Cyclin dependent kinase 9


Yeast Ctk1 kinase, a protein similar to mammalian Cdk9/P-TEFb

The largest subunit of RNA polymerase II contains a unique C-terminal domain important for coupling of transcription and mRNA processing. This domain consists of a repeated heptameric sequence (YSPTSPS) phosphorylated at serines 2 and 5. Serine 5 is phosphorylated during initiation and recruits capping enzyme. Serine 2 is phosphorylated during elongation by Saccharomyces cerevisiae Ctk1 kinase, a protein similar to mammalian Cdk9/P-TEFb. Chromatin immunoprecipitation was used to map positions of transcription elongation and mRNA processing factors in strains lacking Ctk1. Ctk1 is not required for association of elongation factors with transcribing polymerase. However, in ctk1Delta strains, the recruitment of polyadenylation factors to 3' regions of genes is disrupted and changes in 3' ends are seen. Therefore, Serine 2 phosphorylation by Ctk1 recruits factors for cotranscriptional 3' end processing in vivo (Ahn, 2004).

Functional characterization of Cyclin T and CDK9

The transition from abortive into productive elongation is proposed to be controlled by a positive transcription elongation factor b (P-TEFb) through phosphorylation of the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II. Drosophila P-TEFb was identified recently as a cyclin-dependent kinase (CDK9) paired with a cyclin subunit (cyclin T). The cloning of multiple cyclin subunits of human P-TEFb (T1 and T2), is reported. Cyclin T2 has two forms (T2a and T2b) because of alternative splicing. Both cyclin T1 and T2 are ubiquitously expressed. Immunoprecipitation and immunodepletion experiments carried out on HeLa nuclear extract (HNE) indicate that cyclin T1 and T2 are associated with CDK9 in a mutually exclusive manner and that almost all CDK9 is associated with either cyclin T1 or T2. Recombinant CDK9/cyclin T1, CDK9/cyclin T2a, and CDK9/cyclin T2b produced in Sf9 cells possesses DRB-sensitive kinase activity and functions in transcription elongation in vitro. Either cyclin T1 or T2 is required to activate CDK9, and the truncation of the carboxyl terminus of the cyclin reduces, but does not eliminate, P-TEFb activity. Cotransfection experiments indicated that all three CDK9/cyclin combinations dramatically activated the CMV promoter (Peng, 1998b).

CDK9 is the catalytic subunit of a general RNA polymerase II (RNAP II) elongation factor termed p-TEFb, which is targeted by the human immunodeficiency virus (HIV) Tat protein to activate elongation of the integrated proviral genome. CDK9 mRNA and protein levels have been observed to be induced in activated peripheral blood lymphocytes, a cell type relevant to HIV infection. To investigate mechanisms that regulate CDK9 RNA expression, genomic sequences containing the human CDK9 gene were isolated and it was found that CDK9 coding sequences are interrupted by six introns. There is a major transcriptional start site located 79 nucleotides upstream of the ATG initiator codon at nucleotide +1. Nucleotides -352 to -1 contain all the transcriptional regulatory elements needed for full promoter activity in transient expression assays. The CDK9 promoter contains features characteristic of a housekeeping gene, including GC-rich sequences and absence of a functional TATA element. The CDK9 promoter possesses high constitutive activity and may therefore have utility in expression vectors or gene therapy vectors (Liu, 2000).

Phosphorylation of the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAPII) is an important step in transcription and the positive transcription elongation factor b (P-TEFb) has been proposed to facilitate elongation at many genes. The P-TEFb contains a catalytic subunit (Cdk9) that, in association with a cyclin subunit (cyclinT1), has the ability to phosphorylate the CTD substrate in vitro. CyclinT1/Cdk9-mediated transcription requires CTD-containing RNAPII, suggesting that the CTD is the major target of the cyclinT1/Cdk9 complex in vivo. Unlike Cdk7 and Cdk8, two other cyclin-dependent kinases that are capable of phosphorylating the CTD in vitro, only the Cdk9 activates gene expression in a catalysis-dependent manner. Finally, unlike cyclinT1 and T2, the targeted recruitment to promoter DNA of cyclinK (a recently described alternative partner of Cdk9) does not stimulate transcription in vivo. Collectively, these data strongly indicate that the P-TEFb kinase subunits cyclinT/Cdk9 are specifically involved in transcription and the CTD domain of RNAPII is the major functional target of this complex in vivo (Napolitano, 2000).

The chromatin-specific transcription elongation factor FACT functions in conjunction with the RNA polymerase II CTD kinase P-TEFb to alleviate transcription inhibition by DSIF (DRB sensitivity-inducing factor) and NELF (negative elongation factor). The kinase activity of TFIIH is dispensable for this activity, demonstrating that TFIIH-mediated CTD phosphorylation is not involved in the regulation of FACT and DSIF/NELF activities. Thus, a novel transcriptional regulatory network is proposed in which DSIF/NELF inhibition of transcription is prevented by P-TEFb in cooperation with FACT. This study uncovers a novel role for FACT in the regulation of transcription on naked DNA that is independent of its activities on chromatin templates. In addition, this study reveals functional differences between P-TEFb and TFIIH in the regulation of transcription (Wada, 2000).

CDK7, CDK8, and CDK9 are cyclin-dependent kinases (CDKs) that phosphorylate the C-terminal domain (CTD) of RNA polymerase II. They have distinct functions in transcription. Because the three CDKs target only serine 5 in the heptad repeat of model CTD substrates containing various numbers of repeats, the hypothesis was tested that the kinases differ in their ability to phosphorylate CTD heptad arrays. The data show that the kinases display different preferences for phosphorylating individual heptads in a synthetic CTD substrate containing three heptamer repeats and specific regions of the CTD in glutathione S-transferase fusion proteins. They also exhibit differences in their ability to phosphorylate a synthetic CTD peptide that contains Ser-2-PO4. This phosphorylated peptide is a poor substrate for CDK9 complexes. CDK8 and CDK9 complexes, bound to viral activators E1A and Tat, respectively, target only serine 5 for phosphorylation in the CTD peptides, and binding to the viral activators does not change the substrate preference of these kinases. These results imply that the display of different CTD heptads during transcription, as well as their phosphorylation state, can affect their phosphorylation by the different transcription-associated CDKs (Ramanatyhan, 2001).

Protein and RNA interactions of P-TEFb subunits

HIV-1 Tat activates transcription through binding to human cyclin T1, a regulatory subunit of the TAK/P-TEFb CTD kinase complex. The cyclin domain of hCycT1 is necessary and sufficient to interact with Tat and promote cooperative binding to TAR RNA in vitro, as well as mediate Tat transactivation in vivo. A Tat:TAR recognition motif (TRM) was identified at the carboxy-terminal edge of the cyclin domain, and hCycT1 can interact simultaneously with Tat and CDK9 on TAR RNA in vitro. Alanine-scanning mutagenesis of the hCycT1 TRM has identified residues that are critical for the interaction with Tat and others that are required specifically for binding of the complex to TAR RNA. Interestingly, the interaction between Tat and hCycT1 requires zinc as well as essential cysteine residues in both proteins. Cloning and characterization of the murine CycT1 protein reveal that it lacks a critical cysteine residue (C261) and forms a weak, zinc-independent complex with HIV-1 Tat that greatly reduces binding to TAR RNA. A point mutation in mCycT1 (Y261C) restores high-affinity, zinc-dependent binding to Tat and TAR in vitro, and rescues Tat transactivation in vivo. Although overexpression of hCycT1 in NIH3T3 cells strongly enhances transcription from an integrated proviral promoter, this fails to overcome all blocks to productive HIV-1 infection in murine cells (Garber, 1998).

Human immunodeficiency virus type 1 (HIV-1) Tat interacts with cyclin T1 (CycT1), a regulatory partner of CDK9 in the positive transcription elongation factor (P-TEFb) complex, and binds cooperatively with CycT1 to TAR RNA to recruit P-TEFb and promote transcription elongation. Tat also stimulates phosphorylation of affinity-purified core RNA polymerase II and glutathione S-transferase-C-terminal-domain substrates by CycT1-CDK9, but not CycH-CDK7, in vitro. Interestingly, incubation of recombinant Tat-P-TEFb complexes with ATP enhances binding to TAR RNA dramatically, and the C-terminal half of CycT1 masks binding of Tat to TAR RNA in the absence of ATP. ATP incubation leads to autophosphorylation of CDK9 at multiple C-terminal Ser and Thr residues, and full-length CycT1 (amino acids 728) [CycT1(1-728)] is also phosphorylated by CDK9, but not truncated CycT1(1-303). P-TEFb complexes containing a catalytically inactive CDK9 mutant (D167N) bind TAR RNA weakly and independently of ATP, as does a C-terminal truncated CDK9 mutant that is catalytically active but unable to undergo autophosphorylation. Analysis of different Tat proteins reveals that the 101-amino-acid SF2 HIV-1 Tat is unable to bind TAR with CycT1(1-303) in the absence of phosphorylated CDK9, whereas unphosphorylated CDK9 strongly blocks binding of HIV-2 Tat to TAR RNA in a manner that is reversed upon autophosphorylation. Replacement of CDK9 phosphorylation sites with negatively charged residues restores binding of CycT1(1-303)-D167N-Tat, and renders D167N a more potent inhibitor of transcription in vitro. Taken together, these results demonstrate that CDK9 phosphorylation is required for high-affinity binding of Tat-P-TEFb to TAR RNA and that the state of P-TEFb phosphorylation may regulate Tat transactivation in vivo (Garber, 2000).

Important progress in the understanding of elongation control by RNA polymerase II (RNAPII) has come from the recent identification of the positive transcription elongation factor b (P-TEFb) and the demonstration that this factor is a protein kinase that phosphorylates the carboxyl-terminal domain (CTD) of the RNAPII largest subunit. The P-TEFb complex isolated from mammalian cells contains a catalytic subunit (CDK9), a cyclin subunit (cyclin T1 or cyclin T2), and additional, yet unidentified, polypeptides of unknown function. To identify additional factors involved in P-TEFb function, a yeast two-hybrid screen was performed using CDK9 as bait. Cyclin K was found to interact with CDK9 in vivo. Biochemical analyses indicate that cyclin K functions as a regulatory subunit of CDK9. The CDK9-cyclin K complex phosphorylates the CTD of RNAPII and functionally substitutes for P-TEFb, which is comprised of CDK9 and cyclin T, in in vitro transcription reactions (Fu, 1999).

SPT5 and its binding partner SPT4 regulate transcriptional elongation by RNA polymerase II. SPT4 and SPT5 are involved in both 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB)-mediated transcriptional inhibition and the activation of transcriptional elongation by the human immunodeficiency virus type 1 (HIV-1) Tat protein. Recent data suggest that P-TEFb, which is composed of CDK9 and cyclin T1, is also critical in regulating transcriptional elongation by SPT4 and SPT5. The domains of SPT5 that regulate transcriptional elongation in the presence of either DRB or the HIV-1 Tat protein have been analyzed. SPT5 domains that bind SPT4 and RNA polymerase II, in addition to a region in the C terminus of SPT5 that contains multiple heptad repeats and is designated CTR1, are critical for in vitro transcriptional repression by DRB and activation by the Tat protein. Furthermore, the SPT5 CTR1 domain is a substrate for P-TEFb phosphorylation. These results suggest that C-terminal repeats in SPT5, like those in the RNA polymerase II C-terminal domain, are sites for P-TEFb phosphorylation and function in modulating its transcriptional elongation properties (Ivanov, 2000).

AIDS and the bare lymphocyte syndrome (BLS) are severe combined immunodeficiencies. BLS results from mutations in genes that regulate the expression of class II major histocompatibility (MHC II) determinants. One of these is the class II transactivator (CIITA). HIV and its transcriptional transactivator (Tat) also block the expression of MHC II genes. By binding to the same surface in the cyclin T1, which together with CDK9 forms the positive transcription elongation factor b (P-TEFb) complex, Tat inhibits CIITA. CIITA can also activate transcription when tethered artificially to RNA. Moreover, a dominant-negative CDK9 protein inhibits the activity of MHC II promoters. Thus, CIITA is a novel cellular coactivator that binds to P-TEFb for the expression of its target genes (Kanazawa, 2000).

Tat stimulates human immunodeficiency virus type 1 (HIV-1) transcriptional elongation by recruitment of carboxyl-terminal domain (CTD) kinases to the HIV-1 promoter. Using an immobilized DNA template assay, the effect of Tat on kinase activity has been analyzed during the initiation and elongation phases of HIV-1 transcription. Cyclin-dependent kinase 7 (CDK7) (TFIIH) and CDK9 (P-TEFb) both associate with the HIV-1 preinitiation complex. Hyperphosphorylation of the RNA polymerase II (RNAP II) CTD in the HIV-1 preinitiation complex, in the absence of Tat, takes place at CTD serine 2 and serine 5. Analysis of preinitiation complexes formed in immunodepleted extracts suggests that CDK9 phosphorylates serine 2, while CDK7 phosphorylates serine 5. Remarkably, in the presence of Tat, the substrate specificity of CDK9 is altered, such that the kinase phosphorylates both serine 2 and serine 5. Tat-induced CTD phosphorylation by CDK9 is strongly inhibited by low concentrations of 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole, an inhibitor of transcription elongation by RNAP II. Analysis of stalled transcription elongation complexes demonstrates that CDK7 is released from the transcription complex between positions +14 and +36, prior to the synthesis of transactivation response (TAR) RNA. In contrast, CDK9 stays associated with the complex through +79. Analysis of CTD phosphorylation indicates a biphasic modification pattern, one in the preinitiation complex and the other between +36 and +79. The second phase of CTD phosphorylation is Tat-dependent and TAR-dependent. These studies suggest that the ability of Tat to increase transcriptional elongation may be due to its ability to modify the substrate specificity of the CDK9 complex (Zhou, 2000).

To stimulate transcriptional elongation of HIV-1 genes, the transactivator Tat recruits the positive transcription elongation factor b (P-TEFb) to the initiating RNA polymerase II (RNAPII). The activation of transcription by RelA also depends on P-TEFb. Similar to Tat, RelA activates transcription when tethered to RNA. Moreover, TNF-alpha triggers the recruitment of P-TEFb to the NF-kappaB-regulated IL-8 gene. While the formation of the transcription preinitiation complex (PIC) remains unaffected, DRB, an inhibitor of P-TEFb, prevents RNAPII from elongating on the IL-8 gene. Remarkably, DRB inhibition sensitizes cells to TNF-alpha-induced apoptosis. Thus, NF-kappaB requires P-TEFb to stimulate the elongation of transcription and P-TEFb plays an unexpected role in regulating apoptosis (Barboric, 2001).

Findings of the present study add a new dimension to how RelA finalizes its transcriptional tasks. Thus, RelA not only promotes the initiation of transcription, but, through its association with P-TEFb, it also plays an essential role in the elongation of transcription. Importantly, the transactivation domain of RelA activates transcription when tethered artificially to the RNA element in a P-TEFb-dependent manner. Thus, RelA joins the small group of activators that stimulate the elongation of plasmid transcription. They include Tat, VP16, Cyclin T1, Cdk9, and CIITA. Although not determined yet for VP16, all of these proteins associate with or are part of P-TEFb. In contrast, transcriptional activators such as Sp-1 that stimulate only the initiation of transcription are inactive in this assay. This latter group interacts with several factors that are required for PIC assembly as well as with the basal transcription factor TFIIH, whose CTD kinase activity is required for promoter clearance. Notably, it was found that chimeric proteins between Rev and the three subunits of TFIIH, Cdk7, Cyclin H, and Mat1, do not activate plasmid transcription, consistent with their having roles in promoter clearance and not elongation of transcription. Additionally, of the two kinases, only P-TEFb has the ability to confer elongating properties on early transcriptional complexes. Thus, the RNA-tethering system is useful for assessing the ability of transcription factors to stimulate P-TEFb-dependent transcriptional elongation (Barboric, 2001).

Androgen receptor (AR) may communicate with the general transcription machinery on the core promoter to exert its function as a transcriptional modulator. The AR interacts with transcription factor IIH (TFIIH) under physiological conditions and overexpression of Cdk-activating kinase, the kinase moiety of TFIIH, enhances AR-mediated transcription in prostate cancer cells. AR interacts with PITALRE, a kinase subunit of positive elongation factor b (P-TEFb). Cotransfection of the plasmid encoding the mutant PITALRE (mtPITALRE), which is defective in its RNA polymerase II COOH-terminal domain (CTD)-kinase activity, results in preferential inhibition of AR-mediated transactivation. Indeed, AR transactivation in PC-3 cells is preferentially inhibited at the low concentration of 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB), a CTD kinase inhibitor. These results suggest that CTD phosphorylation may play an important role in AR-mediated transcription. Furthermore, a nuclear run-on transcription assay of the prostate-specific antigen gene, an androgen-inducible gene, shows that transcription efficiency of the distal region of the gene is enhanced upon androgen induction. Taken together, these reports suggest that AR interacts with TFIIH and P-TEFb and enhances the elongation stage of transcription (Lee, 2001).

Studying the sensitivity of transcription to the nucleotide analog 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole has led to the discovery of a number of proteins involved in the regulation of transcription elongation by RNA polymerase II. A highly purified elongation control system has been developed, composed of three purified proteins added back to isolated RNA polymerase II elongation complexes. Two of the proteins, 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole sensitivity-inducing factor (DSIF) and negative elongation factor (NELF), act as negative transcription elongation factors by increasing the time the polymerase spends at pause sites. P-TEFb reverses the negative effect of DSIF and NELF through a mechanism dependent on its kinase activity. TFIIF is a general initiation factor that positively affects elongation by decreasing pausing. TFIIF functionally competes with DSIF and NELF, and this competition is dependent on the relative concentrations of TFIIF and NELF (Renner, 2001).

The human positive transcription elongation factor P-TEFb, consisting of a CDK9/cyclin T1 heterodimer, functions as both a general and an HIV-1 Tat-specific transcription factor. P-TEFb activates transcription by phosphorylating RNA polymerase (Pol) II, leading to the formation of processive elongation complexes. As a Tat cofactor, P-TEFb stimulates HIV-1 transcription by interacting with Tat and the transactivating responsive (TAR) RNA structure located at the 5' end of the nascent viral transcript. 7SK, an abundant and evolutionarily conserved small nuclear RNA (snRNA) of unknown function, has been identified as a specific P-TEFb-associated factor. 7SK inhibits general and HIV-1 Tat-specific transcriptional activities of P-TEFb in vivo and in vitro by inhibiting the kinase activity of CDK9 and preventing recruitment of P-TEFb to the HIV-1 promoter. 7SK is efficiently dissociated from P-TEFb by treatment of cells with ultraviolet irradiation and actinomycin D. Since these two agents have been shown to significantly enhance HIV-1 transcription and phosphorylation of Pol II, the data provide a mechanistic explanation for their stimulatory effects. The 7SK/P-TEFb interaction may serve as a principal control point for the induction of cellular and HIV-1 viral gene expression during stress-related responses. These studies demonstrate the involvement of an snRNA in controlling the activity of a Cdk-cyclin kinase (Yang, 2001).

The transcription of eukaryotic protein-coding genes involves complex regulation of RNA polymerase (Pol) II activity in response to physiological conditions and developmental cues. One element of this regulation involves phosphorylation of the carboxy-terminal domain (CTD) of the largest polymerase subunit by a transcription elongation factor, P-TEFb, which comprises the kinase CDK9 and cyclin T1 or T2. In human HeLa cells more than half of the P-TEFb is sequestered in larger complexes that also contain 7SK RNA, an abundant, small nuclear RNA (snRNA) of hitherto unknown function. P-TEFb and 7SK associate in a specific and reversible manner. In contrast to the smaller P-TEFb complexes, which have a high kinase activity, the larger 7SK/P-TEFb complexes show very weak kinase activity. Inhibition of cellular transcription by chemical agents or ultraviolet irradiation trigger the complete disruption of the P-TEFb/7SK complex, and enhance CDK9 activity. The transcription-dependent interaction of P-TEFb with 7SK may therefore contribute to an important feedback loop modulating the activity of RNA Pol II (Nguyen, 2001).

The positive transcriptional elongation factor b (P-TEFb), consisting of CDK9 and cyclin T, stimulates transcription by phosphorylating RNA polymerase II. It becomes inactivated when associated with the abundant 7SK snRNA. The 7SK binding alone is not sufficient to inhibit P-TEFb. P-TEFb is inhibited by the HEXIM1 (hexamethylene bisacetamide-induced protein 1, a novel component of the 7SK snRNP; see Drosophila Hexim) protein in a process that specifically required 7SK for mediating the HEXIM1:P-TEFb interaction. This allows HEXIM1 to inhibit transcription both in vivo and in vitro. P-TEFb dissociated from HEXIM1 and 7SK in cells undergoing stress response, increasing the level of active P-TEFb for stress-induced transcription. P-TEFb is the predominant HEXIM1-associated protein factor, and thus likely to be the principal target of inhibition coordinated by HEXIM1 and 7SK. Since HEXIM1 expression is induced in cells treated with hexamethylene bisacetamide, a potent inducer of cell differentiation, targeting the general transcription factor P-TEFb by HEXIM1/7SK may contribute to the global control of cell growth and differentiation (Yik 2003).

Cyclin T1, together with the kinase CDK9, is a component of the transcription elongation factor P-TEFb which binds the human immunodeficiency virus type 1 (HIV-1) transactivator Tat. P-TEFb facilitates transcription by phosphorylating the carboxy-terminal domain (CTD) of RNA polymerase II. Cyclin T1 is an exceptionally large cyclin and is therefore a candidate for interactions with regulatory proteins. Granulin was identified as a cyclin T1-interacting protein that represses expression from the HIV-1 promoter in transfected cells. The granulins, mitogenic growth factors containing repeats of a cysteine-rich motif, interact with Tat. Granulin formed stable complexes in vivo and in vitro with cyclin T1 and Tat. Granulin binds to the histidine-rich domain of cyclin T1, which binds to the CTD, but not to cyclin T2. Binding of granulin to P-TEFb inhibits the phosphorylation of a CTD peptide. Granulin expression inhibits Tat transactivation, and tethering experiments show that this effect is due, at least in part, to a direct action on cyclin T1 in the absence of Tat. In addition, granulin is a substrate for CDK9 but not for the other transcription-related kinases CDK7 and CDK8. Thus, granulin is a cellular protein that interacts with cyclin T1 to inhibit transcription (Hogue, 2003).

Positive transcription elongation factor b (P-TEFb) comprises a cyclin (T1 or T2) and a kinase, cyclin-dependent kinase 9 (CDK9), which phosphorylates the carboxyl-terminal domain of RNA polymerase II. P-TEFb is essential for transcriptional elongation in human cells. A highly specific interaction among cyclin T1, the viral protein Tat, and the transactivation response (TAR) element RNA determines the productive transcription of the human immunodeficiency virus genome. In growing HeLa cells, half of P-TEFb is kinase inactive and binds to the 7SK small nuclear RNA. A novel protein termed MAQ1 (for menage á quatre) is also present in this complex. Since 7SK RNA is required for MAQ1 to associate with P-TEFb, a structural role for 7SK RNA is proposed. Inhibition of transcription results in the release of both MAQ1 and 7SK RNA from P-TEFb. Thus, MAQ1 cooperates with 7SK RNA to form a novel type of CDK inhibitor. According to yeast two-hybrid analysis and immunoprecipitations from extracts of transfected cells, MAQ1 binds directly to the N-terminal cyclin homology region of cyclins T1 and T2. Since Tat also binds to this cyclin T1 N-terminal domain and since the association between 7SK RNA/MAQ1 and P-TEFb competes with the binding of Tat to cyclin T1, it is speculated that the TAR RNA/Tat lentivirus system has evolved to subvert the cellular 7SK RNA/MAQ1 system (Michels, 2003).

Acidic or type IIB transcriptional activation domains (AADs) increase rates of initiation as well as elongation of transcription. For the former effects, AADs bind general transcription factors and larger coactivator complexes, which position RNA polymerase II (RNAPII) at sites of initiation of transcription. For the latter effects, their ubiquitylation plays an important role. In this study, this posttranslational modification increases the binding between a prototypic AAD and the positive transcription elongation factor b (P-TEFb), which contains a C-type cyclin (CycT1, CycT2, or CycK) and Cdk9. By phosphorylating negative elongation factors and the C-terminal domain of RNAPII, P-TEFb modifies the transcription complex for efficient elongation and cotranscriptional processing of mRNA. Indeed, the activation domain of VP16 and ubiquitin bind the cyclin boxes and the C terminus in CycT1, respectively. Moreover, the artificial fusion of ubiquitin with VP16 not only increases its activity via DNA and RNA, which is reflected in increased ratios of elongated to initiated transcripts, but rescues the deleterious substitution of alanine for phenylalanine at position 442 in its AAD. Thus, the ubiquitylation of AADs increases their interaction with P-TEFb and augments rates of elongation of transcription (Kurosu, 2004).

c-Myc promotes cellular proliferation, sensitizes cells to apoptosis and prevents differentiation. It binds cyclin T1 structurally and functionally from the positive transcription elongation factor b (P-TEFb). The cyclin-dependent kinase 9 (Cdk9) in P-TEFb then phosporylates the C-terminal domain of RNA polymerase II, which is required for the transition from initiation to elongation of eukaryotic transcription. Inhibiting P-TEFb blocks the transcription of its target genes as well as cellular proliferation and apoptosis induced by c-Myc.

To investigate the determinants of promoter-specific gene regulation by the glucocorticoid receptor (GR), the composition and function of regulatory complexes at two NFkappaB-responsive genes that are differentially regulated by GR were compared. Transcription of the IL-8 and IkappaBalpha genes is stimulated by TNFalpha in A549 cells, but GR selectively represses IL-8 mRNA synthesis by inhibiting Ser2 phosphorylation of the RNA polymerase II (pol II) C-terminal domain (CTD). The proximal kappaB elements at these genes differ in sequence by a single base pair, and both recruited RelA and p50. Surprisingly, GR is recruited to both of these elements, despite the fact that GR fails to repress the IkappaBalpha promoter. Rather, the regulatory complexes formed at IL-8 and IkappaBalpha were distinguished by differential recruitment of the Ser2 CTD kinase, P-TEFb. Disruption of P-TEFb function by the Cdk-inhibitor, DRB, or by small interfering RNA selectively blocks TNFalpha stimulation of IL-8 mRNA production. GR competes with P-TEFb recruitment to the IL-8 promoter. Strikingly, IL-8 mRNA synthesis is repressed by GR at a post-initiation step, demonstrating that promoter proximal regulatory sequences assemble complexes that impact early and late stages of mRNA synthesis. Thus, GR accomplishes selective repression by targeting promoter-specific components of NFkappaB regulatory complexes (Luecke, 2005).

Phosphorylated positive transcription elongation factor b (P-TEFb) is tagged for inhibition through association with 7SK snRNA

The positive transcription elongation factor b (P-TEFb), comprising CDK9 and cyclin T, stimulates transcription of cellular and viral genes by phosphorylating RNA polymerase II. A major portion of nuclear P-TEFb is sequestered and inactivated by the coordinated actions of the 7SK snRNA and the HEXIM1 protein, whose induced dissociation from P-TEFb is crucial for stress-induced transcription and pathogenesis of cardiac hypertrophy. The 7SK.P-TEFb interaction, which can occur independently of HEXIM1 and does not by itself inhibit P-TEFb, recruits HEXIM1 for P-TEFb inactivation. To study the control of this interaction, an in vitro system was established that reconstituted the specific interaction of P-TEFb with 7SK but not other snRNAs. Using this system, together with an in vivo binding assay, it has been show that the phosphorylation of CDK9, on possibly the conserved Thr-186 in the T-loop, is crucial for the 7SK.P-TEFb interaction. This phosphorylation is not caused by CDK9 autophosphorylation or the general CDK-activating kinase CAK, but rather by a novel HeLa nuclear kinase. Furthermore, the stress-induced disruption of the 7SK.P-TEFb interaction is not caused by any prohibitive changes in 7SK but by the dephosphorylation of P-TEFb, leading to the loss of the key phosphorylation important for 7SK binding. Thus, the phosphorylated P-TEFb is tagged for inhibition through association with 7SK. The implications of this mechanism in controlling P-TEFb activity during normal and stress-induced transcription are discussed (Chen, 2004).

PP2B and PP1alpha cooperatively disrupt 7SK snRNP to release P-TEFb for transcription in response to Ca2+ signaling

The positive transcription elongation factor b (P-TEFb), consisting of Cdk9 and cyclin T, stimulates RNA polymerase II elongation and cotranscriptional pre-mRNA processing. To accommodate different growth conditions and transcriptional demands, a reservoir of P-TEFb is kept in an inactive state in the multisubunit 7SK snRNP. Under certain stress or disease conditions, P-TEFb is released to activate transcription, although the signaling pathway(s) that controls this is largely unknown. Through analyzing the UV- or hexamethylene bisacetamide (HMBA)-induced release of P-TEFb from 7SK snRNP, this study found an essential role for the calcium ion (Ca2+)-calmodulin-protein phosphatase 2B (PP2B) signaling pathway. However, Ca2+ signaling alone is insufficient, and PP2B must act sequentially and cooperatively with protein phosphatase 1{alpha} (PP1{alpha}) to disrupt 7SK snRNP. Activated by UV/HMBA and facilitated by a PP2B-induced conformational change in 7SK snRNP, PP1{alpha} releases P-TEFb through dephosphorylating phospho-Thr186 in the Cdk9 T-loop. This event is also necessary for the subsequent recruitment of P-TEFb by the bromodomain protein Brd4 to the preinitiation complex, where Cdk9 remains unphosphorylated and inactive until after the synthesis of a short RNA. Thus, through cooperatively dephosphorylating Cdk9 in response to Ca2+ signaling, PP2B and PP1{alpha} alter the P-TEFb functional equilibrium through releasing P-TEFb from 7SK snRNP for transcription (Chen, 2008).

Inactivation of P-TEFb with Flavopiridol

Flavopiridol (L86-8275, HMR1275) is a cyclin-dependent kinase (Cdk) inhibitor that is in clinical trials as a cancer treatment because of its antiproliferative properties. The flavonoid potently inhibits transcription by RNA polymerase II in vitro by blocking the transition into productive elongation, a step controlled by P-TEFb. The ability of P-TEFb to phosphorylate the carboxyl-terminal domain of the large subunit of RNA polymerase II is inhibited by flavopiridol with a K(i) of 3 nm. Interestingly, the drug is not competitive with ATP. P-TEFb composed of Cdk9 and cyclin T1 is a required cellular cofactor for the human immunodeficiency virus (HIV-1) transactivator, Tat. Consistent with its ability to inhibit P-TEFb, flavopiridol blocks Tat transactivation of the viral promoter in vitro. Furthermore, flavopiridol blocks HIV-1 replication in both single-round and viral spread assays with an IC(50) of less than 10 nm (Chao, 2000).

Flavopiridol (L86-8275, HMR1275) is a cyclin-dependent kinase (Cdk) inhibitor in clinical trials as a cancer therapy that has been recently shown to block human immunodeficiency virus Tat transactivation and viral replication through inhibition of positive transcription elongation factor b (P-TEFb). Flavopiridol is the most potent P-TEFb inhibitor reported and the first Cdk inhibitor that is not competitive with ATP. The ability of flavopiridol to inhibit P-TEFb (Cdk9/cyclin T1) phosphorylation of both RNA polymerase II and the large subunit of the 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor was investigated; the IC(50) determined was directly related to the concentration of the enzyme. It is concluded that the flavonoid associates with P-TEFb with 1:1 stoichiometry even at concentrations of enzyme in the low nanomolar range. These results indicate that the apparent lack of competition with ATP could be caused by a very tight binding of the drug. A novel immobilized P-TEFb assay was developed and it was demonstrated that the drug remains bound for minutes even in the presence of high salt. Flavopiridol remains bound in the presence of a 1000-fold excess of the commonly used inhibitor DRB, suggesting that the immobilized P-TEFb can be used in a simple screening assay that would allow the discovery or characterization of compounds with binding properties similar to flavopiridol. Finally, the ability of flavopiridol and DRB to inhibit transcription in vivo was compared using nuclear run-on assays and it was concluded that P-TEFb is required for transcription of most RNA polymerase II molecules in vivo (Chao, 2001).

Recruitment of P-TEFb via Tat to HIV-1 LTR

To identify novel inhibitors of transcriptional activation by the HIV Tat protein, a combination of in vitro and in vivo Tat-dependent transcription assays were used to screen >100,000 compounds. All compounds identified block Tat-dependent stimulation of transcriptional elongation. Analysis of a panel of structurally diverse inhibitors indicates that their target is the human homolog of Drosophila positive transcription elongation factor b (P-TEFb). Loss of Tat transactivation in extracts depleted of the kinase subunit of human P-TEFb, PITALRE, is reversed by addition of partially purified human P-TEFb. Transfection experiments with wild-type or kinase knockout PITALRE demonstrate that P-TEFb is required for Tat function. These results suggest that P-TEFb represents an attractive target for the development of novel HIV therapeutics (Mancebo, 1997).

The cloning of the small subunit of Drosophila P-TEFb is reported in this study; it encodes a Cdc2-related protein kinase. Sequence comparison suggests that a protein with 72% identity, PITALRE, could be the human homolog of the Drosophila protein. Functional homology was suggested by transcriptional analysis of an RNA polymerase II promoter with HeLa nuclear extract depleted of PITALRE. Because the depleted extract lost the ability to produce long DRB-sensitive transcripts and this loss was reversed by the addition of purified Drosophila P-TEFb, it is proposed that PITALRE is a component of human P-TEFb. In addition, it was found that PITALRE associates with the activation domain of HIV-1 Tat, indicating that P-TEFb is a Tat-associated kinase (TAK). An in vitro transcription assay demonstrates that the effect of Tat on transcription elongation requires P-TEFb and suggests that the enhancement of transcriptional processivity by Tat is attributable to enhanced function of P-TEFb on the HIV-1 LTR (Zhu, 1997).

HIV-1 Tat greatly stimulates gene expression from the viral promoter located in the long terminal repeat (LTR) of the human immunodeficiency virus (HIV) genome. The primary effect of Tat is to increase the processivity of RNA polymerases that otherwise would prematurely terminate after the synthesis of short nascent transcripts. The elongation effect requires an RNA element, transactivation response (TAR), that forms a stem and loop structure with which Tat can associate. Consistent with its effect on elongation Tat has been found to be an integral component of activated elongation complexes. The function of Tat in elongation is similar to the function of P-TEFb in that the transcriptional stimulation by Tat is sensitive to DRB and requires the CTD of the largest subunit of RNA polymerase II. In vitro Tat specifically associates with a serine/threonine kinase through its activation domain. Like P-TEFb, this Tat-associated kinase (TAK) is sensitive to DRB. It has been suggested that Tat might function by recruiting TAK to phosphorylate the CTD. Tat has also been shown to affect initiation. Tat can associate with transcription preinitiation complexes and this association does not require the TAR element. Consistent with a function in initiation, recent results have shown that Tat can interact with the RNA polymerase II holoenzyme in the absence of TAR (Zhu, 1997 and references therein).

Transcriptional activation of the HIV type 1 (HIV-1) long terminal repeat (LTR) promoter element by the viral Tat protein is an essential step in the HIV-1 life cycle. Tat function is mediated by the TAR RNA target element encoded within the LTR and is known to require the recruitment of a complex consisting of Tat and the cyclin T1 (CycT1) component of P-TEFb to TAR. Both TAR and Tat become entirely dispensable for activation of the HIV-1 LTR promoter when CycT1/P-TEFb is artificially recruited to a heterologous promoter proximal RNA target. The level of activation observed is indistinguishable from the level induced by Tat and is neither inhibited nor increased when Tat is expressed in trans. Activation by artificially recruited CycT1 depends on the ability to bind the CDK9 component of P-TEFb. In contrast, although binding to both Tat and TAR is essential for the ability of CycT1 to act as a Tat cofactor, these interactions become dispensable when CycT1 is directly recruited to the LTR. Importantly, activation of the LTR both by Tat and by directly recruited CycT1 was found to be at the level of transcription elongation. Together, these data demonstrate that recruitment of CycT1/P-TEFb to the HIV-1 LTR is fully sufficient to activate this promoter element and imply that the sole role of the Tat/TAR axis in viral transcription is to permit the recruitment of CycT1/P-TEFb (Bieniasz, 1999).

Human immunodeficiency virus, type 1 (HIV-1) Tat protein activates transcription from the HIV-1 long terminal repeat. Tat interacts with TFIIH and Tat-associated kinase (a transcription elongation factor P-TEFb) and requires the carboxyl-terminal domain of the largest subunit of RNA polymerase II (pol II) for transactivation. A stepwise RNA pol II walking approach has been developed and Western blotting has been used to determine the role of TFIIH and P-TEFb in HIV-1 transcription elongation. P-TEFb is a component of the preinitiation complex and travels with the elongating RNA pol II, whereas TFIIH is released from the elongation complexes before the trans-activation responsive region RNA is synthesized. These results suggest that TFIIH and P-TEFb are involved in the clearance of promoter-proximal pausing of RNA pol II on the HIV-1 long terminal repeat at different stages (Ping, 1999).

Tat stimulation of human immunodeficiency virus type 1 (HIV-1) transcription requires Tat-dependent recruitment of human positive transcription elongation factor b (P-TEFb) to the HIV-1 promoter and the formation on the trans-acting response element (TAR) RNA of a P-TEFb-Tat-TAR ternary complex. The P-TEFb heterodimer of Cdk9-cyclin T1 is intrinsically incapable of forming a stable complex with Tat and TAR due to two built-in autoinhibitory mechanisms in P-TEFb. Both mechanisms exert little effect on the P-TEFb-Tat interaction but prevent the P-TEFb-Tat complex from binding to TAR RNA. The first autoinhibition arises from the unphosphorylated state of Cdk9, which establishes a P-TEFb conformation unfavorable for TAR recognition. Autophosphorylation of Cdk9 overcomes this inhibition by inducing conformational changes in P-TEFb, thereby exposing a region in cyclin T1 for possible TAR binding. An intramolecular interaction between the N- and C-terminal regions of cyclin T1 sterically blocks the P-TEFb-TAR interaction and constitutes the second autoinhibitory mechanism. This inhibition is relieved by the binding of the C-terminal region of cyclin T1 to the transcription elongation factor Tat-SF1 and perhaps other cellular factors. Upon release from the intramolecular interaction, the C-terminal region also interacts with RNA polymerase II and is required for HIV-1 transcription, suggesting its role in bridging the P-TEFb-Tat-TAR complex and the basal elongation apparatus. These data reveal novel control mechanisms for the assembly of a multicomponent transcription elongation complex at the HIV-1 promoter (Fong, 2000).

The elongation of transcription is a highly regulated process that requires negative and positive effectors. By binding the double-stranded stem in the transactivation response (TAR) element, RD protein from the negative transcription elongation factor (NELF) inhibits basal transcription from the long terminal repeat of the human immunodeficiency virus type 1 (HIVLTR). Tat and its cellular cofactor, the positive transcription elongation factor b (P-TEFb), overcome this negative effect. Cdk9 in P-TEFb also phosphorylates RD at sites next to its RNA recognition motif. A mutant RD protein that mimics its phosphorylated form no longer binds TAR nor represses HIV transcription. In sharp contrast, a mutant RD protein that cannot be phosphorylated by P-TEFb functions as a dominant-negative effector and inhibits Tat transactivation. These results better define the transition from abortive to productive transcription and thus replication of HIV (Fujinaga, 2004).

The elongation of transcription from the human immunodeficiency virus type 1 long terminal repeat (HIVLTR) is regulated negatively and positively by cellular factors and the viral transactivator Tat. In the absence of Tat, the elongating RNA polymerase II (RNAPII) is arrested by the negative transcriptional elongation factor (N-TEF), which includes the DRB sensitivity-inducing factor (DSIF) and the negative elongation factor (NELF), resulting in the accumulation of short transcripts. However, in the presence of Tat, the positive transcription elongation factor b (P-TEFb), consisting of the cyclin-dependent kinase 9 (Cdk9) and cyclin T1 (CycT1), is recruited to the transactivation response (TAR) element, which forms a stable RNA stem-loop at the 5' end of all viral transcripts. Cdk9 then phosphorylates DSIF and the C-terminal domain (CTD) of RNAPII, which is essential for the productive elongation of transcription (Fujinaga, 2004 and references therein).

Both DSIF and NELF are found on the HIVLTR after the initiation of viral transcription. DSIF is composed of Spt4 and Spt5. Spt5 binds the unphosphorylated but not the phosphorylated form of the CTD (CTDa of RNAPIIa but not CTDo from RNAPIIo). Thus, P-TEFb directly regulates the interaction between DSIF and RNAPII. NELF is comprised of four subunits, NELF-A or WHSC, NELF-B, alternatively spliced NELF-C/D, and NELF-E or RD. NELF-A and RD contain RNA recognition motifs (RRM) and bind a number of RNA elements, which are required for the inhibitory effect of NELF on transcription. Of importance, RD binds TAR via its RRM. This interaction could contribute to low basal levels of viral transcription, and therefore, to the proviral transcriptional latency in infected cells. Although P-TEFb can alleviate negative effects of NELF in vitro, no mechanism exists for this transition from negative to positive regulation of transcriptional elongation (Fujinaga, 2004 and references therein).

This study provides such a mechanism, taken from HIV. First, by binding the bottom stem in TAR, RD from NELF and Spt5 from DSIF cooperatively help to arrest RNAPII on the HIVLTR. Next, the complex between P-TEFb and Tat is recruited to the 5' bulge and central loop in TAR. Finally, Cdk9 phosphorylates RD, Spt5, and RNAPII, thus removing N-TEF from TAR. As a consequence, productive elongation of HIV transcription ensues (Fujinaga, 2004 and references therein).

Cdk9 and transcriptional elongation in C. elegans

The metazoan transcription elongation factor P-TEFb (CDK-9/cyclin T) is essential for HIV transcription, and is recruited by some cellular activators. P-TEFb promotes elongation in vitro by overcoming pausing that requires the SPT-4/SPT-5 complex, but considerable evidence indicates that SPT-4/SPT-5 facilitates elongation in vivo. Double stranded RNA interference to investigate P-TEFb functions in vivo, in the C. elegans embryo. P-TEFb is found to be broadly essential for expression of early embryonic genes. P-TEFb is required for phosphorylation of Ser 2 of the RNA Polymerase II C-terminal domain (CTD) repeat, but not for most CTD Ser 5 phosphorylation, supporting the model that P-TEFb phosphorylates CTD Ser 2 during elongation. Remarkably, although heat shock genes are cdk-9-dependent, they can be activated when spt-4 and spt-5 expression is inhibited along with cdk-9. This observation suggests that SPT-4/SPT-5 has an inhibitory function in vivo, and that mutually opposing influences of P-TEFb and SPT-4/SPT-5 may combine to facilitate elongation, or ensure fidelity of mRNA production. Other genes are not expressed when cdk-9, spt-4, and spt-5 are inhibited simultaneously, suggesting that these genes require P-TEFb in an additional mechanism, and that they and heat shock genes are regulated through different P-TEFb-dependent elongation pathways (Shim, 2002).

The metazoan kinase CDK-9 and its cyclin partners (T1, T2 or K) form the transcription factor P-TEFb, which prevents transcription from stalling in vitro. CDK-9 is similar to and may be functionally related to yeast Ctk1. Stimulation of elongation by P-TEFb is required for human immunodeficiency virus (HIV) transcription, during which P-TEFb is recruited directly to the nascent mRNA by the transactivator Tat. P-TEFb preferentially phosphorylates CTD Ser 2 in vitro, but when associated with Tat P-TEFb, also phosphorylates Ser 5, and may thereby bypass the requirement for CDK-7. P-TEFb is recruited by cellular activators that include NF-kappaB and c-Myc, and it is present at sites of active transcription in Drosophila. Treatment of mammalian cells with a pharmacological inhibitor of CDK-9 appears to block most Pol II transcription, but P-TEFb functions have not been investigated in a genetic system. It is an important issue for HIV therapeutics to understand how broadly P-TEFb is required for transcription of cellular genes, because CDK-9 inhibition is a preeminent strategy for interfering with Tat (Shim, 2002).

Transcription proceeds independently of P-TEFb in vitro in the absence of the factor DSIF, which cooperates with the negative factor NELF to inhibit elongation. While this argues that P-TEFb is required simply to overcome DSIF/NELF-dependent pausing, DSIF increases elongation in vitro under conditions that enhance pausing, suggesting that it has a dual function. The bulk of in vivo evidence also suggests that DSIF facilitates elongation, possibly in concert with P-TEFb. DSIF consists of the proteins SPT-4 and SPT-5, which yeast and zebrafish genetic experiments indicate are positive elongation factors. SPT-5 is related to Escherichia coli NusG, an antiterminator that increases elongation. Accordingly, SPT-5 is associated with elongating RNA Pol II in vivo and is required for HIV Tat to stimulate elongation. To understand how P-TEFb promotes transcription elongation, it is critical to determine whether an inhibitory effect of SPT-4/SPT-5 can be identified in vivo, and to elucidate how P-TEFb interacts functionally with SPT-4/SPT-5 in vivo (Shim, 2002).

Using RNA interference (RNAi), the requirements for P-TEFb for CTD Ser 2 and Ser 5 phosphorylation, and for early embryonic transcription has been investigated. Whether depletion of SPT-4/SPT-5 relieves any requirements for P-TEFb was also tested. The results indicate that P-TEFb is required for phosphorylation of CTD Ser 2 but not for most Ser 5 phosphorylation. In apparent contrast to yeast Ctk1, P-TEFb is also broadly essential for transcription in the early embryo. Simultaneous inhibition of spt-4 and spt-5 restores heat shock gene transcription in cdk-9(RNAi) embryos, suggesting that P-TEFb counteracts an inhibitory function of SPT-4/SPT-5 in vivo. Significantly, other genes still required P-TEFb when spt-4 and spt-5 are inhibited, suggesting that they and heat shock genes maintain transcription elongation through different P-TEFb-dependent pathways (Shim, 2002).

In this first genetics-based study of P-TEFb, it was determined that C. elegans P-TEFb is broadly required for embryonic transcription. In cdk-9(RNAi) embryos, terminal arrest phenotypes are indistinguishable from those caused by lack of the essential transcription factors ama-1 or ttb-1, and expression of each gene that was analyzed was reduced to background levels. Simultaneous inhibition of the cyclin T genes cit-1.1 and cit-1.2 has similar effects, suggesting that in the embryo these cyclin T proteins function redundantly. This broad requirement for P-TEFb suggests that a significant number of transcription activators or coactivators may recruit P-TEFb directly. P-TEFb-dependent elongation pathways might be especially important in rapidly dividing early embryonic cells, but it is considered more likely that P-TEFb is also generally essential for mRNA transcription in other metazoan cell types, a model that is consistent with pharmacologic inhibitor studies. Although Tat-dependent transcription is particularly sensitive to CDK-9 inhibition, treatment with highly specific CDK-9 inhibitors may significantly impair transcription of important host cell genes (Shim, 2002).

C. elegans P-TEFb also appears to be essential for most embryonic CTD Ser 2 phosphorylation. RNAi embryos in which either CDK-9 or cyclin T was depleted lack detectable specific alpha-PSer 2 staining, comparably to ama-1(RNAi) embryos. Depletion of CDK-9 does not appear to reduce the overall level of CTD Ser 5 phosphorylation however. The results suggest that P-TEFb is the major embryonic CTD Ser 2 kinase, and that CTD Ser 2 phosphorylation by P-TEFb may be generally important for metazoan transcription. An analysis of two genes in S. cerevisiae suggests that during transcription, the CDK-9-related kinase Ctk1 phosphorylates Ser 2 after Ser 5 phosphorylation has occurred. In the C. elegans embryo, appearance of both the alpha-PSer 5 and alpha-Ser 2 CTD phosphoepitopes depends on the CTD Ser 5 kinase CDK-7, and on essential initiation factors such as ttb-1. The finding that P-TEFb may not be required for most CTD Ser 5 phosphorylation suggests that a sequential ordering of CTD Ser 5 and Ser 2 phosphorylation may be a general characteristic of Pol II transcription (Shim, 2002).

In S. cerevisiae, the CTD Ser 2 kinase Ctk1 is not essential for viability, in apparent contrast to the importance of C. elegans P-TEFb for embryonic transcription. Yeast genes on average are shorter than metazoan genes, but some genes analyzed in this study are less than 2 kb in length (med-1; hsp-16.2), arguing against a significant difference in intrinsic elongation requirements. Alternatively, differences in biological contexts may be involved, or in S. cerevisiae, a different kinase might compensate for Ctk1 at some genes or substrates. One candidate is the related kinase Bur1, which promotes elongation but preferentially phosphorylates CTD Ser 5. Finally, given that mRNA processing is coupled to elongation, a broader requirement for a CTD Ser 2 kinase in metazoans might be related to the greater relative importance of mRNA splicing (Shim, 2002).

In light of the considerable evidence that SPT-4/SPT-5 promotes transcription elongation, it seems paradoxical that in vitro SPT-4/SPT-5 induces polymerase pausing that must be overcome by P-TEFb. In vivo evidence has been obtained to support the latter model. In cdk-9(RNAi) embryos, transcription of two different heat shock genes was significantly restored when spt-4 and spt-5 were also inhibited by RNAi. This recovery of heat shock gene expression in spt-4; spt-5; cdk-9(RNAi) embryos is not associated with a global activation of an alternative CTD Ser 2 phosphorylation pathway by heat shock, strongly suggesting that removal of an inhibitory effect of SPT-4/SPT-5 is involved. HSP-16.2::GFP expression is restored to a lesser extent in spt-5; cdk-9(RNAi) embryos, indicating that both spt-4 and spt-5 contribute to this inhibition. Although these effects could derive from unanticipated functions of P-TEFb and SPT-4/SPT-5, a simpler explanation is that these factors have opposing influences on transcription. Biochemical evidence indicates that P-TEFb counteracts SPT-4/SPT-5 by phosphorylating the CTD, but its phosphorylation of SPT-5 might also be important. By demonstrating an antagonistic relationship between cdk-9 and spt-4/spt-5 directly in a genetic system, the data strongly support the model that overcoming SPT-4/SPT-5-dependent inhibition is a critical function of P-TEFb. At the same time, the model that SPT-4/SPT-5 has both negative and positive effects may explain elevated HSP-16.2::GFP expression was not observed in spt-4; spt-5(RNAi) embryos (Shim, 2002).

A model that accommodates both positive and negative roles is that SPT-4/SPT-5 monitors recruitment of elongation or mRNA processing factors at the Pol II complex. By enforcing this transcription 'checkpoint,' SPT-4/SPT-5 may enhance the efficiency of elongation, or the fidelity of cotranscriptional mRNA processing. This scheme is consistent with evidence that SPT-5 directly promotes mRNA capping. SPT-5 may also promote elongation by acting as an antiterminator, presumably after SPT-4/SPT-5-dependent pausing has been relieved by P-TEFb. It has been proposed that SPT-4/SPT-5 and NELF together induce 5' pausing of Pol II prior to elongation or capping, but the evidence that SPT-4/SPT-5 is opposed by P-TEFb suggests that SPT-4/SPT-5-dependent pausing might also occur more distally within a gene. In yeast, CTD Ser 2 phosphorylation predominates >500 bp from the promoter, where Ctk1 and the CTD phosphatase Fcp1 engage in an ongoing process of CTD Ser 2 phosphorylation and dephosphorylation. SPT-5 similarly appears to be present along actively transcribed metazoan genes. If SPT-5-dependent pausing is regulated by a dynamic modulation of CTD Ser 2 phosphorylation, this process may monitor recruitment of elongation or mRNA processing factors until late stages of the transcription cycle (Shim, 2002).

cdk-9; spt-4; spt-5(RNAi) embryos expressed two heat shock genes, but not the early genes med-1 or pes-10. cdk-9; spt-4; spt-5(RNAi) embryos also are not distinguishably different in their terminal arrest phenotype from cdk-9(RNAi) or ama-1(RNAi) embryos, suggesting that they fail to transcribe many or possibly most other genes. In yeast, heat shock genes differ from most cellular genes in that they can be transcribed independently of the CTD, and do not require activation mechanisms that depend upon kin28 or certain Mediator components. These findings indicate that heat shock genes are also distinct in their requirements for P-TEFb, suggesting that metazoans are able to sustain transcription elongation through two different P-TEFb-dependent pathways (Shim, 2002).

What distinguishes these P-TEFb-dependent postinitiation pathways from each other? It is possible that heat shock genes are highly resistant to SPT-4/SPT-5-dependent pausing, and that sufficient residual SPT-4/SPT-5 remain in cdk-9; spt-4; spt-5(RNAi) embryos to prevent expression of med-1, pes-10, and most other embryonic genes. This seems unlikely however, given that spt-4; spt-5(RNAi) embryos are characterized by consistent and severe developmental abnormalities. Alternatively, at most cellular genes P-TEFb may be required in an SPT-4/SPT-5-independent mechanism that was not predicted by in vitro experiments that were performed using naked DNA. For example, P-TEFb may be needed to overcome an SPT-4/SPT-5-independent elongation block that is specific to a chromatin environment. Consistent with this model, the FACT complex is required along with P-TEFb for elongation to occur on chromatin templates in vitro. P-TEFb might also activate other positive elongation factors. Alternatively, evidence that the phosphorylated CTD stimulates certain mRNA processing steps suggests that these steps may be facilitated by P-TEFb, an effect that could increase elongation rates. Elucidation of this P-TEFb-dependent mechanism, and of why it is regulated differently at heat shock genes, should provide important and perhaps unexpected insights into postinitiation levels of transcription regulation (Shim, 2002).

The positive transcription elongation factor b (P-TEFb) contains cyclin T1 (CycT1) and cyclin-dependent kinase 9 (Cdk9). For activating the expression of eukaryotic genes, the histidine-rich sequence in CycT1 binds the heptapeptide repeats in the C-terminal domain (CTD) of RNA polymerase II (RNAPII), whereupon Cdk9 phosphorylates the CTD. Alanine-substituted heptapeptide repeats that cannot be phosphorylated also bind CycT1. When placed near transcription units, these CTD analogs block effects of P-TEFb. Remarkably, the transcriptional repressor PIE-1 from Caenorhabditis elegans behaves analogously. It binds CycT1 via an alanine-containing heptapeptide repeat and inhibits transcriptional elongation. Thus, these findings reveal a new mechanism by which repressors inhibit eukaryotic transcription (Zhang, 2003).

Nuclear CDKs drive Smad transcriptional activation and turnover in BMP and TGF-beta pathways

TGF-beta and BMP receptor kinases activate Smad transcription factors by C-terminal phosphorylation. This study identified a subsequent agonist-induced phosphorylation that plays a central dual role in Smad transcriptional activation and turnover. As receptor-activated Smads form transcriptional complexes, they are phosphorylated at an interdomain linker region by CDK8 and CDK9, which are components of transcriptional mediator and elongation complexes. These phosphorylations promote Smad transcriptional action, which in the case of Smad1 is mediated by the recruitment of YAP (Drosophila homolog: Yorkie) to the phosphorylated linker sites. An effector of the highly conserved Hippo organ size control pathway, YAP supports Smad1-dependent transcription and is required for BMP suppression of neural differentiation of mouse embryonic stem cells. The phosphorylated linker is ultimately recognized by specific ubiquitin ligases, leading to proteasome-mediated turnover of activated Smad proteins. Thus, nuclear CDK8/9 drive a cycle of Smad utilization and disposal that is an integral part of canonical BMP and TGF-beta pathways (Alarcon, 2009).

The present findings reveal a remarkable integration of Smad regulatory functions by agonist-induced, CDK8/9-mediated phosphorylation of the linker region and highlight this event as an integral feature of the transcriptional action and turnover of receptor-activated Smad proteins. Agonist-induced linker phosphorylation of R-Smads is a general feature of BMP and TGF-β pathways, occurring in all the responsive cell types examined, shortly after Smad tail phosphorylation. The evidence identifies CDK9 as the kinases involved and does not support a major role for MAPKs or cell-cycle-regulatory CDKs in this process. CDK8 and cyclinC are components of the Mediator complex that couples enhancer-binding transcriptional activators to RNAP II for transcription initiation. CDK9 and cyclinT1 constitute the P-TEFb complex, which promotes transcriptional elongation. CDK8 and CDK9 phosphorylate overlapping serine clusters in the C-terminal domain of RNAP II, a region which integrates regulatory inputs by binding proteins involved in mRNA biogenesis. Thus, CDK8 and CDK9 may provide coordinated regulation of Smad transcriptional activators and RNAP II (Alarcon, 2009).

Precedent exists for the ability of CDK8 to phosphorylate enhancer-binding transcription factors. The CDK8 ortholog Srb10 in budding yeast phosphorylates Gcn4 marking this transcriptional activator of amino acid biosynthesis for recognition by the SCF(Cdc4) ubiquitin ligase. In mammalian cells, CDK8 phosphorylates the ICD signal transduction component of Notch, targeting it to the Fbw7/Sel10 ubiquitin ligase. However, whereas CDK8-mediated phosphorylation inhibits Gcn4 and Notch activity, this study shows that phosphorylation of agonist-activated Smads by CDK8/9 enables Smad-dependent transcription before triggering Smad turnover (Alarcon, 2009).

Activated Smads undergo proteasome-mediated degradation as well as phosphatase-mediated tail dephosphorylation to keep signal transduction closely tied to receptor activation. This study shows that BMP-induced Smad1-ALP generates binding sites for Smurf1, accomplishing in the nucleus what MAPK-mediated phosphorylation of basal-state Smad1 accomplishes in the cytoplasm. Similarly, TGF-β-induced linker phosphorylation of Smad2/3 provides a binding site for Nedd4L (Alarcon, 2009).

The results also reveal a positive role for ALP in Smad-dependent transcription. Smad proteins with phosphorylation-resistant linker mutations are more stable as receptor-activated signal transducers than their wild-type counterparts, yet they are transcriptionally less active. Indeed, mutation of Smad1 linker phosphorylation sites (in a wild-type Smad5 background) does not result in a straight BMP gain-of-function phenotype but rather in an unforeseen gastric epithelial phenotype. Although the interpretation of this phenotype is confounded by the contribution of MAPK signaling to linker phosphorylation, it is consistent with the present evidence that Smad1 linker phosphorylation plays an active role in BMP signaling (Alarcon, 2009).

Focusing on Smad1 to define this dual role, it was found that the phosphorylated linker sites, together with a neighboring PY motif, are recognized also by the transcriptional coactivator YAP. Smurf1 and YAP present closely related WW domains with a similar selectivity toward linker-phosphorylated Smad1. YAP is recruited with Smad1 to BMP responsive enhancers and knockdown of YAP inhibits BMP-induced Id gene responses in mouse embryonic stem cells. Both BMP and YAP act as suppressors of neural differentiation in specific contexts. This study shows that YAP supports the ability of BMP to block neural lineage commitment through the induction of Id family members, creating a link between YAP-dependent BMP transcriptional output and ES cell fate determination (Alarcon, 2009).

Thus, a common structure fulfills two opposite functions -- Smad1 transcriptional action and turnover -- by recruiting different proteins, YAP and Smurf1, at different stages of the signal transduction cycle. The cyclic recruitment and continuous turnover of transcription factors on target enhancers are required for the proper response of cells to developmental and homeostatic cues. It is proposed that Smad activation by TGF-β family agonists accomplishes this important requirement through linker phosphorylation that triggers transcriptional action and messenger turnover in one stroke (Alarcon, 2009).

Activation of the Hippo pathway by cell density cues triggers a kinase cascade that culminates in the inactivation of YAP (Yorkie in Drosophila), a transcriptional coactivator that acts through interactions with enhancer-binding factors, including TEAD/scalloped, Runx, p73, and others. Yorkie/YAP promotes cell proliferation and survival and organ growth, whereas the upstream components of the inhibitory kinase cascade constrain organ size and act as tumor suppressors. Elucidating the links between the Hippo pathway and other signaling cascades is an important open question. The evidence that YAP is recruited to BMP-activated Smad1 reveals a link between the BMP and the Hippo pathways. Both these signaling cascades have the capacity to control organ size and do so in a manner suggestive of interactions with other patterned signals. An example is the regulation of imaginal disc growth by Dpp via cell competition, a process by which slow proliferating cells are eliminated in favor of their higher-proliferating neighbors. A genetic screen for negative regulators of Dpp signaling that protect cells from being outcompeted identified upstream components of the Hippo pathway. Inactivation of these factors elevated Dpp target gene expression, presumably by failing to inhibit Yorkie, and allowed cells to outcompete their neighbors, suggesting a functional convergence of the Hippo and BMP pathways that foreshadowed these findings (Alarcon, 2009).

Although ALP is a general event in Smad activation, YAP may not be a universal partner of linker-phosphorylated Smad1. Smad ALP likely plays a wider role potentially acting to recruit coactivators other than YAP, depending on the cellular context or the target gene. Also of interest is the identity of factors that may play an analogous role in linker-phosphorylated Smad2/3 in the TGF-β pathway. The linker phosphorylation sites and PY motifs of Smad1 and Smad2/3 are conserved in the otherwise divergent linker regions of the Drosophila orthologs Mad/dSmad1 and dSmad2, respectively. Although the contribution of the MAPK pathway in linker phosphorylation precludes a clearcut genetic investigation of these functions, they are probably conserved across metazoans. A concerted search for Smad phospholinker interacting factors would answer many of these questions and would fully elucidate the role of the Smad linker region as a centerpiece in the function, regulation, and connectivity of Smad transcription factors (Alarcon, 2009).

A Smad action turnover switch operated by WW domain readers of a phosphoserine code

When directed to the nucleus by TGF-β or BMP signals, Smad proteins undergo cyclin-dependent kinase 8/9 (CDK8/9) and glycogen synthase kinase-3 (GSK3) phosphorylations that mediate the binding of YAP and Pin1 for transcriptional action, and of ubiquitin ligases Smurf1 and Nedd4L for Smad destruction. This study demonstrates that there is an order of events-Smad activation first and destruction later-and that it is controlled by a switch in the recognition of Smad phosphoserines by WW domains in their binding partners. In the BMP pathway, Smad1 phosphorylation by CDK8/9 creates binding sites for the WW domains of YAP, and subsequent phosphorylation by GSK3 switches off YAP binding and adds binding sites for Smurf1 WW domains. Similarly, in the TGF-β pathway, Smad3 phosphorylation by CDK8/9 creates binding sites for Pin1 and GSK3, then adds sites to enhance Nedd4L binding. Thus, a Smad phosphoserine code and a set of WW domain code readers (see A Smad action turnover switch operated by WW domain readers of a phosphoserine code) provide an efficient solution to the problem of coupling TGF-β signal delivery to turnover of the Smad signal transducers (Aragón, 2011).

Cross-talk among RNA polymerase II kinases modulates C-terminal domain phosphorylation

The RNA polymerase II (Pol II) C-terminal domain (CTD) serves as a docking site for numerous proteins, bridging various nuclear processes to transcription. The recruitment of these proteins is mediated by CTD phospho-epitopes generated during transcription. The mechanisms regulating the kinases that establish these phosphorylation patterns on the CTD are not known. This study reports that three CTD kinases, CDK7, CDK9, and BRD4, engage in cross-talk, modulating their subsequent CTD phosphorylation. BRD4 phosphorylates PTEFb/CDK9 at either Thr-29 or Thr-186, depending on its relative abundance, which represses or activates CDK9 CTD kinase activity, respectively. Conversely, CDK9 phosphorylates BRD4 enhancing its CTD kinase activity. The CTD Ser-5 kinase CDK7 also interacts with and phosphorylates BRD4, potently inhibiting BRD4 kinase activity. Additionally, the three kinases regulate each other indirectly through the general transcription factor TAF7. An inhibitor of CDK9 and CDK7 CTD kinase activities, TAF7 also binds to BRD4 and inhibits its kinase activity. Each of these kinases phosphorylates TAF7, affecting its subsequent ability to inhibit the other two. Thus, a complex regulatory network governs Pol II CTD kinases (Devaiah, 2012).

CDK9-mediated transcription elongation is required for MYC addiction in hepatocellular carcinoma

One-year survival rates for newly diagnosed hepatocellular carcinoma (HCC) are <50%, and unresectable HCC carries a dismal prognosis owing to its aggressiveness and the undruggable nature of its main genetic drivers. By screening a custom library of shRNAs directed toward known drug targets in a genetically defined Myc-driven HCC model, cyclin-dependent kinase 9 (Cdk9) was identified as required for disease maintenance. Pharmacological or shRNA-mediated CDK9 inhibition led to robust anti-tumor effects that correlated with MYC expression levels and depended on the role that both CDK9 and MYC exert in transcription elongation. These results establish CDK9 inhibition as a therapeutic strategy for MYC-overexpressing liver tumors and highlight the relevance of transcription elongation in the addiction of cancer cells to MYC (Huang, 2014).

Phase-separation mechanism for C-terminal hyperphosphorylation of RNA polymerase II

Hyperphosphorylation of the C-terminal domain (CTD) of the RPB1 subunit of human RNA polymerase (Pol) II is essential for transcriptional elongation and mRNA processing (see Drosophila Pol II). The CTD contains 52 heptapeptide repeats of the consensus sequence YSPTSPS. The highly repetitive nature and abundant possible phosphorylation sites of the CTD exert special constraints on the kinases that catalyse its hyperphosphorylation. Positive transcription elongation factor b (P-TEFb)-which consists of CDK9 (see Drosophila Cdk9) and cyclin T1-is known to hyperphosphorylate the CTD and negative elongation factors to stimulate Pol II elongation. The sequence determinant on P-TEFb that facilitates this action is currently unknown. This study identified a histidine-rich domain in cyclin T1 that promotes the hyperphosphorylation of the CTD and stimulation of transcription by CDK9. The histidine-rich domain markedly enhances the binding of P-TEFb to the CTD and functional engagement with target genes in cells. In addition to cyclin T1, at least one other kinase -- DYRK1A (see Drosophila Minibrain) -- also uses a histidine-rich domain to target and hyperphosphorylate the CTD. As a low-complexity domain, the histidine-rich domain also promotes the formation of phase-separated liquid droplets in vitro, and the localization of P-TEFb to nuclear speckles that display dynamic liquid properties and are sensitive to the disruption of weak hydrophobic interactions. The CTD-which in isolation does not phase separate, despite being a low-complexity domain-is trapped within the cyclin T1 droplets, and this process is enhanced upon pre-phosphorylation by CDK7 of transcription initiation factor TFIIH. By using multivalent interactions to create a phase-separated functional compartment, the histidine-rich domain in kinases targets the CTD into this environment to ensure hyperphosphorylation and efficient elongation of Pol II (Lu, 2018).

Cyclin dependent kinase 9: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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