Controlling the Elongation Phase of Transcription with P-TEFb

The positive transcription elongation factor b (P-TEFb) is a cyclin-dependent kinase that controls the elongation phase of transcription by RNA polymerase II (RNAPII). This process is made possible by the reversal of effects of negative elongation factors that include NELF and DSIF. In complex organisms, elongation control is critical for the regulated expression of most genes. In those organisms, the function of P-TEFb is influenced negatively by HEXIM proteins and 7SK snRNA and positively by a variety of recruiting factors. Phylogenetic analyses of the components of the human elongation control machinery indicate that the number of mechanisms utilized to regulate P-TEFb function increased as organisms developed more complex developmental patterns. The positive transcription elongation factor b (P-TEFb) is a cyclin-dependent kinase that controls the elongation phase of transcription by RNA polymerase II (RNAPII). This process is made possible by the reversal of effects of negative elongation factors that include NELF and DSIF. In complex organisms, elongation control is critical for the regulated expression of most genes. In those organisms, the function of P-TEFb is influenced negatively by HEXIM proteins and 7SK snRNA and positively by a variety of recruiting factors. Phylogenetic analyses of the components of the human elongation control machinery indicate that the number of mechanisms utilized to regulate P-TEFb function increased as organisms developed more complex developmental patterns. Some of the most important mechanisms regulating prokaryotic and eukaryotic gene expression target the movement of RNA polymerases. For example, in bacteria, termination, antitermination, and pausing mechanisms dictate mRNA levels (Henkin and Yanofsky, 2002Henkin T.M. Yanofsky C. Regulation by transcription attenuation in bacteria: how RNA provides instructions for transcription termination/antitermination decisions.Bioessays. 2002; 24: 700-707Crossref PubMed Scopus (209) Google Scholar). In eukaryotes, early studies indicated that elongation control mechanisms were associated with expression of c-myc, the human immunodeficiency virus (HIV), and heat shock genes. Initial results indicated that a block to elongation rather than initiation was responsible for repressing the c-myc gene (Bentley and Groudine, 1986Bentley D.L. Groudine M. A block to elongation is largely responsible for decreased transcription of c-myc in differentiated HL60 cells.Nature. 1986; 321: 702-706Crossref PubMed Scopus (485) Google Scholar). Additionally, the transcriptional transactivator Tat was found to affect elongation rather than initiation during its regulation of HIV transcription (Kao et al., 1987Kao S.Y. Calman A.F. Luciw P.A. Peterlin B.M. Anti-termination of transcription within the long terminal repeat of HIV-1 by tat gene product.Nature. 1987; 330: 489-493Crossref PubMed Scopus (622) Google Scholar). In another study, it was found that RNAPII is paused close to the HSP70 promoter at normal temperatures in D. melanogaster (Rougvie and Lis, 1988Rougvie A.E. Lis J.T. The RNA polymerase II molecule at the 5′ end of the uninduced hsp70 gene of D. melanogaster is transcriptionally engaged.Cell. 1988; 54: 795-804Abstract Full Text PDF PubMed Scopus (464) Google Scholar). Events promoted by temperature shift then release RNAPII to synthesize full-length HSP70 transcripts (Rougvie and Lis, 1990Rougvie A.E. Lis J.T. Postinitiation transcriptional control in Drosophila melanogaster.Mol. Cell. Biol. 1990; 10: 6041-6045Crossref PubMed Scopus (134) Google Scholar). Over the past two decades, it has become clear that what happens on the c-myc and HSP70 genes as well as on the HIV long terminal repeat (LTR) is more the rule than the exception. Indeed, reversing an early block to RNAPII elongation is how many genes are regulated in organisms from flies to humans (Chao and Price, 2001Chao S.H. Price D.H. Flavopiridol inactivates P-TEFb and blocks most RNA polymerase II transcription in vivo.J. Biol. Chem. 2001; 276: 31793-31799Crossref PubMed Scopus (543) Google Scholar, Price, 2000Price D.H. P-TEFb, a cyclin-dependent kinase controlling elongation by RNA polymerase II.Mol. Cell. Biol. 2000; 20: 2629-2634Crossref PubMed Scopus (568) Google Scholar). A significant advance in our understanding of this process came from studies utilizing the ATP analog 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB). They demonstrated that DRB leads to a dramatic reduction in mRNA levels and the appearance of short capped transcripts (Sehgal et al., 1976Sehgal P.B. Darnell Jr., J.E. Tamm I. The inhibition by DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) of hnRNA and mRNA production in HeLa cells.Cell. 1976; 9: 473-480Abstract Full Text PDF PubMed Scopus (152) Google Scholar). Importantly, DRB inhibited transcription elongation in vitro (Chodosh et al., 1989Chodosh L.A. Fire A. Samuels M. Sharp P.A. 5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole inhibits transcription elongation by RNA polymerase II in vitro.J. Biol. Chem. 1989; 264: 2250-2257Abstract Full Text PDF PubMed Google Scholar). Subsequently, studies using a fly transcription system led to the formulation of a model for controlling the movement of RNAPII (Marshall and Price, 1992Marshall N.F. Price D.H. Control of formation of two distinct classes of RNA polymerase II elongation complexes.Mol. Cell. Biol. 1992; 12: 2078-2090Crossref PubMed Scopus (241) Google Scholar) (Figure 1). It states that after initiation, RNAPII comes under the control of negative transcription elongation factors (N-TEF) and the elongation complex is trapped near the promoter. However, P-TEFb overcomes the influences of the negative factors and RNAPII enters productive elongation. Indeed, P-TEFb rather than RNAPII is the target of DRB (Marshall and Price, 1995Marshall N.F. Price D.H. Purification of P-TEFb, a transcription factor required for the transition into productive elongation.J. Biol. Chem. 1995; 270: 12335-12338Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). Over the past decade, the identity of P-TEFb was revealed. It is a kinase that phosphorylates the C-terminal domain (CTD) of the large subunit (RPB1) of RNAPII (Marshall et al., 1996Marshall N.F. Peng J. Xie Z. Price D.H. Control of RNA polymerase II elongation potential by a novel carboxyl-terminal domain kinase.J. Biol. Chem. 1996; 271: 27176-27183Crossref PubMed Scopus (525) Google Scholar). Subsequent cloning of the two subunits of P-TEFb from flies and humans revealed it to be a cyclin-dependent kinase. Although first identified as PITALRE (Grana et al., 1994Grana X. De Luca A. Sang N. Fu Y. Claudio P.P. Rosenblatt J. Morgan D.O. Giordano A. PITALRE, a nuclear CDC2-related protein kinase that phosphorylates the retinoblastoma protein in vitro.Proc. Natl. Acad. Sci. USA. 1994; 91: 3834-3838Crossref PubMed Scopus (205) Google Scholar), because it requires a C type cyclin subunit, it was later renamed Cdk9. Flies have one cyclin T (CycT) (Peng et al., 1998aPeng J. Marshall N.F. Price D.H. Identification of a cyclin subunit required for the function of Drosophila P-TEFb.J. Biol. Chem. 1998; 273: 13855-13860Crossref PubMed Scopus (150) Google Scholar), but humans express cyclins T1 and T2 (CycT1 and CycT2) (Peng et al., 1998bPeng J. Zhu Y. Milton J.T. Price D.H. Identification of multiple cyclin subunits of human P-TEFb.Genes Dev. 1998; 12: 755-762Crossref PubMed Scopus (450) Google Scholar). The human Cdk9 protein also binds cyclin K (CycK) (Fu et al., 1999Fu T.J. Peng J. Lee G. Price D.H. Flores O. Cyclin K functions as a CDK9 regulatory subunit and participates in RNA polymerase II transcription.J. Biol. Chem. 1999; 274: 34527-34530Crossref PubMed Scopus (176) Google Scholar). Two negative factors with properties consistent with N-TEF were also identified. First, the DRB-sensitivity inducing factor (DSIF) is required for the effects of DRB and P-TEFb on transcription in vitro (Wada et al., 1998Wada T. Takagi T. Yamaguchi Y. Watanabe D. Handa H. Evidence that P-TEFb alleviates the negative effect of DSIF on RNA polymerase II-dependent transcription in vitro.EMBO J. 1998; 17: 7395-7403Crossref PubMed Scopus (280) Google Scholar), in part via its interactions with RNAPII (Yamaguchi et al., 1999bYamaguchi Y. Wada T. Watanabe D. Takagi T. Hasegawa J. Handa H. Structure and function of the human transcription elongation factor DSIF.J. Biol. Chem. 1999; 274: 8085-8092Crossref PubMed Scopus (117) Google Scholar). It contains two subunits, which are similar to yeast transcription factors Spt4 and Spt5 (Winston, 2001Winston F. Control of eukaryotic transcription elongation.Genome Biol. 2001; 2 (REVIEWS 1006)Crossref PubMed Google Scholar). Subsequently, the negative elongation factor (NELF) was found to be necessary for the negative function of DSIF (Yamaguchi et al., 1999aYamaguchi Y. Takagi T. Wada T. Yano K. Furuya A. Sugimoto S. Hasegawa J. Handa H. NELF, a multisubunit complex containing RD, cooperates with DSIF to repress RNA polymerase II elongation.Cell. 1999; 97: 41-51Abstract Full Text Full Text PDF PubMed Scopus (616) Google Scholar). Following their characterization, recombinant DSIF and affinity-purified NELF were used in a defined in vitro transcription system along with recombinant P-TEFb to recapitulate essential features of elongation control (Renner et al., 2001Renner D.B. Yamaguchi Y. Wada T. Handa H. Price D.H. A highly purified RNA polymerase II elongation control system.J. Biol. Chem. 2001; 276: 42601-42609Crossref PubMed Scopus (144) Google Scholar). Although neither factor had any effect alone, the combination of DSIF and NELF slowed the elongation of RNAPII and this effect was eliminated by P-TEFb. In a similar in vitro system, NELF and DSIF also inhibited the transcript cleavage factor TFIIS (Palangat et al., 2005Palangat M. Renner D.B. Price D.H. Landick R. A negative elongation factor for human RNA polymerase II inhibits the anti-arrest transcript-cleavage factor TFIIS.Proc. Natl. Acad. Sci. USA. 2005; 102: 15036-15041Crossref PubMed Scopus (49) Google Scholar). Because the function of TFIIS is to relieve strong pauses and arrests, this inhibitory property could stabilize the paused RNAPII conformation induced by NELF and DSIF. The function of NELF and DSIF was confirmed by studies that demonstrated that these two factors induce promoter proximal pausing on the HSP70 gene from D. melanogaster in vitro (Wu et al., 2003Wu C.H. Yamaguchi Y. Benjamin L.R. Horvat-Gordon M. Washinsky J. Enerly E. Larsson J. Lambertsson A. Handa H. Gilmour D. NELF and DSIF cause promoter proximal pausing on the hsp70 promoter in Drosophila.Genes Dev. 2003; 17: 1402-1414Crossref PubMed Scopus (220) Google Scholar). What are the targets of P-TEFb? Many studies indicate that P-TEFb phosphorylates the C-terminal domain (CTD) of RNAPII primarily on serines at position 2 (serine 2) of its heptapeptide (YSPTSPS) repeats (Garriga and Grana, 2004Garriga J. Grana X. Cellular control of gene expression by T-type cyclin/CDK9 complexes.Gene. 2004; 337: 15-23Crossref PubMed Scopus (142) Google Scholar, Price, 2000Price D.H. P-TEFb, a cyclin-dependent kinase controlling elongation by RNA polymerase II.Mol. Cell. Biol. 2000; 20: 2629-2634Crossref PubMed Scopus (568) Google Scholar). The phosphorylation of the CTD, which in humans contains 52 such repeats, is a complicated process. At least two different serine/threonine kinases are required (Figure 1). First, Cdk7 from TFIIH, which is a general transcription factor, phosphorylates serines at position 5 (serine 5). Next, Cdk9 phosphorylates serine 2 (Figure 1). This posttranslational modification increases the diameter and rigidity of the CTD. In the process, most proteins in the preinitation complex, the Mediator, and general transcription factors, which comprise 100 or more proteins, are removed from RNAPII. The phosphorylated CTD now increases the affinity of human capping enzymes (HCEs) for the elongation complex and acts as a scaffold for splicing (SR) and polyadenylation (pA) machineries (Figure 1). In addition, chromatin-modifying enzymes and other elongation factors also travel with the phosphorylated RNAPII. As presented in Figure 1, these steps are sequential and have been subdivided into the formation of the preinitiation complex (PIC), promoter clearance, 5′ capping, and pausing followed by productive elongation (Sims et al., 2004Sims 3rd, R.J. Belotserkovskaya R. Reinberg D. Elongation by RNA polymerase II: the short and long of it.Genes Dev. 2004; 18: 2437-2468Crossref PubMed Scopus (568) Google Scholar). The last step requires P-TEFb. Indeed, when Cdk9 or its two cyclin T subunits are genetically inactivated in C. elegans, serine 2 phosphorylation is lost (Shim et al., 2002Shim E.Y. Walker A.K. Shi Y. Blackwell T.K. CDK-9/cyclin T (P-TEFb) is required in two postinitiation pathways for transcription in the C. elegans embryo.Genes Dev. 2002; 16: 2135-2146Crossref PubMed Scopus (164) Google Scholar). The development of these worms is arrested at the 100 cell stage, which is identical to that observed when a subunit of RNAPII is knocked down. P-TEFb also phosphorylates the human Spt5 protein (Kim and Sharp, 2001Kim J.B. Sharp P.A. Positive transcription elongation factor B phosphorylates hSPT5 and RNA polymerase II carboxyl-terminal domain independently of cyclin-dependent kinase-activating kinase.J. Biol. Chem. 2001; 276: 12317-12323Crossref PubMed Scopus (152) Google Scholar). Thus modified, DSIF functions as a positive elongation factor (Yamada et al., 2006Yamada T. Yamaguchi Y. Inukai N. Okamoto S. Mura T. Handa H. P-TEFb-mediated phosphorylation of hSpt5 C-terminal repeats is critical for processive transcription elongation.Mol. Cell. 2006; 21: 227-237Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). Finally, P-TEFb phosphorylates NELFe (RD). This phosphorylation releases NELF from the double-stranded RNA and leads to some read through transcription (Fujinaga et al., 2004Fujinaga K. Irwin D. Huang Y. Taube R. Kurosu T. Peterlin B.M. Dynamics of human immunodeficiency virus transcription: P-TEFb phosphorylates RD and dissociates negative effectors from the transactivation response element.Mol. Cell. Biol. 2004; 24: 787-795Crossref PubMed Scopus (256) Google Scholar). For its effects on RNAPII, P-TEFb must be recruited to transcription units. Some specific activators are known to recruit P-TEFb as well as at least one general chromatin remodeling protein, Brd4 (Jang et al., 2005Jang M.K. Mochizuki K. Zhou M. Jeong H.S. Brady J.N. Ozato K. The bromodomain protein Brd4 is a positive regulatory component of P-TEFb and stimulates RNA polymerase II-dependent transcription.Mol. Cell. 2005; 19: 523-534Abstract Full Text Full Text PDF PubMed Scopus (917) Google Scholar, Yang et al., 2005Yang Z. Yik J.H. Chen R. He N. Jang M.K. Ozato K. Zhou Q. Recruitment of P-TEFb for stimulation of transcriptional elongation by the bromodomain protein Brd4.Mol. Cell. 2005; 19: 535-545Abstract Full Text Full Text PDF PubMed Scopus (815) Google Scholar) (Figure 2). However, certain specific or general repressors, such as PIE-1 from C. elegans, can block these effects (Figure 3), keeping RNAPII arrested despite the presence of P-TEFb (Zhang et al., 2003Zhang F. Barboric M. Blackwell T.K. Peterlin B.M. A model of repression: CTD analogs and PIE-1 inhibit transcriptional elongation by P-TEFb.Genes Dev. 2003; 17: 748-758Crossref PubMed Scopus (91) Google Scholar). The first example of interactions between P-TEFb and transcription units was found to be via RNA (Selby et al., 1989Selby M.J. Bain E.S. Luciw P.A. Peterlin B.M. Structure, sequence, and position of the stem-loop in tar determine transcriptional elongation by tat through the HIV-1 long terminal repeat.Genes Dev. 1989; 3: 547-558Crossref PubMed Scopus (245) Google Scholar), but later P-TEFb was found to function also via DNA (Majello et al., 1999Majello B. Napolitano G. Giordano A. Lania L. Transcriptional regulation by targeted recruitment of cyclin-dependent CDK9 kinase in vivo.Oncogene. 1999; 18: 4598-4605Crossref PubMed Scopus (59) Google Scholar, Taube et al., 2002Taube R. Lin X. Irwin D. Fujinaga K. Peterlin B.M. Interaction between P-TEFb and the C-terminal domain of RNA polymerase II activates transcriptional elongation from sites upstream or downstream of target genes.Mol. Cell. Biol. 2002; 22: 321-331Crossref PubMed Scopus (95) Google Scholar).Figure 3Active Repression of P-TEFbShow full captionThe left panel of the diagram depicts the typical activation of transcriptional elongation by P-TEFb, where CycT1 mediates interactions with the CTD of RNAPII. The panel on the right demonstrates how a repressor can block this activation by binding CycT1 and preventing the subsequent phosphorylation of RNAPII, DSIF, and NELF.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The left panel of the diagram depicts the typical activation of transcriptional elongation by P-TEFb, where CycT1 mediates interactions with the CTD of RNAPII. The panel on the right demonstrates how a repressor can block this activation by binding CycT1 and preventing the subsequent phosphorylation of RNAPII, DSIF, and NELF. The clearest example of recruitment of P-TEFb is at the HIV LTR, where Tat binds TAR with the help of CycT1 (Wei et al., 1998Wei P. Garber M.E. Fang S.M. Fischer W.H. Jones K.A. A novel CDK9-associated C-type cyclin interacts directly with HIV-1 Tat and mediates its high-affinity, loop-specific binding to TAR RNA.Cell. 1998; 92: 451-462Abstract Full Text Full Text PDF PubMed Scopus (1048) Google Scholar, Zhu et al., 1997Zhu Y. Pe'ery T. Peng J. Ramanathan Y. Marshall N. Marshall T. Amendt B. Mathews M.B. Price D.H. Transcription elongation factor P-TEFb is required for HIV-1 tat transactivation in vitro.Genes Dev. 1997; 11: 2622-2632Crossref PubMed Scopus (611) Google Scholar) (Figure 2). In the absence of Tat, HIV transcription initiates efficiently and RNAPII clears the promoter, synthesizing short nonpolyadenylated transcripts (Kao et al., 1987Kao S.Y. Calman A.F. Luciw P.A. Peterlin B.M. Anti-termination of transcription within the long terminal repeat of HIV-1 by tat gene product.Nature. 1987; 330: 489-493Crossref PubMed Scopus (622) Google Scholar). However, it does not elongate past the HIV LTR, in part because TAR is also very efficient at recruiting N-TEF (Fujinaga et al., 2004Fujinaga K. Irwin D. Huang Y. Taube R. Kurosu T. Peterlin B.M. Dynamics of human immunodeficiency virus transcription: P-TEFb phosphorylates RD and dissociates negative effectors from the transactivation response element.Mol. Cell. Biol. 2004; 24: 787-795Crossref PubMed Scopus (256) Google Scholar). Whereas Tat binds the 5′ bulge in TAR via its arginine-rich motif, CycT1 binds the central loop via its basic Tat•TAR recognition motif, which is also its nuclear localization signal (Garber et al., 1998Garber M.E. Wei P. KewalRamani V.N. Mayall T.P. Herrmann C.H. Rice A.P. Littman D.R. Jones K.A. The interaction between HIV-1 Tat and human cyclin T1 requires zinc and a critical cysteine residue that is not conserved in the murine CycT1 protein.Genes Dev. 1998; 12: 3512-3527Crossref PubMed Scopus (383) Google Scholar). Upon this recruitment, P-TEFb phosphorylates Spt5 (Ivanov et al., 2000Ivanov D. Kwak Y.T. Guo J. Gaynor R.B. Domains in the SPT5 protein that modulate its transcriptional regulatory properties.Mol. Cell. Biol. 2000; 20: 2970-2983Crossref PubMed Scopus (177) Google Scholar), RD (Fujinaga et al., 2004Fujinaga K. Irwin D. Huang Y. Taube R. Kurosu T. Peterlin B.M. Dynamics of human immunodeficiency virus transcription: P-TEFb phosphorylates RD and dissociates negative effectors from the transactivation response element.Mol. Cell. Biol. 2004; 24: 787-795Crossref PubMed Scopus (256) Google Scholar), and serine 2 in the CTD of RNAPII (Isel and Karn, 1999Isel C. Karn J. Direct evidence that HIV-1 Tat stimulates RNA polymerase II carboxyl-terminal domain hyperphosphorylation during transcriptional elongation.J. Mol. Biol. 1999; 290: 929-941Crossref PubMed Scopus (100) Google Scholar). RD dissociates from TAR (Fujinaga et al., 2004Fujinaga K. Irwin D. Huang Y. Taube R. Kurosu T. Peterlin B.M. Dynamics of human immunodeficiency virus transcription: P-TEFb phosphorylates RD and dissociates negative effectors from the transactivation response element.Mol. Cell. Biol. 2004; 24: 787-795Crossref PubMed Scopus (256) Google Scholar), then P-TEFb and phosphorylated DSIF travel with the elongating RNAPII (Ping and Rana, 2001Ping Y.H. Rana T.M. DSIF and NELF interact with RNA polymerase II elongation complex and HIV-1 Tat stimulates P-TEFb-mediated phosphorylation of RNA polymerase II and DSIF during transcription elongation.J. Biol. Chem. 2001; 276: 12951-12958Crossref PubMed Scopus (158) Google Scholar). Because Tat requires the presence of a promoter and functions synergistically with DNA bound activators, TAR has also been called an RNA enhancer (Sharp and Marciniak, 1989Sharp P.A. Marciniak R.A. HIV TAR: an RNA enhancer?.Cell. 1989; 59: 229-230Abstract Full Text PDF PubMed Scopus (109) Google Scholar). Such recruitment of transcription factors via RNA had not been observed previously in eukaryotic systems. Thus, several strategies were developed to examine whether other activators can also function via RNA. They were fused to the coat protein of the bacteriophage MS2/R17, which binds its operator RNA to block translation in bacteria (Selby and Peterlin, 1990Selby M.J. Peterlin B.M. Trans-activation by HIV-1 Tat via a heterologous RNA binding protein.Cell. 1990; 62: 769-776Abstract Full Text PDF PubMed Scopus (158) Google Scholar) or to the regulator of viral gene expression (Rev), which binds the Rev response element (RRE) RNA in the env gene of HIV (Tiley et al., 1992Tiley L.S. Madore S.J. Malim M.H. Cullen B.R. The VP16 transcription activation domain is functional when targeted to a promoter-proximal RNA sequence.Genes Dev. 1992; 6: 2077-2087Crossref PubMed Scopus (93) Google Scholar). Because Rev forms oligomers on its preferred binding site, the stem loop II B (SLIIB), the latter RNA tethering is more efficient. Importantly, studies indicate that only proteins that interact with P-TEFb activate transcription in this assay. Thus, RNA-tethering represents a genetic assay for activators that recruit P-TEFb for their effects (Barboric and Peterlin, 2005Barboric M. Peterlin B.M. A new paradigm in eukaryotic biology: HIV Tat and the control of transcriptional elongation.PLoS Biol. 2005; 3: e76https://doi.org/10.1371/journal.pbio.0030076Crossref PubMed Scopus (82) Google Scholar). However, for this regulation of HIV transcription via RNA, Tat has to be synthesized first. Thus, HIV must use an alternative strategy to modify RNAPII for initial rounds of elongation. To this end, it was demonstrated first that heterologous DNA tethering of Tat could also lead to efficient elongation of transcription (Southgate and Green, 1991Southgate C.D. Green M.R. The HIV-1 Tat protein activates transcription from an upstream DNA-binding site: implications for Tat function.Genes Dev. 1991; 5: 2496-2507Crossref PubMed Scopus (182) Google Scholar). Once P-TEFb was found to be the coactivator of Tat (Zhu et al., 1997Zhu Y. Pe'ery T. Peng J. Ramanathan Y. Marshall N. Marshall T. Amendt B. Mathews M.B. Price D.H. Transcription elongation factor P-TEFb is required for HIV-1 tat transactivation in vitro.Genes Dev. 1997; 11: 2622-2632Crossref PubMed Scopus (611) Google Scholar), heterologous tethering of CycT1, CycT2, and Cdk9 gave identical results (Majello et al., 1999Majello B. Napolitano G. Giordano A. Lania L. Transcriptional regulation by targeted recruitment of cyclin-dependent CDK9 kinase in vivo.Oncogene. 1999; 18: 4598-4605Crossref PubMed Scopus (59) Google Scholar) (Figure 2). Of interest, the smaller CycK protein could not affect transcription via DNA (Lin et al., 2002Lin X. Taube R. Fujinaga K. Peterlin B.M. P-TEFb containing cyclin K and Cdk9 can activate transcription via RNA.J. Biol. Chem. 2002; 277: 16873-16878Crossref PubMed Scopus (52) Google Scholar). Further mapping revealed that a histidine-rich stretch in C-terminal halves of CycT1 and CycT2 as well as a leucine-rich sequence next to the cyclin boxes in CycT2 were required for this transcriptional activation (Kurosu et al., 2004Kurosu T. Zhang F. Peterlin B.M. Transcriptional activity and substrate recognition of cyclin T2 from P-TEFb.Gene. 2004; 343: 173-179Crossref PubMed Scopus (13) Google Scholar, Taube et al., 2002Taube R. Lin X. Irwin D. Fujinaga K. Peterlin B.M. Interaction between P-TEFb and the C-terminal domain of RNA polymerase II activates transcriptional elongation from sites upstream or downstream of target genes.Mol. Cell. Biol. 2002; 22: 321-331Crossref PubMed Scopus (95) Google Scholar). These motifs interact with the CTD of RNAPII. They can thus be viewed as the substrate recognition motifs of P-TEFb. Importantly, when the histidine-rich stretch from CycT1 was fused with CycK, thus chimera activated transcription via DNA (Lin et al., 2002Lin X. Taube R. Fujinaga K. Peterlin B.M. P-TEFb containing cyclin K and Cdk9 can activate transcription via RNA.J. Biol. Chem. 2002; 277: 16873-16878Crossref PubMed Scopus (52) Google Scholar). These studies suggested that DNA elements to which P-TEFb was recruited could satisfy the classical criteria for enhancers, as elements that can function to promote transcription from reiterated motifs upstream or downstream from target genes (Muller et al., 1988Muller M.M. Gerster T. Schaffner W. Enhancer sequences and the regulation of gene transcription.Eur. J. Biochem. 1988; 176: 485-495Crossref PubMed Scopus (100) Google Scholar). Of interest, the locus control regions of globin genes were later found also to affect the process of elongation rather than initiation of transcription (Sawado et al., 2003Sawado T. Halow J. Bender M.A. Groudine M. The beta -globin locus control region (LCR) functions primarily by enhancing the transition from transcription initiation to elongation.Genes Dev. 2003; 17: 1009-1018Crossref PubMed Scopus (149) Google Scholar), and P-TEFb is recruited to heat shock-regulated genes on the polytene chromosomes of D. melanogaster with temperature shift (Lis et al., 2000Lis J.T. Mason P. Peng J. Price D.H. Werner J. P-TEFb kinase recruitment and function at heat shock loci.Genes Dev. 2000; 14: 792-803PubMed Google Scholar). Subsequent studies focused on specific DNA bound activators that mediate the enhancer function of their DNA elements (Figure 2). Because NF-κB contributes to the activation of the HIV LTR and synergizes with Tat (Nabel and Baltimore, 1987Nabel G. Baltimore D. An inducible transcription factor activates expression of human immunodeficiency virus in T cells.Nature. 1987; 326: 711-713Crossref PubMed Scopus (1451) Google Scholar), its p65 (RelA) subunit was examined first. Indeed, RelA could function when tethered to RNA as a fusion protein with Rev (Barboric et al., 2001Barboric M. Nissen R.M. Kanazawa S. Jabrane-Ferrat N. Peterlin B.M. NF-kappaB binds P-TEFb to stimulate transcriptional elongation by RNA polymerase II.Mol. Cell. 2001; 8: 327-337Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar). Moreover, a dominant-negative Cdk9 protein and/or inhibitors of Cdk9 blocked its activity. Additionally, chromatin immunoprecipitation assays revealed that NF-κB recruits P-TEFb and increases transcriptional elongation of a target gene (Barboric et al., 2001Barboric M. Nissen R.M. Kanazawa S. Jabrane-Ferrat N. Peterlin B.M. NF-kappaB binds P-TEFb to stimulate transcriptional elongation by RNA polymerase II.Mol. Cell. 2001; 8: 327-337Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar). This finding also addressed the conundrum of how Tat is made in the first place. In this simplified scheme, HIV promoter elements assemble the PIC and RNAPII clears the promoter and transcribes TAR, where it is paused. Upon its binding the HIV LTR, NF-κB recruits P-TEFb, which allows for the first rounds of viral transcription and the synthesis of Tat. After sufficient amounts of Tat are produced, the recruitment of P-TEFb via Tat and TAR takes over and results in abundant replication of HIV and the eventual demise of the infected cell. NF-κB is not the only activator that interacts with P-TEFb to mediate the effect of an enhancer. Indeed, other activators, such as, c-Myc (Eberhardy and Farnham, 2002Eberhardy S.R. Farnham P.J. Myc recruits P-TEFb to mediate the final step in the transcriptional activation of the cad promoter.J. Biol. Chem. 2002; 277: 40156-40162Crossref PubMed Scopus (187) Google Scholar, Kanazawa et al., 2003Kanazawa S. Soucek L. Evan G. Okamoto T. Peterlin B.M. c-Myc recruits P-TEFb for transcription, cellular proliferation and apoptosis.Oncogene. 2003; 22: 5707-5711Crossref PubMed Scopus (138) Google Scholar), the class II transactivator (CIITA) (Kanazawa et al., 2000Kanazawa S. Okamoto T. Peterlin B.M. Tat competes with CIITA for the binding to P-TEFb and blocks the expression of MHC class II genes in HIV infection.Immunity. 2000; 12: 61-70Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar), MyoD (Simone et al., 2002Simone C. Stiegler P. Bagella L. Pucci B. Bellan C. De Falco G. De Luca A. Guanti G. Puri P.L. Giordano A. Activation of MyoD-dependent transcription by cdk9/cyclin T2.Oncogene. 2002; 21: 4137-4148Crossref PubMed Scopus (99) Google Scholar), steroid hormone receptors (Lee et al., 2001Lee D.K. Duan H.O. Chang C. Androgen receptor interacts with the positive elongation factor P-TEFb and enhances the efficiency of transcr