TRIM28 promotes HIV-1 latency by SUMOylating CDK9 and inhibiting P-TEFb

Comprehensively elucidating the molecular mechanisms of human immunodeficiency virus type 1 (HIV-1) latency is a priority to achieve a functional cure. As current 'shock' agents failed to efficiently reactivate the latent reservoir, it is important to discover new targets for developing more efficient latency-reversing agents (LRAs). Here, we found that TRIM28 potently suppresses HIV-1 expression by utilizing both SUMO E3 ligase activity and epigenetic adaptor function. Through global site-specific SUMO-MS study and serial SUMOylation assays, we identified that P-TEFb catalytic subunit CDK9 is significantly SUMOylated by TRIM28 with SUMO4. The Lys44, Lys56 and Lys68 residues on CDK9 are SUMOylated by TRIM28, which inhibits CDK9 kinase activity or prevents P-TEFb assembly by directly blocking the interaction between CDK9 and Cyclin T1, subsequently inhibits viral transcription and contributes to HIV-1 latency. The manipulation of TRIM28 and its consequent SUMOylation pathway could be the target for developing LRAs.

Cdk9 regulates a promoter-proximal checkpoint to modulate RNA Polymerase II elongation rate

Multiple kinases modify RNA Polymerase II (Pol II) and its associated pausing and elongation factors to regulate Pol II transcription and transcription-coupled mRNA processing1,2. The conserved Cdk9 kinase is essential for regulated eukaryotic transcription3, but its mechanistic role remains incompletely understood. Here, we use altered-specificity kinase mutations and highly-specific inhibitors in fission yeast, Schizosaccharomyces pombe to examine the role of Cdk9, and related Cdk7 and Cdk12 kinases, on transcription at base-pair resolution using Precision Run-On sequencing (PRO-seq). Within a minute, Cdk9 inhibition causes a dramatic reduction in the phosphorylation of Pol II-associated factor, Spt5. The effects of Cdk9 inhibition on transcription are the more severe than inhibition of Cdk7 and Cdk12 and result in a shift of Pol II towards the transcription start site (TSS). A kinetic time course of Cdk9 inhibition reveals that early transcribing Pol II is the most compromised, with a measured rate of only ~400 bp/min, while Pol II that is already well into the gene continues rapidly to the end of genes with a rate > 1 kb/min. Our results indicate that while Pol II in S. pombe can escape promoter-proximal pausing in the absence of Cdk9 activity, it is impaired in elongation, suggesting the existence of a conserved global regulatory checkpoint that requires Cdk9 kinase activity.

Bromodomain Protein Brd4 Regulates Human Immunodeficiency Virus Transcription through Phosphorylation of CDK9 at Threonine 29

ABSTRACT Positive transcription elongation factor b (P-TEFb), composed of cyclin-dependent kinase 9 (CDK9) and cyclin T, is a global transcription factor for eukaryotic gene expression, as well as a key factor for human immunodeficiency virus (HIV) transcription elongation. P-TEFb phosphorylates the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II (RNAP II), facilitating the transition from nonprocessive to processive transcription elongation. Recently, the bromodomain protein Brd4 has been shown to interact with the low-molecular-weight, active P-TEFb complex and recruit P-TEFb to the HIV type 1 long terminal repeat (LTR) promoter. However, the subsequent events through which Brd4 regulates CDK9 kinase activity and RNAP II-dependent transcription are not clearly understood. Here we provide evidence that Brd4 regulates P-TEFb kinase activity by inducing a negative pathway. Moreover, by analyzing stepwise initiation and elongation complexes, we demonstrate that P-TEFb activity is regulated in the transcription complex. Brd4 induces phosphorylation of CDK9 at threonine 29 (T29) in the HIV transcription initiation complex, inhibiting CDK9 kinase activity. P-TEFb inhibition is transient, as Brd4 is released from the transcription complex between positions +14 and +36. Removal of the phosphate group at T29 by an incoming phosphatase released P-TEFb activity, resulting in increased RNAP II CTD phosphorylation and transcription. Finally, we present chromatin immunoprecipitation studies showing that CDK9 with phosphorylated T29 is associated with the HIV promoter region in the integrated and transcriptionally silent HIV genome.

Abstract 1470: The Kinase Activity of Cyclin-Dependent Kinase-9 is Required for Phosphorylation of p300 and its Histone Acetyltransferase Activity during Cardiomyocyte Hypertrophy

Introduction: A zinc finger protein GATA4 is one of the factors involved in transcriptional regulation during myocardial cell hypertrophy and forms a complex with an intrinsic histone acetyltransferase (HAT), p300. HAT activity of p300 is required for acetylation and the transcriptional activity of GATA4 as well as for cardiomyocyte hypertrophy and the development of heart failure in vivo . By tandem affinity purification and mass spectrometric analyses, we identified cyclin-dependent kinase-9 (Cdk9), a component of positive transcription elongation factor b, as a novel GATA4-binding partner. Cdk9 also formed a complex with p300 as well as GATA4. However, the precise functional relationships among p300, GATA4, and Cdk9 remain unknown. Methods and Results: A series of GST pull-down assays revealed that the N-terminal of Cdk9 bound to the N-terminal zinc finger domain of GATA4 and C/H-3 domain of p300, respectively. By chromatin immunoprecipitation and DNA pull-down assay, we showed that GATA4 recruited Cdk9 onto GATA elements within the endothelin-1 promoter. Immuno-precipitation followed by Western blotting demonstrated that intact p300 induced not only the acetylation of GATA4, but also the interaction between GATA4 and Cdk9. Furthermore, p300 induced the hyperphosphorylation of RNA Pol II, suggesting that p300 is involved in the regulation of Cdk9 kinase activity. All of these effects were inhibited by the co-expression of a dominant-negative form (DN-) of p300. Conversely, Cdk9 induced not only the hyperphos-phorylation of RNA Pol II, but also the phosphorylation of p300. Notably, Cdk9 induced the acetylation and DNA binding of GATA4. However, DN-Cdk9, which loses its kinase activity by a single amino acid substitution, was unable to achieve these, suggesting the requirement of Cdk9 kinase activity for p300 HAT activity. Finally, a Cdk9 kinase inhibitor, DRB, inhibited phenylephrine-induced hypertrophic responses as well as the acetylation of GATA4 in cardiomyocytes. Conclusion: These findings demonstrate that Cdk9 is required for the phosphorylation of p300 and its HAT activity, and forms a functional complex with p300/GATA4 during cardiomyocyte hypertrophy.

P-TEFb kinase recruitment and function at heat shock loci

P-TEFb, a heterodimer of the kinase Cdk9 and cyclin T, was isolated as a factor that stimulates formation of productive transcription elongation complexes in vitro. Here, we show that P-TEFb is located at >200 distinct sites on Drosophila polytene chromosomes. Upon heat shock, P-TEFb, like the regulatory factor HSF, is rapidly recruited to heat shock loci, and this recruitment is blocked in an HSF mutant. Yet, HSF binding to DNA is not sufficient to recruit P-TEFb in vivo, and HSF and P-TEFb immunostainings within a heat shock locus are not coincident. Insight to the function of P-TEFb is offered by experiments showing that the direct recruitment of a Gal4-binding domain P-TEFb hybrid to an hsp70 promoter in Drosophilacells is sufficient to activate transcription in the absence of heat shock. Analyses of point mutants show this P-TEFb stimulation is dependent on Cdk9 kinase activity and on Cdk9's interaction with cyclin T. These results, coupled with the frequent colocalization of P-TEFb and the hypophosphorylated form of RNA polymerase II (Pol II) found at promoter-pause sites, support a model in which P-TEFb acts to stimulate promoter-paused Pol II to enter into productive elongation.