Explore the vast range of titles available.

The positive transcription elongation factor b (P‐TEFb) (CDK9/cyclin T (CycT)) promotes mRNA transcriptional elongation through phosphorylation of elongation repressors and RNA polymerase II. To understand the regulation of a transcriptional CDK by its cognate cyclin, we have determined the structures of the CDK9/CycT1 and free cyclin T2. There are distinct differences between CDK9/CycT1 and the cell cycle CDK CDK2/CycA manifested by a relative rotation of 26° of CycT1 with respect to the CDK, showing for the first time plasticity in CDK cyclin interactions. The CDK9/CycT1 interface is relatively sparse but retains some core CDK–cyclin interactions. The CycT1 C‐terminal helix shows flexibility that may be important for the interaction of this region with HIV TAT and HEXIM. Flavopiridol, an anticancer drug in phase II clinical trials, binds to the ATP site of CDK9 inducing unanticipated structural changes that bury the inhibitor. CDK9 activity and recognition of regulatory proteins are governed by autophosphorylation. We show that CDK9/CycT1 autophosphorylates on Thr186 in the activation segment and three C‐terminal phosphorylation sites. Autophosphorylation on all sites occurs in cis .

Production of mRNA depends critically on the rate of RNA polymerase II (Pol II) elongation. To dissect Pol II dynamics in mouse ES cells, we inhibited Pol II transcription at either initiation or promoter-proximal pause escape with Triptolide or Flavopiridol, and tracked Pol II kinetically using GRO-seq. Both inhibitors block transcription of more than 95% of genes, showing that pause escape, like initiation, is a ubiquitous and crucial step within the transcription cycle. Moreover, paused Pol II is relatively stable, as evidenced from half-life measurements at ∼3200 genes. Finally, tracking the progression of Pol II after drug treatment establishes Pol II elongation rates at over 1000 genes. Notably, Pol II accelerates dramatically while transcribing through genes, but slows at exons. Furthermore, intergenic variance in elongation rates is substantial, and is influenced by a positive effect of H3K79me2 and negative effects of exon density and CG content within genes. Many different factors determine how quickly the DNA in genes is transcribed to produce molecules of messenger RNA. The start of the transcription process features two milestones: first, an enzyme called RNA Polymerase II starts the process; shortly afterwards, however, the process pauses and only starts again when other proteins are recruited. This provides two levels of control over the production of messenger RNA and, it also allows the transcription process to be interrupted in order to study the rate of transcription. Here, Jonkers, Kwak and Lis used two drugs to block either the start of transcription or the release from the paused state in mouse cells. Both drugs prevented new transcription and disrupted about 95% of the total number of genes. However, RNA Polymerase II that was already copying DNA could continue to copy, and did so at an average rate of 2000 bases per minute. Transcription rates were, however, shown to vary between different genes—highly active genes are transcribed faster. Transcription rates also varied within individual genes, with the enzyme accelerating as it moves along the gene. This suggests that the transcription machinery, including other proteins that improve the enzyme’s efficiency, are recruited or modified after transcription has already started, and that these proteins help the enzyme to reach its maximum transcription speed. Other factors also affected the transcription rate: the genetic code is written in four letters—A, C, G and T—and genes that contained more Cs and Gs were transcribed slower than those with lots of As and Ts. Genes also contain regions called exons that code for proteins, and regions called introns that do not: Jonkers, Kwak and Lis found that genes with lots of exons were transcribed slower. Furthermore, DNA is wrapped around proteins into a compacted structure, and genes that had certain chemical markings added to these proteins were transcribed faster. The work of Jonkers, Kwak and Lis is the first in-depth look at how transcription is affected by gene structure, and leads the way to uncovering how transcription rates throughout genes are regulated to influence production of messenger RNA.

Current treatment for acute myeloid leukemia (AML) is less than optimal, but increased understanding of disease pathobiology and genomics has led to clinical investigation of novel targeted therapies and rational combinations. Targeting the cyclin-dependent kinase 9 (CDK9) pathway, which is dysregulated in AML, is an attractive approach. Inhibition of CDK9 leads to downregulation of cell survival genes regulated by super enhancers such as MCL-1 , MYC , and cyclin D1. As CDK9 inhibitors are nonselective, predictive biomarkers that may help identify patients most likely to respond to CDK9 inhibitors are now being utilized, with the goal of improving efficacy and safety.

The positive transcription elongation factor b (P-TEFb) was first identified as a general factor that stimulates transcription elongation by RNA polymerase II (RNAPII), but soon afterwards it turned out to be an essential cellular co-factor of human immunodeficiency virus (HIV) transcription mediated by viral Tat proteins. Studies on the mechanisms of Tat-dependent HIV transcription have led to radical advances in our knowledge regarding the mechanism of eukaryotic transcription, including the discoveries that P-TEFb-mediated elongation control of cellular transcription is a main regulatory step of gene expression in eukaryotes, and deregulation of P-TEFb activity plays critical roles in many human diseases and conditions in addition to HIV/AIDS. P-TEFb is now recognized as an attractive and promising therapeutic target for inflammation/autoimmune diseases, cardiac hypertrophy, cancer, infectious diseases, etc. In this review article, I will summarize our knowledge about basic P-TEFb functions, the regulatory mechanism of P-TEFb-dependent transcription, P-TEFb’s involvement in biological processes and diseases, and current approaches to manipulating P-TEFb functions for the treatment of these diseases.

The bromodomain and extraterminal (BET) domain family of proteins binds to acetylated lysines on histones and regulates gene transcription. Recently, BET inhibitors (BETi) have been developed that show promise as potent anticancer drugs against various solid and hematological malignancies. Here we show that the structurally novel and orally bioavailable BET inhibitor RVX2135 inhibits proliferation and induces apoptosis of lymphoma cells arising in Myc-transgenic mice in vitro and in vivo. We find that BET inhibition exhibits broad transcriptional effects in Myc-transgenic lymphoma cells affecting many transcription factor networks. By examining the genes induced by BETi, which have largely been ignored to date, we discovered that these were similar to those induced by histone deacetylase inhibitors (HDACi). HDACi also induced cell-cycle arrest and cell death of Myc-induced murine lymphoma cells and synergized with BETi. Our data suggest that BETi sensitize Myc-overexpressing lymphoma cells partly by inducing HDAC-silenced genes, and suggest synergistic and therapeutic combinations by targeting the genetic link between BETi and HDACi.

Transcriptionally latent forms of replication-competent proviruses, present primarily in a small subset of memory CD4 + T cells, pose the primary barrier to a cure for HIV-1 infection because they are the source of the viral rebound that almost inevitably follows the interruption of antiretroviral therapy. Over the last 30 years, many of the factors essential for initiating HIV-1 transcription have been identified in studies performed using transformed cell lines, such as the Jurkat T-cell model. However, as highlighted in this review, several poorly understood mechanisms still need to be elucidated, including the molecular basis for promoter-proximal pausing of the transcribing complex and the detailed mechanism of the delivery of P-TEFb from 7SK snRNP. Furthermore, the central paradox of HIV-1 transcription remains unsolved: how are the initial rounds of transcription achieved in the absence of Tat? A critical limitation of the transformed cell models is that they do not recapitulate the transitions between active effector cells and quiescent memory T cells. Therefore, investigation of the molecular mechanisms of HIV-1 latency reversal and LRA efficacy in a proper physiological context requires the utilization of primary cell models. Recent mechanistic studies of HIV-1 transcription using latently infected cells recovered from donors and ex vivo cellular models of viral latency have demonstrated that the primary blocks to HIV-1 transcription in memory CD4 + T cells are restrictive epigenetic features at the proviral promoter, the cytoplasmic sequestration of key transcription initiation factors such as NFAT and NF-κB, and the vanishingly low expression of the cellular transcription elongation factor P-TEFb. One of the foremost schemes to eliminate the residual reservoir is to deliberately reactivate latent HIV-1 proviruses to enable clearance of persisting latently infected cells—the “Shock and Kill” strategy. For “Shock and Kill” to become efficient, effective, non-toxic latency-reversing agents (LRAs) must be discovered. Since multiple restrictions limit viral reactivation in primary cells, understanding the T-cell signaling mechanisms that are essential for stimulating P-TEFb biogenesis, initiation factor activation, and reversing the proviral epigenetic restrictions have become a prerequisite for the development of more effective LRAs.

The positive transcription elongation factor b (P‐TEFb) is a heterodimeric complex composed of cyclin‐dependent kinase 9 and its regulator cyclin T1/2. It stimulates transcription elongation by phosphorylation of serine 2 residues in the carboxy‐terminal domain of polymerase II. 7SK RNA and HEXIM proteins can antagonize transcriptional stimulation by sequestering P‐TEFb in a catalytically inactive ribonucleoprotein (RNP). Here, we show that the previously uncharacterized La‐related protein 7 (LARP7) has a role in 7SK‐mediated regulation of transcription. LARP7 binds to the highly conserved 3′‐terminal U‐rich stretch of 7SK RNA and is an integral part of the 7SK RNP. On stimulation, LARP7 remains associated with 7SK RNA, whereas P‐TEFb is released. Interestingly, reduction of LARP7 by RNA interference enhances transcription from cellular polymerase II promoters, as well as a TAT‐dependent HIV‐1 promoter. Thus, LARP7 is a negative transcriptional regulator of polymerase II genes, acting by means of the 7SK RNP system.

Pyrimidine nucleotide biosynthesis is a druggable metabolic dependency of cancer cells, and chemotherapy agents targeting pyrimidine metabolism are the backbone of treatment for many cancers. Dihydroorotate dehydrogenase (DHODH) is an essential enzyme in the de novo pyrimidine biosynthesis pathway that can be targeted by clinically approved inhibitors. However, despite robust preclinical anticancer efficacy, DHODH inhibitors have shown limited single-agent activity in phase 1 and 2 clinical trials. Therefore, novel combination therapy strategies are necessary to realize the potential of these drugs. To search for therapeutic vulnerabilities induced by DHODH inhibition, we examined gene expression changes in cancer cells treated with the potent and selective DHODH inhibitor brequinar (BQ). This revealed that BQ treatment causes upregulation of antigen presentation pathway genes and cell surface MHC class I expression. Mechanistic studies showed that this effect is (1) strictly dependent on pyrimidine nucleotide depletion, (2) independent of canonical antigen presentation pathway transcriptional regulators, and (3) mediated by RNA polymerase II elongation control by positive transcription elongation factor B (P-TEFb). Furthermore, BQ showed impressive single-agent efficacy in the immunocompetent B16F10 melanoma model, and combination treatment with BQ and dual immune checkpoint blockade (anti-CTLA-4 plus anti-PD-1) significantly prolonged mouse survival compared to either therapy alone. Our results have important implications for the clinical development of DHODH inhibitors and provide a rationale for combination therapy with BQ and immune checkpoint blockade.

Human T-cell leukemia virus type 1 (HTLV-1), the cause of adult T-cell leukemia/lymphoma (ATLL), is a retrovirus, which integrates into the host genome and persistently infects CD4+ T-cells. Virus propagation is stimulated by (1) clonal expansion of infected cells and (2) de novo infection. Viral gene expression is induced by the transactivator protein Tax, which recruits host factors like positive transcription elongation factor b (P-TEFb) to the viral promoter. Since HTLV-1 gene expression is repressed in vivo by viral, cellular, and epigenetic mechanisms in late phases of infection, HTLV-1 avoids an efficient CD8+ cytotoxic T-cell (CTL) response directed against the immunodominant viral Tax antigen. Hence, therapeutic strategies using latency reversing agents (LRAs) sought to transiently activate viral gene expression and antigen presentation of Tax to enhance CTL responses towards HTLV-1, and thus, to expose the latent HTLV-1 reservoir to immune destruction. Here, we review strategies that aimed at enhancing Tax expression and Tax-specific CTL responses to interfere with HTLV-1 latency. Further, we provide an overview of LRAs including (1) histone deacetylase inhibitors (HDACi) and (2) activators of P-TEFb, that have mainly been studied in context of human immunodeficiency virus (HIV), but which may also be powerful in the context of HTLV-1.

Protein arginine deimination leading to the non-coded amino acid citrulline remains a key question in the field of post-translational modifications ever since its discovery by Rogers and Simmonds in 1958. Citrullination is catalyzed by a family of enzymes called peptidyl arginine deiminases (PADIs). Initially, increased citrullination was associated with autoimmune diseases, including rheumatoid arthritis and multiple sclerosis, as well as other neurological disorders and multiple types of cancer. During the last decade, research efforts have focused on how citrullination contributes to disease pathogenesis by modulating epigenetic events, pluripotency, immunity and transcriptional regulation. However, our knowledge regarding the functional implications of citrullination remains quite limited, so we still do not completely understand its role in physiological and pathological conditions. Here, we review the recently discovered functions of PADI2-mediated citrullination of the C-terminal domain of RNA polymerase II in transcriptional regulation in breast cancer cells and the proposed mechanisms to reshape the transcription regulatory network that promotes cancer progression.