. Recruited by either the bromodomain protein Brd4 to cellular promoters for general transcription or the HIV-1 Tat protein to the viral LTR for activated HIV-1 transcription, P-TEFb stimulates the processivity of Pol II through phosphorylating the C-terminal domain of Pol II and a pair of negative elongation factors, leading to the synthesis of full-length transcripts. However, abundant evidence indicates that P-TEFb does not act alone in the cell and that all of its known biological functions are likely mediated through the interactions with various regulators. Although a number of P-TEFb-associated factors have already been identified, there are likely more yet to be discovered. Given that P-TEFb plays an essential role in HIV-1 transcription, a major challenge facing the field is to identify all the P-TEFb-associated factors and determine how they may modulate Tat-transactivation and HIV-1 replication. Described here is a set of experimental procedures that have not only enabled us to isolate and identify several P-TEFb-associated factors, but also provided the means to characterize their biochemical functions in HIV-1 transcriptional control. In light of the recent demonstrations that transcriptional elongation plays a much more important role in controlling metazoan gene expression than previously thought, the techniques presented here will also be useful for analyzing Pol II elongation of cellular genes.