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Previous Article | Next Article 
Molecular and Cellular Biology, January 2002, p. 321-331, Vol. 22, No. 1
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.22.1.321-331.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Howard Hughes Medical Institute, Departments of Medicine, Microbiology and Immunology, University of California at San Francisco, San Francisco, California 94143-0703
Received 18 June 2001/ Returned for modification 23 August 2001/ Accepted 1 October 2001
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ABSTRACT |
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INTRODUCTION |
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Transcriptional enhancer sequences (enhancers) are defined as cis-acting DNA elements that increase levels of gene expression (4, 6, 36). Their effects are dependent on PPR and CP but independent of location, orientation, and distance with respect to the site of initiation of transcription (4, 6, 36). They can function independently of chromatin, i.e., on episomal plasmids and in in vitro transcription systems (49). Moreover, their effects are dependent on the CTD of RNAPII, which in primates contains 52 repeats of the heptapeptide YSPTSPS. Whereas sequences with 52 and 31 repeats support effects of different enhancers, those with 5 repeats have been found to be inactive in these assays (14). Thus, the CTD functions as a surface for the integration of different signals from activators and repressors on transcription. When unphosphorylated, the CTD is covered by mediators and recruits coactivators (50, 51). In sharp contrast, the phosphorylated CTD plays a central role in the cotranscriptional processing of mRNA by binding to the elongator complex (2). To date, an accepted model of how enhancers function is not available. The looping model suggests that proteins bound to enhancers interact directly with the PIC on promoters. Alternatively, the scanning model speculates that enhancer-binding proteins travel along the DNA until they meet their cognate promoter (4).
A well-defined system that incorporates important features of eukaryotic promoters and enhancers and reveals the central role that P-TEFb plays in the elongation of transcription by RNAPII is represented by the human immunodeficiency virus (HIV). On the HIV long terminal repeat (LTR) are found binding sites for TFE3, LEF-1, NF-AT, NF-
B, Sp1, TFIID, TFIII, and LBP-1 (24). Thus, there exists a classical arrangement of the enhancer, PPR, and CP (24). In the absence of enhancer binding proteins and Tat, the transcription of HIV is arrested at the transactivation response (TAR) element stem loop RNA, which is present at the 5' ends of all viral transcripts (24). With the help of P-TEFb, Tat binds to TAR and promotes the elongation rather than the initiation of HIV transcription (3, 9, 13, 22, 24, 29, 59). NF-B and other activators that bind to the HIV enhancer can substitute for the function of Tat (18) and/or act synergistically with it to increase levels of viral replication (40, 52). NF-B also increases the expression of the immunoglobulin light chain kappa (Ig) gene (43). As in HIV, RNAPII is engaged but does not elongate past this cellular promoter. The binding of NF-B to Ig enhancer then releases RNAPII from its pausing site (46). A similar regulation of transcription is found in the heat-shock genes from Drosophila melanogaster and the human proto-oncogene c-myc (32, 55). Tat transactivation also resembles enhancers in its requirement for the CTD of RNAPII for its effects (38). Thus, it is not surprising that the major histocompatibility complex class II (MHC II) transactivator (CIITA), which mediates effects of B-cell-specific and gamma interferon-inducible enhancers (54) and NF-B (1), also binds P-TEFb.
Thus, we hypothesized that P-TEFb plays a general role in mediating effects of enhancers. Indeed, in this report, we demonstrate that P-TEFb activates transcription from proximal and distal sites upstream and downstream of the promoter and coding sequence of a reporter gene. PPR, CP, and the CTD of RNAPII were required for these effects of P-TEFb. A direct correlation between the binding of a histidine-rich stretch in the C-terminal region of CycT1 and the CTD could be made with this transcriptional activation, which also required the kinase activity of Cdk9. Importantly, adding these sequences of CycT1 to CycK, which lacks a long C-terminal extension, allowed this P-TEFb complex to activate transcription from the same distal sites. These effects were on the elongation rather than initiation of transcription, indicating that PPR and CP were sufficient to recruit and position RNAPII. We conclude that the interaction between C-terminal regions of RNAPII, CycT1, and distal sites enables P-TEFb to act as a transcriptional coactivator. Thus, P-TEFb can link activators at enhancers and PIC at promoters, which results in the productive elongation of transcription.
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MATERIALS AND METHODS |
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The different plasmid effectors of Gal-CycT1 code for the chimeras of the full-length CycT1 [Gal-CycT(1–726)] and truncated forms Gal-CycT(1–590), Gal-CycT(1–420), and Gal-CycT(1–300) (see Fig. 1A), all fused at their N termini to the DBD of Gal4(1–147) and inserted at the BamHI/XbaI sites of pEF-Bos-myc(EF). pSG424-Gal-CycK(1–357) and pSG424-Gal-CycK(1–357)T(300–726) code for the chimeras of residues 1 to 357 CycK and of the same portion of CycK fused to CycT1 residues 300 to 726, respectively, linked to the Gal4 DBD protein at their N termini, and inserted at the EcoRI/XbaI sites of pEF-Bos-myc(EF). The plasmid effectors Gal-Cdk9 and Gal-Cdk9(D167N) expressed the chimeras of Cdk9 and its kinase-deficient mutant Cdk9(D167N) fused at their N termini to the DBD of Gal4 and inserted at the BamHI/XbaI sites of pEF-Bos-myc(EF). pSG424-Gal-VP16 codes for the activation domain of VP16 fused to Gal4 DBD at its N terminus. For in vitro transcription and translation, the different CycT1 truncation forms were subcloned into pEF-Bos-T7 in the BamHI/EcoRI sites. For in vitro glutathione S-transferase (GST) pull-down assays, CycT1 and its C-terminal truncation forms were cloned into pGEX-2TK (Amersham Pharmacia Biotech, Piscataway, N. J., Madison, Wis.) in the BamHI/EcoRI sites to express GST fusion proteins of CycT1 as described elsewhere (10).
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For interaction between CycT1 and the endogenous RNAPII, HeLa nuclear extracts were incubated with the different GST-CycT1 fusion proteins at 4°C for 3 h in ELB binding buffer (50 mM HEPES-KOH [pH 7.9], 0.1% Triton X-100, 5 mM dithiothreitol, 5 mM EDTA, 150 mM NaCl). Following incubation, reaction products were washed extensively with ELB binding buffer containing 500 mM NaCl. Beads were subjected to 5% SDS-PAGE, followed by Western blotting with anti-RNAPII antibody (8WG16; BAbCO, Richmond, Calif.).
For interaction between CycT1 and the CTD of RNAPII, Raji cells (35) (kind gift of D. Eick) expressing hemagglutnin (HA) epitope-tagged RNAPII, where the CTD contains 52 or 5 repeats of the heptapeptide sequence, were transfected with 30 µg of effector plasmids. Lysates were prepared from these cells with ELB buffer and were immunoprecipitated with anti-Myc antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.). Proteins were subjected to SDS-PAGE, transferred to a nitrocellulose membrane, and probed with anti-HA antibody (Santa Cruz Biotechnology).
Transient-transfection assays. HeLa cells were transfected with target plasmids (0.2 µg) with or without the different effector plasmids (1 µg) with Lipofectamine as recommended by the manufacturer (GIBCO/BRL, Gaithersburg, Md.). All transfections were balanced to total of 1.2 µg of DNA with pEF-Bos-myc plasmid DNA. At 48 h after transfection, cells were lysed in lysis buffer (250 mM Tris-HCl [pH 7.5] and 0.1% Triton X-100), and CAT activities were measured by a liquid scintillation assay as described previously (9). Western blot analysis was performed to evaluate expression levels of the proteins (see Fig. 1D). For the inhibition of kinase activity of Cdk9, cells were treated with DRB (Sigma, St. Louis, Mo.) (diluted in dimethyl sulfoxide) at various concentrations, 24 h before and 48 h after transfection. CAT activities were measured from cell lysates as described previously (9).
Raji cells expressing the different RNAPII CTD were grown in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 100 µg of streptomycin per ml, 2 mM L-glutamine, 1 mg of G418 per ml, and 0.1 µg of tetracycline per ml. Before transfection, cells were washed three times with medium containing only 1% FCS and were grown for an additional 1 day with medium containing 10% FCS but no tetracycline. This enabled the expression of the different RNAPII-CTD forms. The next day, the different CycT1 plasmid effectors were transfected by electroporation (250V; 950 µF), and cells were grown for an additional 2 days. Cells were lysed with ELB buffer, and the lysates were subjected to immunoprecipitation with the anti-Myc antibody followed by Western blotting with anti-HA antibody (35).
Immunoprecipitation and Western blotting. For determining the expression levels of proteins, 293T cells were transfected with various plasmids (2 µg). At 24 h after transfection, cells were lysed with ELB buffer. Proteins were subjected to SDS-PAGE, transferred onto Immobilon-NC membranes (Millipore, Bedford, Mass.), and reacted with the appropriate antibodies. Proteins were visualized by enhanced chemiluminescence (Amersham Inc., Evanston, Ill.).
For immunoprecipitation experiments, cells were lysed with ELB buffer and immunoprecipitated with the appropriate antibody. Following binding to the antibody, reaction mixtures were incubated with protein A-Sepharose beads at 4°C. Immunoprecipitate reaction products were washed extensively with ELB buffer and subjected to SDS-PAGE followed by Western blotting.
RPA.
COS cells were transfected with pG6TAR(5'Pro) (10 µg) and with Gal-CycT1 fusion proteins and C-terminal-truncation forms (10 µg). At 24 h after transfection, cells were harvested and RNA was purified. A 30-µg portion of RNA was used for RNase protection assays (RPA). Antisense probes MTX-147 and MTX-89 (kind gift of J. Karn) were linearized with XbaI and BamHI, respectively. Probes were transcribed in the presence of [
-32P]UTP by using MAXIscript (Ambion, Austin, Tex.) with T3 RNA polymerase to produce labeled RNA probe. Following synthesis, probes were subjected to polyacrylamide-urea gel electrophoresis and purified before use. RPA were performed using the RPAIII kit as recommended by the manufacturer (Ambion). Protected RNA fragments were separated on 11% polyacrylamide–urea sequencing gels. Gels were dried and subjected to autoradiography (38, 60).
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B binding sites, which represent the enhancer in the HIV LTR. Gal-CycT1 chimeras containing the wild-type 726 (Fig. 1B, lane 3) or N-terminal 590 (lane 4) residues activated transcription from pG6(5'Pro) 35- to 40-fold above basal levels. Further C-terminal truncations of CycT1 reduced the ability of the hybrid Gal-CycT1 proteins to activate transcription from pG6(5'Pro). Thus, Gal-CycT1 fusion proteins containing the N-terminal 420 (Fig. 1B, lane 5) or 300 (lane 6) residues displayed little or no activity on pG6(5'pro). Importantly, levels of expression of our mutant chimeras were even higher than that of the wild-type fusion protein (Fig. 1D, compare lanes 4, 5, and 6 to lane 3). We conclude that the C-terminal region of CycT1 is essential for the ability of CycT1 to activate transcription via DNA. Furthermore, the hybrid Gal-Cdk9 protein (Fig. 1B, lane 7) but not a kinase-deficient hybrid Gal-Cdk9 protein (lane 8) activated transcription 50-fold from the same reporter gene. Levels of activation by the Gal-Cdk9 chimera were similar to levels of activation by the Gal-VP16 fusion protein (data not presented). These results demonstrate that the direct recruitment of an active Cdk9 to a site upstream of a promoter activates transcription. We conclude that long CycT1 as well as Cdk9 can function when presented via DNA, indicating that P-TEFb mediates transcriptional effects via the C-terminal region of CycT1. Given these results and since there is no directionality to UAS repeats, we hypothesized that P-TEFb should also function when placed at some distance downstream of a target gene. To examine this notion, six UAS repeats were positioned 3' of the CAT gene, distant from the promoter and coding sequence [Fig. 1A, pG6(3'Pro)]. In this system, only the Gal-CycT(1–726) (Fig. 1C, lane 3) and Gal-CycT(1–590) (lane 4) chimeras activated transcription via pG6(3'Pro). Levels of activation were 25- to 30-fold above basal levels. C-terminal deletions of CycT1, i.e., the hybrids Gal-CycT(1–420) (Fig. 1C, lane 5) and Gal-CycT(1–300) (lane 6), had lower or no activity on this plasmid target. Moreover, the Gal-Cdk9 chimera (Fig. 1C, lane 7) but not the kinase-deficient Gal-Cdk9 protein (lane 8) also supported transcriptional activation from this distal site. Similar to the results with pG6(5'Pro), the C-terminal region of CycT1 was required for effects of our chimeras on pG6(3'Pro). We conclude that P-TEFb can function via sites which are located upstream or downstream of and at a distance from the promoter and coding sequence.
Sp1 is required for the activation of transcription by P-TEFb when presented via DNA.
As P-TEFb could replace NF-B when UAS were placed just upstream of the three Sp1 sites on the HIV LTR, we examined whether PPR is required for P-TEFb to mediate its effects. It should be noted that Sp1 sites are required for NF-B to activate HIV transcription (24) and for the function of the 72-bp repeats of simian virus 40 (20). To this end, five UAS repeats were placed next to the CP (E1b TATA box) and upstream of the CAT reporter gene (Fig. 2A, pG5). Importantly, no Sp1 sites were present in pG5. None of our Gal-CycT1 fusion proteins activated transcription from pG5 (Fig. 2B, lanes 1 to 6). However, the Gal-VP16 chimera activated transcription via the same plasmid target (Fig. 2B, lane 7). Moreover, deletions of Sp1 sites in pG6(5'Pro) gave the same result, with three sites functioning optimally and their progressive removal resulting in no activity (data not presented). This suggests that Sp1 and CP recruit the PIC to initiate transcription. We conclude that PPR and CP are essential for the ability of P-TEFb to activate transcription by RNAPII via DNA.
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To evaluate this hypothesis, we examined whether P-TEFb interacts with RNAPII. Indeed, both CycT1 and Cdk9 coimmunoprecipitated this polymerase in HeLa cells (Fig. 3A, lanes 2 and 3). Moreover, we mapped the region of CycT1 that binds RNAPII (Fig. 3B). In vitro binding reactions using nuclear extracts and GST-CycT1 fusion proteins demonstrated that neither GST alone (Fig. 3B, lane 1) nor the fusion protein containing the N-terminal 300 residues (lane 8) interacted with RNAPII. The same was true for GST-CycT1 chimeras containing the first 480 residues (Fig. 3B, lane 7) and residues from positions 611 to 726 (lane 9). In contrast, full-length CycT1 (Fig. 3B, lane 2) and GST-CycT1 fusion proteins containing the 551 N-terminal residues (lane 3) or only sequences from positions 231 to 611 (lane 4) and 350 to 611 (lane 5) bound RNAPII. Moreover, a region of the hybrid GST-CycT1 protein from positions 481 to 551 (Fig. 3B, lane 6) still interacted with RNAPII. This region contains a histidine-rich stretch that could mediate the binding between CycT1 and RNAPII. Other GST-CycT1 chimeras that contained these 70 residues also binds RNAPII (Fig. 3B, lanes 4 and 5). We conclude that the C-terminal region of CycT1 between positions 481 and 551 interacts with RNAPII in vivo and with nuclear extracts in vitro.
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C-terminal region of CycT1 rescues the ability of CycK to mediate transcriptional activation. Based on these results, the CTD appeared to be the key target for the C-terminal region of CycT1. The binding of these two domains was documented in several different ways and mapped to the histidine-rich stretch in CycT1. To prove the functional significance of this interaction, we examined the ability of a Gal-CycK chimera to activate transcription from distal sites via DNA. The same plasmid targets were used (Fig. 5A). By binding Cdk9, CycK forms an alternative P-TEFb complex (8). However, it contains only 357 residues, which include the N-terminal cyclin boxes but not the long C-terminal region of CycT1 and CycT2. Neither Gal-CycT(1–300) (Fig. 5B and C, lanes 4) nor Gal-CycK (lanes 5) activated transcription. In contrast, a Gal-CycK chimera which was fused to the C-terminal region of CycT1 (Fig. 5B and C, lanes 6) activated transcription when presented via DNA to levels of the Gal-CycT(1–726) fusion protein (lanes 3). Thus, the C-terminal residues of CycT1 rescued the ability of Gal-CycK fusion protein to support transcriptional activation from distal sites upstream and downstream of the promoter and coding sequence.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our experiments confirm and extend previous studies on P-TEFb, where Gal-P-TEFb chimeras could activate transcription via UAS (34, 37). Distinct from promoter effects, our study revealed that P-TEFb can also function as a coactivator from sites distal to the target gene. Additionally, we documented that the CTD is the target of P-TEFb. Most importantly, the ability of P-TEFb to bind the CTD could be correlated directly with its effects on the elongation of transcription. Our study also extends data from D. melanogaster, where the CTD is essential for effects of P-TEFb (41). In this system, following heat shock, P-TEFb is recruited to regions upstream of promoters of over 200 transcriptional units, implying its requirement for the activated transcription of genes, where RNAPII is paused (33).
Although CycT1 and Cdk9 were tethered artificially to DNA, this situation only mimicked several strong activators that can function on enhancers and which bind and recruit P-TEFb to these sequences. Previously, we made this connection with CIITA, which is the coactivator of MHC II genes (25). It functions from conserved upstream sequences in these promoters or from distal enhancers of these genes (54). Additionally, we found that NF-B also recruits P-TEFb to the interleukin 8 gene (1). A recent report made the same connection with the androgen receptor (30). Thus, the recruitment of P-TEFb represents an essential step for transcriptional effects of these activators. Although the direct tethering to DNA made our analyses simpler, additional insights into complex DNA-protein assembly, looping, and transcriptional activation will come from more detailed studies with these individual cellular and viral activators.
This study also addresses two important issues regarding HIV replication. First, it supports the notion that DNA-bound activators, such as NF-B, recruit P-TEFb (1). This interaction explains the ability of HIV to start transcribing its genome in the absence of Tat. Thus, cellular activation releases NF-B to the nucleus, where it binds to two repeated sequences upstream of the three Sp1 sites. The recruitment of P-TEFb then leads to the synthesis of Tat. Second, P-TEFb on NF-B could promote the autophosphorylation of Cdk9, which is necessary for productive interactions between Tat, TAR, and P-TEFb. This posttranslational modification is required for the creation of the optimal RNA-binding surface of the complex between Tat and P-TEFb (7, 12). Finally, this tripartite complex leads to sustained high levels of viral replication (59).
In fulfilling the criteria of enhancers, this study advances the concept that P-TEFb is an important coactivator for distal activators, i.e., that its interaction with the CTD of RNAPII can convey signals over long distances. Thus, our study suggests a model of how some enhancers could function (Fig. 8). First, the PIC is assembled and positioned on promoters. Proteins bound to TATA box, INR, and Sp1 sequences play important roles in this process (step 1). Other activators that bind to promoter and enhancer elements also remodel chromatin and recruit P-TEFb (step 2). Some proteins also bend DNA, enabling the C-terminal region of CycT1 to interact with the CTD of RNAPII (step 3). Cdk9 then phosphorylates the CTD (step 4), and possibly other proteins that interact with the paused RNAPII, resulting in the elongation of transcription (step 5). In summary, the interaction between the C-terminal region of CycT1 and the CTD of RNAPII allows P-TEFb to act as a coactivator that connects enhancer-binding proteins with the paused RNAPII at promoters, which leads to the elongation of transcription. This model brings closer the worlds of prokaryotic and eukaryotic transcription, where transcriptional antitermination, i.e., elongation, regulates among others the expression of bacteriophage 
genes (16).
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ACKNOWLEDGMENTS |
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Koh Fujinaga was supported by a fellowship from the Universitywide AIDS Research Program (F99-SF-118). This work was supported by the Howard Hughes Medical Institute.
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FOOTNOTES |
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5]) repeats of the CTD. Arrows indicate the wild-type and truncated large subunit of RNAPII (RPB1). Below the Western blots are the wild-type HA epitope-tagged RPB1 and 
