PKC phosphorylates HEXIM1 and regulates P-TEFb activity

WOS:000309927100040Peer reviewe

Tat competes with HEXIM1 to increase the active pool of P-TEFb for HIV-1 transcription

Human immunodeficiency virus type 1 (HIV-1) transcriptional transactivator (Tat) recruits the positive transcription elongation factor b (P-TEFb) to the viral promoter. Consisting of cyclin dependent kinase 9 (Cdk9) and cyclin T1, P-TEFb phosphorylates RNA polymerase II and the negative transcription elongation factor to stimulate the elongation of HIV-1 genes. A major fraction of nuclear P-TEFb is sequestered into a transcriptionally inactive 7SK small nuclear ribonucleoprotein (snRNP) by the coordinated actions of the 7SK small nuclear RNA (snRNA) and hexamethylene bisacetamide (HMBA) induced protein 1 (HEXIM1). In this study, we demonstrate that Tat prevents the formation of and also releases P-TEFb from the 7SK snRNP in vitro and in vivo. This ability of Tat depends on the integrity of its N-terminal activation domain and stems from the high affinity interaction between Tat and cyclin T1, which allows Tat to directly displace HEXIM1 from cyclin T1. Furthermore, we find that in contrast to the Tat-independent activation of the HIV-1 promoter, Tat-dependent HIV-1 transcription is largely insensitive to the inhibition by HEXIM1. Finally, primary blood lymphocytes display a reduced amount of the endogenous 7SK snRNP upon HIV-1 infection. All these data are consistent with the model that Tat not only recruits but also increases the active pool of P-TEFb for efficient HIV-1 transcription

RNA polymerase II stalling promotes nucleosome occlusion and pTEFb recruitment to drive immortalization by Epstein-Barr virus

Epstein-Barr virus (EBV) immortalizes resting B-cells and is a key etiologic agent in the development of numerous cancers. The essential EBV-encoded protein EBNA 2 activates the viral C promoter (Cp) producing a message of ~120 kb that is differentially spliced to encode all EBNAs required for immortalization. We have previously shown that EBNA 2-activated transcription is dependent on the activity of the RNA polymerase II (pol II) C-terminal domain (CTD) kinase pTEFb (CDK9/cyclin T1). We now demonstrate that Cp, in contrast to two shorter EBNA 2-activated viral genes (LMP 1 and 2A), displays high levels of promoter-proximally stalled pol II despite being constitutively active. Consistent with pol II stalling, we detect considerable pausing complex (NELF/DSIF) association with Cp. Significantly, we observe substantial Cp-specific pTEFb recruitment that stimulates high-level pol II CTD serine 2 phosphorylation at distal regions (up to +75 kb), promoting elongation. We reveal that Cp-specific pol II accumulation is directed by DNA sequences unfavourable for nucleosome assembly that increase TBP access and pol II recruitment. Stalled pol II then maintains Cp nucleosome depletion. Our data indicate that pTEFb is recruited to Cp by the bromodomain protein Brd4, with polymerase stalling facilitating stable association of pTEFb. The Brd4 inhibitor JQ1 and the pTEFb inhibitors DRB and Flavopiridol significantly reduce Cp, but not LMP1 transcript production indicating that Brd4 and pTEFb are required for Cp transcription. Taken together our data indicate that pol II stalling at Cp promotes transcription of essential immortalizing genes during EBV infection by (i) preventing promoter-proximal nucleosome assembly and ii) necessitating the recruitment of pTEFb thereby maintaining serine 2 CTD phosphorylation at distal regions

miR-198 Inhibits HIV-1 Gene Expression and Replication in Monocytes and Its Mechanism of Action Appears To Involve Repression of Cyclin T1

Cyclin T1 is a regulatory subunit of a general RNA polymerase II elongation factor known as P-TEFb. Cyclin T1 is also required for Tat transactivation of HIV-1 LTR-directed gene expression. Translation of Cyclin T1 mRNA has been shown to be repressed in human monocytes, and this repression is relieved when cells differentiate to macrophages. We identified miR-198 as a microRNA (miRNA) that is strongly down-regulated when monocytes are induced to differentiate. Ectopic expression of miR-198 in tissue culture cells reduced Cyclin T1 protein expression, and plasmid reporter assays verified miR-198 target sequences in the 3′ untranslated region (3′UTR) of Cyclin T1 mRNA. Cyclin T1 protein levels increased when an inhibitor of miR-198 was transfected into primary monocytes, and overexpression of miR-198 in primary monocytes repressed the normal up-regulation of Cyclin T1 during differentiation. Expression of an HIV-1 proviral plasmid and HIV-1 replication were repressed in a monocytic cell line upon overexpression of miR-198. Our data indicate that miR-198 functions to restrict HIV-1 replication in monocytes, and its mechanism of action appears to involve repression of Cyclin T1 expression

Invertebrate 7SK snRNAs

7SK RNA is a highly abundant noncoding RNA in mammalian cells whose function in transcriptional regulation has only recently been elucidated. Despite its highly conserved sequence throughout vertebrates, all attempts to discover 7SK RNA homologues in invertebrate species have failed so far. Here we report on a combined experimental and computational survey that succeeded in discovering 7SK RNAs in most of the major deuterostome clades and in two protostome phyla: mollusks and annelids. Despite major efforts, no candidates were found in any of the many available ecdysozoan genomes, however. The additional sequence data confirm the evolutionary conservation and hence functional importance of the previously described 3′ and 5′ stem-loop motifs, and provide evidence for a third, structurally well-conserved domain

A Cyclin-Dependent Kinase that Promotes Cytokinesis through Modulating Phosphorylation of the Carboxy Terminal Domain of the RNA Pol II Rpb1p Sub-Unit

In Schizosaccharomyces pombe, the nuclear-localized kinase, Lsk1p, promotes cytokinesis by positively regulating the Septation Initiation Network (SIN). Although a member of the cyclin-dependent kinase (CDK) family, neither a cyclin partner nor a physiological target has been identified. In this report we identify a cyclin, Lsc1p, that physically interacts and co-localizes with Lsk1p. Furthermore, lsk1Δ, lsc1Δ, as well as kinase-dead lsk1-K306R mutants, display highly similar cytokinesis defects. Lsk1p is related to CDKs that phosphorylate the carboxy-terminal domain (CTD) of the largest sub-unit of RNA polymerase II (Rpb1p). Interestingly, we find that Lsk1p and Lsc1p are required for phosphorylation of Ser-2 residues found in the heptad repeats of the CTD. To determine if Rpb1p could be a physiological target, we replaced the native rpb1 gene with a synthetic gene encoding a Rpb1p protein in which Ser-2 was substituted with the non-phosphorylatable amino-acid alanine in all heptads. Cells carrying this allele were similar to lsk1Δ mutants: They were viable, displayed genetic interactions with the SIN, and were unable to complete cytokinesis upon perturbation of the cell division machinery. We conclude that Ser-2 phosphorylation of the CTD heptads plays a novel physiological role in the regulation of cytokinesis

Transcription Inhibition by DRB Potentiates Recombinational Repair of UV Lesions in Mammalian Cells

Homologous recombination (HR) is intricately associated with replication, transcription and DNA repair in all organisms studied. However, the interplay between all these processes occurring simultaneously on the same DNA molecule is still poorly understood. Here, we study the interplay between transcription and HR during ultraviolet light (UV)-induced DNA damage in mammalian cells. Our results show that inhibition of transcription with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) increases the number of UV-induced DNA lesions (γH2AX, 53BP1 foci formation), which correlates with a decrease in the survival of wild type or nucleotide excision repair defective cells. Furthermore, we observe an increase in RAD51 foci formation, suggesting HR is triggered in response to an increase in UV-induced DSBs, while inhibiting transcription. Unexpectedly, we observe that DRB fails to sensitise HR defective cells to UV treatment. Thus, increased RAD51 foci formation correlates with increased cell death, suggesting the existence of a futile HR repair of UV-induced DSBs which is linked to transcription inhibition

Caffeine Prevents Transcription Inhibition and P-TEFb/7SK Dissociation Following UV-Induced DNA Damage

Background: The mechanisms by which DNA damage triggers suppression of transcription of a large number of genes are poorly understood. DNA damage rapidly induces a release of the positive transcription elongation factor b (P-TEFb) from the large inactive multisubunit 7SK snRNP complex. P-TEFb is required for transcription of most class II genes through stimulation of RNA polymerase II elongation and cotranscriptional pre-mRNA processing. Methodology/Principal Findings: We show here that caffeine prevents UV-induced dissociation of P-TEFb as well as transcription inhibition. The caffeine-effect does not involve PI3-kinase-related protein kinases, because inhibition of phosphatidylinositol 3-kinase family members (ATM, ATR and DNA-PK) neither prevents P-TEFb dissociation nor transcription inhibition. Finally, caffeine prevention of transcription inhibition is independent from DNA damage. Conclusion/Significance: Pharmacological prevention of P-TEFb/7SK snRNP dissociation and transcription inhibitio

Zelda Binding in the Early Drosophila melanogaster Embryo Marks Regions Subsequently Activated at the Maternal-to-Zygotic Transition

The earliest stages of development in most metazoans are driven by maternally deposited proteins and mRNAs, with widespread transcriptional activation of the zygotic genome occurring hours after fertilization, at a period known as the maternal-to-zygotic transition (MZT). In Drosophila, the MZT is preceded by the transcription of a small number of genes that initiate sex determination, patterning, and other early developmental processes; and the zinc-finger protein Zelda (ZLD) plays a key role in their transcriptional activation. To better understand the mechanisms of ZLD activation and the range of its targets, we used chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) to map regions bound by ZLD before (mitotic cycle 8), during (mitotic cycle 13), and after (late mitotic cycle 14) the MZT. Although only a handful of genes are transcribed prior to mitotic cycle 10, we identified thousands of regions bound by ZLD in cycle 8 embryos, most of which remain bound through mitotic cycle 14. As expected, early ZLD-bound regions include the promoters and enhancers of genes transcribed at this early stage. However, we also observed ZLD bound at cycle 8 to the promoters of roughly a thousand genes whose first transcription does not occur until the MZT and to virtually all of the thousands of known and presumed enhancers bound at cycle 14 by transcription factors that regulate patterned gene activation during the MZT. The association between early ZLD binding and MZT activity is so strong that ZLD binding alone can be used to identify active promoters and regulatory sequences with high specificity and selectivity. This strong early association of ZLD with regions not active until the MZT suggests that ZLD is not only required for the earliest wave of transcription but also plays a major role in activating the genome at the MZT

Limited redundancy in genes regulated by Cyclin T2 and Cyclin T1

<p>Abstract</p> <p>Background</p> <p>The elongation phase, like other steps of transcription by RNA Polymerase II, is subject to regulation. The positive transcription elongation factor b (P-TEFb) complex allows for the transition of mRNA synthesis to the productive elongation phase. P-TEFb contains Cdk9 (Cyclin-dependent kinase 9) as its catalytic subunit and is regulated by its Cyclin partners, Cyclin T1 and Cyclin T2. The HIV-1 Tat transactivator protein enhances viral gene expression by exclusively recruiting the Cdk9-Cyclin T1 P-TEFb complex to a RNA element in nascent viral transcripts called TAR. The expression patterns of Cyclin T1 and Cyclin T2 in primary monocytes and CD4⁺T cells suggests that Cyclin T2 may be generally involved in expression of constitutively expressed genes in quiescent cells, while Cyclin T1 may be involved in expression of genes up-regulated during macrophage differentiation, T cell activation, and conditions of increased metabolic activity To investigate this issue, we wished to identify the sets of genes whose levels are regulated by either Cyclin T2 or Cyclin T1.</p> <p>Findings</p> <p>We used shRNA lentiviral vectors to stably deplete either Cyclin T2 or Cyclin T1 in HeLa cells. Total RNA extracted from these cells was subjected to cDNA microarray analysis. We found that 292 genes were down- regulated by depletion of Cyclin T2 and 631 genes were down-regulated by depletion of Cyclin T1 compared to cells transduced with a control lentivirus. Expression of 100 genes was commonly reduced in either knockdown. Additionally, 111 and 287 genes were up-regulated when either Cyclin T2 or Cyclin T1 was depleted, respectively, with 45 genes in common.</p> <p>Conclusions</p> <p>These results suggest that there is limited redundancy in genes regulated by Cyclin T1 or Cyclin T2.</p