INTRODUCTION

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Regulation of transcriptional elongation is a critical process in the control of viral and cellular gene expression (reviewed in references 3 and 28). A number of cellular factors that regulate transcriptional elongation have been defined using both biochemical and genetic techniques. These factors include the general transcription factors TFIIF and TFIIS, as well as other factors including the elongin and ELL proteins (20, 41, 48).

In addition, cellular kinases play an important role in the control of transcriptional elongation based on their ability to phosphorylate the RNA polymerase II C-terminal domain (CTD) (27). One of these kinases, CDK-activating kinase (CAK), is composed of the CDK7 kinase in addition to cyclin H and MAT1. CAK is contained in the multiprotein TFIIH complex and is involved in modulating promoter clearance of specific promoters (13, 45, 47). A second kinase complex, P-TEFb, is composed of cyclin T1 and CDK9 and also phosphorylates the RNA polymerase II CTD and stimulates transcriptional elongation (18, 32, 33, 36, 64). The Tat protein, which is a potent stimulator of transcriptional elongation, interacts with P-TEFb to stimulate human immunodeficiency virus type 1 (HIV-1) gene expression (4, 7, 17-19, 25, 26, 30, 31, 55, 56, 62, 64).

SPT4 and SPT5 are highly conserved proteins which are present in a variety of species from yeast to humans and are involved in the regulation of transcriptional elongation (23, 53, 58, 60, 61). Genetic assays in yeast demonstrate that SPT5 conditional mutants can be suppressed by mutations in the genes encoding two largest subunits of RNA polymerase II (23). Furthermore, SPT5 interacts directly with RNA polymerase II via a domain in SPT5 that has homology to the Escherichia coli transcription elongation factor NusG (23, 53, 61). The human homologues of the SPT4 and SPT5 proteins have also been characterized (8, 9, 22, 49). These proteins were also isolated independently by two groups based on their ability to either mediate the inhibition of transcriptional elongation in the presence of the ATP analogue 5,6-dichloro-1--D-ribofuranosylbenzimidazole (DRB) (10, 53) or rescue Tat activation in fractionated HeLa extract that does not otherwise support this process (58). Although SPT4 and SPT5 are required for DRB-mediated inhibition of transcriptional elongation, these proteins also can stimulate transcriptional elongation in in vitro transcription assay mixtures containing limiting concentrations of ribonucleoside triphosphates (53). Thus, SPT4 and SPT5 can regulate transcriptional elongation in both a positive and negative manner depending on the experimental conditions.

The mechanism by which SPT4 and SPT5 regulate transcriptional elongation has recently been investigated. SPT5 contains a number of distinct domains including an acidic amino terminus, four KOW repeats that have homology to the E. coli transcriptional regulator NusG (23, 53, 61), and two C-terminal repeat elements designated CTR1 and CTR2 (49). These latter domains contain multiple amino acid repeats that are rich in serine and threonine residues and may serve as potential sites for phosphorylation by cellular kinases. Recent data indicate that SPT4 and SPT5 function at an early step in the transcriptional elongation process that is regulated by P-TEFb (37, 54). For example, immunodepletion of P-TEFb from HeLa nuclear extract greatly reduces the production of full-length transcripts in in vitro transcription assays, while immunodepletion of both P-TEFb and SPT5 restores transcription to control levels. However, the addition of SPT4 and SPT5 to extract that is immunodepleted of both SPT5 and P-TEFb results in transcriptional repression. The subsequent addition of P-TEFb to this extract is able to alleviate the inhibitory effect of the SPT4 and SPT5 proteins (54). Therefore, immunodepletion of P-TEFb from HeLa nuclear extract results in a similar effect to the addition of DRB, demonstrating the negative effect of the SPT4-SPT5 complex on transcriptional elongation. These data suggest that P-TEFb is probably the target of DRB-inhibitory effects on transcriptional elongation mediated by SPT4 and SPT5.

In this study, the functional interaction between SPT4 and SPT5 and P-TEFb in DRB-mediated transcriptional repression and Tat activation was studied. We demonstrated that SPT5 domains that bind SPT4 and RNA polymerase II in addition to the CTR1 domain are critical for mediating DRB inhibition and Tat activation. Furthermore, we found that P-TEFb phosphorylates the CTR1 domain in SPT5. These studies suggest that P-TEFb phosphorylation of both RNA polymerase II and SPT5 may be critical for the regulation of transcriptional elongation.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Materials. DRB was purchased from CalBiochem and dissolved in 50% ethanol-10 mM HEPES (pH 7.9) at 10 mM. Prior to the addition of DRB to in vitro transcription reaction mixtures, a 10 mM stock was diluted with 10% ethanol-10 mM HEPES (pH 7.9) to a concentration of 1.5 mM.

Nuclear-extract preparation. Nuclear extract was prepared as previously described (12). It was then fractionated on a phosphocellulose column as previously described (58, 63). The column was washed with buffer A (20 mM Tris HCl [pH 7.9], 20% glycerol, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride [PMSF], 2 mM dithiothreitol [DTT]) containing 0.1 M KCl and 0.5 M potassium acetate and eluted with buffer containing 0.1 M KCl and 1.0 M potassium acetate. The eluate was dialyzed against buffer A containing 0.1 M KCl and used in partially reconstituted transcription reactions as a fraction that can support basal transcription but not DRB-sensitive transcription or Tat transactivation.

In vitro transcription assays. In vitro transcription reactions assaying the sensitivity of transcription to DRB were carried out as described previously (53) with modifications. For the partially reconstituted in vitro transcription reactions, 15 µl (~15 µg) of 1.0 M potassium acetate phosphocellulose eluate supplemented with recombinant human TATA binding protein (25 to 50 ng) purified from E. coli was incubated with 250 ng of circular pTF3-6C₂AT template (a generous gift of H. Handa). This template contained a 380-bp G-less cassette immediately following the promoter. These reactions were performed in the presence of 4 mM MgCl₂ and 10 µl of purified SPT4 and SPT5 for 45 min at 30°C. Reaction mixtures with nuclear extract contained 6.5 µl (~30 µg) of unfractionated nuclear extract. DRB was then added to a final concentration of 50 µM, and this was followed by the addition of 38 µl of TRX buffer (25 mM HEPES [pH 7.9], 10% glycerol, 50 mM KCl, 6 mM MgCl₂, 0.5 mM DTT, 0.5 mM PMSF, 60 µM ATP, 5 µM CTP, 600 µM GTP, 600 µM UTP) containing [-³²P]CTP (NEN) (800Ci/mmol; 1 µl per reaction). After a 10-min reaction, 250 U of RNase T₁ (GIBCO) was added and the incubation was continued at 37°C for an additional 10 min. Reactions were then stopped by the addition of 350 µl of termination mixture (0.1 M Tris HCl [pH 7.5], 7 M urea, 0.35 M NaCl, 10 mM EDTA, 1% sodium dodecyl sulfate [SDS], 25 µg of yeast tRNA per ml). Samples were extracted with a mixture of phenol and chloroform, ethanol precipitated, dissolved in gel-loading buffer (8 mM EDTA, 0.3 mg of bromphenol blue per ml, and 0.3 mg of xylene cyanol per ml in deionized formamide), heat treated for 3 min at 100°C, and separated on a 6% denaturing gel. Autoradiography was performed at 70°C with two intensifying screens for 12 to 24 h.

Construction of GST fusion expression vectors. The full-length SPT5 cDNA (58) was cloned into pGEX-KG vector at the EcoRI site. An StuI-EcoRI fragment containing CTR2 sequences (encoding amino acids [aa] 852 to 1087), a SmaI-StuI fragment containing CTR1 repeats (encoding aa 760 to 852), and an SmaI-EcoRI fragment containing CTR1 and CTR2 repeats (encoding aa 760 to 1087) were each individually subcloned into the pGEX-KG vector. To make a construct devoid of CTR sequences at the C terminus, a nonsense mutation substituting an amber stop codon (TGA) for a Ser codon (TCA) at position 755 was introduced by PCR mutagenesis using a QuickChange site-directed mutagenesis kit (Stratagene). Vectors encoding the GST fusion proteins were transformed into the BL21 pLysE strain of E. coli, and proteins were induced and purified by affinity chromatography on a glutathione-agarose column as described previously (26).

Purification of recombinant proteins from baculovirus. Baculovirus expression vectors containing either CDK9, CDK9, and cyclin T1, CDK9 and cyclin T2a, or CDK9 and cyclin T2b were a generous gift from David Price (36). Recombinant His₆-tagged proteins were purified by Ni²⁺-nitrilotriacetic acid (NTA)-agarose and S-Sepharose chromatography as described previously (36). Baculoviruses expressing CDK7, cyclin H, and MAT1, were a generous gift of David Morgan. Cyclin H had an amino-terminal His₆ tag. Trimeric CAK was purified by chromatography on Ni²⁺-NTA agarose, Q-Sepharose, and Superdex 200 columns as described previously (11).

In vitro kinase assays. RNA polymerase II used as a substrate in kinase reactions was purified from HeLa cells as described previously (26, 34, 39). GST-SPT5 fusion proteins, which were purified as described elsewhere (26), were also analyzed in kinase assays. Kinase assays (in a 15-µl volume) were performed for 2 h at room temperature in reaction buffer containing 20 mM HEPES (pH 7.9), 15% glycerol, 0.15 mM EDTA, 7 mM MnCl₂, 2 mM DTT, 0.7 mM PMSF, 7 mM NaF, 0.7 mM Na₃VO₄, 2 µM ATP, and 0.04 µl of [-³²P]ATP (ICN) (7,000 Ci/mmol).

Immunoprecipitation of SPT5 from nuclear extract. Rabbit polyclonal antibody was generated against a GST-SPT5 fusion protein (aa 1 to 73) or GST-CTR2 (aa 852 to 1087) (58). Antibodies were purified following two fractionation steps on a protein A-Sepharose column and covalently coupled to a protein A-Sepharose matrix using dimethylpimelimidate (21). Nuclear extract (0.5 ml) was incubated with 75 µl of the beads for 2 h at 4°C with gentle shaking. The beads were washed three times with 10 bed volumes of buffer containing 20 mM Tris HCl (pH 7.9), 1 mM EDTA, 100 mM NaCl, 1% NP-40, 1 mM DTT, and 0.5 mM PMSF. They were then resuspended in 150 µl of SDS-polyacrylamide gel electrophoresis (PAGE) gel-loading buffer. One-third of the sample (50 µl) was separated on an SDS-polyacrylamide gel. Western blot analysis was performed using rabbit polyclonal antibody against CDK9 (Santa Cruz) or mouse monoclonal antibody against the CTD of the largest subunit of RNA polymerase II (8WG16; BAbCo). To deplete nuclear extract of endogenous SPT5, 1 mg of anti-CTR2 rabbit polyclonal affinity-purified antibody was chemically coupled to 200 µl of protein A-containing beads. Nuclear extract (1 ml) was incubated with the beads overnight and then with fresh beads for an additional 4 h.

Construction of SPT5 and SPT4 expression vectors. A wild-type SPT5 cDNA was cloned into the EcoRI and SalI endonuclease restriction sites of pCMV2-pFlag (Sigma). C-terminal deletion mutants of SPT5 were prepared using a QuickChange site-directed mutagenesis kit with pCMV2-pFlag-SPT5 as a template. SPT5 constructs containing aa 1 to 837, 1 to 754, 1 to 518, and 1 to 229 were constructed using oligonucleotide primers 5'-GAA GAA TAT GAG TAG GCT TTC GAT GAT-3', 5'-CAC GGT GGG CTG ACG GCG CCC GGG CGG CA-3', 5'-CCG GGA CCT GTA GCT CTG CTC AGA GAC AG-3', and 5'-GTG GAG GCC TAG AAG CAG ACC CAC GTG A-3', respectively, and complementary oligonucleotides. In each case, a stop codon (underlined) was inserted in the sequence. The N-terminal deletion mutants of SPT5 extending from aa 176 to 1087, 519 to 1087, 755 to 1087, and 838 to 1087 were constructed using the 5' PCR primers 5'-CGG AAT TCC GAT CCC AAT CTG TGG ACT GTC AA-3', 5'-CGG AAT TCC CAG CTC TGC TCA GAG ACA GCA TCA-3', 5'-CGG AAT TCC TCA CGG CGC CCG GGC GGC ATG AC-3', and 5'-CGG AAT TCC TAT GCT TTC GAT GAT GAG CCC ACC-3', respectively, and the 3' PCR primer 5'-GCG TCG ACT CAG GCT TCC AGG AGC TTC CCC AG-3'.

Affinity purification of Flag-tagged SPT5 proteins from COS cells. Expression vectors containing Flag-tagged SPT5 cDNA either alone or in combination with expression vectors containing Myc-tagged SPT4 cDNA were transfected into COS cells using Fugene 6 (Roche). At 48 h posttransfection, the cells were harvested and homogenized in Tris-buffered saline (50 mM Tris HCl [pH 7.4], 150 mM NaCl). After centrifugation at 3,000 rpm for 10 min in a CS-6R Beckman centrifuge, supernatant was applied to an anti-Flag M2 affinity gel column (Sigma) as specified by the manufacturer. Flag-tagged fusion proteins were eluted with a Tris-buffered saline solution containing Flag peptide (Sigma) at 100 µg/ml. Proteins were dialysed against buffer D and used in the in vitro transcription assays.

Immunoprecipitation and Western blotting. SPT5 and SPT4 expression vectors were transfected into COS cells with Fugene 6 (Roche). At 48 h posttransfection, the cells were harvested in phosphate-buffered saline and subjected to centrifugation for 2 min at 2,000 rpm at 4°C in a CS-6R Beckman centrifuge. The cell pellets were resuspended in 600 µl of buffer B (10 mM HEPES [pH 7.9], 10 mM KCl, 1.5 mM MgCl₂, 0.5 mM DTT, and 0.5 mM PMSF) and allowed to swell on ice for 15 min. Cells were then lysed by being rapidly pushed through a 25-gauge 5/8 BD hypodermic needle eight times. The cell homogenates were then centrifuged for 20 s at 12,000 rpm in a 5415 C-Eppendorf microcentrifuge. The lysates were incubated with anti-Flag M2 monoclonal antibody (Sigma) or anti-HA monoclonal antibody (Roche) for 1 h at 4°C. Then 20 µl of protein G-agarose beads was added and mixed for 1 h at 4°C. After the mixture was washed three times with ELB buffer (50 mM HEPES [pH 7.9], 250 mM NaCl, 5 mM EDTA, 1% NP-40, 0.5 mM DTT, and 0.5 mM PMSF), the immunoprecipitates were analyzed on an SDS-polyacrylamide gel. Western blotting was performed using an enhanced chemiluminescence agent (Amersham).

Construction of SPT5 GST-CTR1 mutants. Sequences encoding two of the CTR1 repeats (aa 772 to 787) were incorporated into the 3' oligonucleotide primer that was used in the PCR to amplify a fragment of polylinker of pGEX-KG vector. For the wild-type construct, the sequence of the 3' primer was TTC CCA AGC TTA [GTA CAT GGG TGT TCG GGA GCC AGA GCC ATA CAT GGG CGT CTG GGA GCC] ACC CAT GGA GTC TAG. The portion of the primer corresponding to the CTR1 repeat is shown in brackets. The 5' portion of the primer contains a HindIII site and the stop codon (UAA) immediately following the coding sequence. The 5' oligonucleotide primer CCG CGT GGA TCC CCG GGA anneals to pGEX-KG and contains a BamHI site. The PCR product was then digested with HindIII and BamHI and cloned into pGEX-KG vector. Mutant CTR1 repeats were also generated which contained alanine in place of either threonine (at aa 775 and 784) or serine (at aa 773 and 782). All the constructs were confirmed by DNA sequencing. The construct encoding GST fused to two of the RNA polymerase II CTD repeats was generated using a similar approach and was described previously (26).

Phosphorylation of SPT5. Recombinant SPT4-SPT5 complex purified from baculovirus was incubated with recombinant P-TEFb also expressed in baculovirus in a total volume of 20 µl. Kinase reactions were performed in buffer (15 mM HEPES [pH 7.9], 0.1 mM EDTA, 1.5 mM DTT, 0.5 mM DTT, 10% glycerol) supplemented with 5 mM MnCl₂ and 2.5 mM MgCl₂ in either the presence or absence of 5 mM ATP at room temperature for 30 min. Samples were then dialyzed against buffer D at 4°C to remove excess ATP. In vitro transcription reactions were performed as described above, except that DRB was added at the start of the incubation.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Domains in the SPT5 protein. Several distinct domains in the human SPT5 protein have been delineated based on their homology to the yeast SPT5 protein and the E. coli transcriptional regulator NusG (Fig. 1A). The amino terminus of SPT5 extending between aa 1 and 118 is highly charged, being abundant in the acidic amino acid residues aspartic acid and glutamic acid (53, 58). Four regions in the middle of the SPT5 protein extending between residues 421 and 447, 473 and 499, 595 and 621, and 705 and 731 have homology to KOW motifs present in the procaryotic transcription termination and antitermination factor NusG (23, 53). There are two clusters of different repeats in the C-terminal 280 residues of SPT5 which are rich in serine, threonine, proline, and tyrosine but are unrelated to C-terminal repeat motifs in the yeast SPT5 protein (49). The first motif, which has been designated CTR1, contains 9 repeats of the consensus sequence GS(R/Q)TPXY, while a less highly conserved motif, designated CTR2, contains 10 repeats of the consensus sequence P(T/S)PSP(Q/A)(S/G)Y, which has weak homology to the repeats found in the CTD of RNA polymerase II.

View larger version (16K): [in this window] [in a new window]   FIG. 1.   Schematic of the SPT5 protein. (A) A schematic of the SPT5 protein with the positions of the acidic domain, the four KOW domains that have homology to the E. coli NusG protein, and two domains, CTR1 and CTR2, each of which has multiple amino acid repeats, is shown. (B) A variety of contructs with mutations in different domains of the SPT5 protein are shown. These mutants were analyzed for their function using both in vivo interaction and in vitro transcription assays.

Domains in SPT5 that interact with SPT4. Previously, in vitro interaction studies with a GST-SPT4 fusion protein and in vitro-translated SPT5 proteins labeled with [³⁵S]methionine were used to map SPT5 interactions with SPT4 (61). To map the in vivo interactions between these proteins, we used expression vectors containing a Myc-tagged SPT4 protein and Flag-tagged SPT5 proteins. Following transfection of COS cells with these epitope-tagged SPT4 and SPT5 constructs, extracts were prepared and the association of SPT4 and SPT5 was assayed using immunoprecipitation followed by Western blot analysis.

View larger version (32K): [in this window] [in a new window]   FIG. 2.   SPT5 association with SPT4, RNA polymerase II, and CDK9. (A) Expression vectors containing either a Myc-tagged SPT4 construct or the indicated Flag-tagged SPT5 constructs were cotransfected into COS cells. Extracts were prepared from each of these transfections, and the SPT4 levels were analyzed by Western blot analysis with antibody directed against the Myc epitope (upper panel). Extracts prepared from the SPT4-SPT5-cotransfected cells were immunoprecipitated with M2 monoclonal antibody to isolate the Flag-tagged SPT5 proteins, and then Western blot analysis was performed with monoclonal antibody directed against the Myc epitope to detect the epitope-tagged SPT4 protein (lower panel). In lane 2, an expression vector containing the SPT4 cDNA was transfected into COS cells alone without cotransfection of the SPT5 cDNA. (B) Western blot analysis of COS cell extracts prepared following transfection of expression vectors containing Flag-tagged SPT5 cDNA constructs (lanes 1 to 17) or a Flag-tagged SPT4 cDNA (lane 18) was performed using the M2 monoclonal antibody. In lane 1, an expression vector containing SPT4 was cotransfected with the wild-type SPT5 construct. MW, molecular weight in thousands. (C and D) Flag-tagged SPT5 constructs were each transfected into COS cells, and extracts were prepared. Immunoprecipitation of these extracts was performed with antibodies directed against either the RNA polymerase II CTD (C) or CDK9 (D). Western blot analysis with the M2 monoclonal antibody was then performed to detect the Flag-tagged SPT5.

SPT5 interactions with RNA polymerase II and CDK9. Next we addressed the domains in SPT5 that are responsible for in vivo interactions with RNA polymerase II and potentially CDK9 (Fig. 2B to D). Flag-tagged SPT5 constructs were transfected into COS cells, and their association with both endogenous RNA polymerase II and CDK9 was assayed. Western blot analysis of the COS cell extracts revealed roughly equivalent expression of each of the epitope-tagged SPT5 constructs (Fig. 2B, lanes 1 to 17), except for the SPT5 construct extending from aa 1 to 229 (lane 10), which had consistently low levels of expression. A Flag-tagged SPT4 construct that was transfected alone (lane 18) or cotransfected with SPT5 (lane 1) was also assayed. COS extracts were then immunoprecipitated with antibodies directed against either the CTD of RNA polymerase II (Fig. 2C) or CDK9 (Fig. 2D) followed by Western blot analysis with the M2 monoclonal antibody which detects the Flag-tagged SPT5 proteins. For RNA polymerase II, the monoclonal antibody 8WG16 (anti-CTD), which recognizes both the phosphorylated and unphosphorylated forms of this enzyme, was used for immunoprecipitation. CDK9 was immunoprecipitated with a rabbit polyclonal antibody directed against this kinase.

View larger version (21K): [in this window] [in a new window]   FIG. 3.   Association of SPT5 with RNA polymerase II and CDK9 in vivo and in vitro. (A) HeLa nuclear extract was immunoprecipitated with either preimmune sera (lanes 2 and 5) or rabbit polyclonal antibody directed against SPT5 (lanes 3 and 6). A total of 20% of the HeLa nuclear extract that was subjected to immunoprecipitation is shown (lanes 1 and 4). Following immunoprecipitation, Western blot analysis was performed with antibody directed against the RNA polymerase II (Pol II) CTD (lanes 1 to 3) or CDK9 (lanes 4 to 6). (B) Purified RNA polymerase II (lanes 1 to 4) or baculovirus-purified CDK9/cyclin T1 (lanes 5 to 8) was either untreated (30% of the input is shown in lanes 1 and 5) or incubated with glutathione-agarose beads containing GST (lanes 2 and 6), GST-SPT5 (lanes 3 and 7), or a GST-SPT5 fusion protein extending from aa 1 to 754 (lanes 4 and 8). Following extensive washing, Western blot analysis was performed with antibodies directed against the RNA polymerase II CTD (lanes 1 to 4) or CDK9 (lanes 5 to 8).

The CTR1 domain of SPT5 is critical for DRB-mediated repression. Previously, we and others have demonstrated that fractionation of HeLa nuclear extract on a phosphocellulose column followed by washing with 0.5 M potassium acetate and elution with 1.0 M potassium acetate resulted in extract that is unable to support Tat activation (58, 63). Addition of a highly purified cellular fraction that contained SPT5 restored Tat activation (58). First, we analyzed this 1.0 M potassium acetate fraction for the presence of cyclin T1, CDK9, and SPT5. The 1.0 M potassium acetate phosphocellulose fraction contained the P-TEFb components, cyclin T1 and CDK9, but not SPT5 (Fig. 4A, lanes 4 and 5). Baculovirus-produced and purified SPT4 and SPT5 (lane 3) and affinity-purified SPT4 and SPT5 proteins isolated from COS cells transfected with expression vectors containing these epitope-tagged cDNAs (lane 2) were also assayed by Western blot analysis.

View larger version (39K): [in this window] [in a new window]   FIG. 4.   Reconstitution of DRB inhibition and Tat activation by recombinant SPT5 proteins. (A) Western blot analysis was performed with antibodies directed against cyclin T1, CDK9, and SPT5 using unfractionated HeLa nuclear extract (Nuc Ext) (lane 1), affinity-purified SPT4-SPT5 complex produced following transfection of epitope-tagged SPT4 and SPT5 cDNAs into COS cells (lane 2), baculovirus-expressed and purified SPT4-SPT5 (lane 3), or 10-µl (lane 4) or 50-µl (lane 5) aliquots of a 1.0 M potassium acetate fraction of HeLa nuclear extract obtained following phosphocellulose (PC) chromatography. (B) Western blot analysis of affinity-purified SPT4 and SPT5 proteins. Expression vectors containing the different Flag-tagged SPT5 cDNA constructs and a Myc-tagged SPT4 cDNA construct were cotransfected into COS cells. The Flag epitope-tagged SPT5 and associated SPT4 protein in these extracts were purified by binding to protein G beads containing M2 monoclonal antibody followed by elution with Flag peptide. Western blot analysis was performed with anti-Flag monoclonal antibody to detect SPT5 proteins (top panel) or anti-Myc monoclonal antibody to detect the associated SPT4 (lower panel). (C) In vitro transcription analysis was performed with the pTF3-6C2AT template using unfractionated HeLa nuclear extract (NE) (lanes 1 and 2), or the 1.0 M potassium acetate fraction of HeLa nuclear extract eluted from the phosphocellulose (PC) column (lanes 3 to 24). The 1.0 M potassium acetate fraction was assayed either alone (lanes 3 and 4), with affinity-purified SPT4 added alone (lanes 5 and 6), with affinity-purified SPT5 added alone (lanes 7 and 8), or with both SPT4 and the different affinity-purified SPT5 proteins shown in panel B added as indicated (lanes 9 to 24). DRB was added to the even-numbered lanes in the in vitro transcription assays. (D) In vitro transcription analysis was performed with the HIV-1 LTR wild-type (top panel) or the HIV-1 LTR loop mutant (lower panel) templates using unfractionated HeLa nuclear extract (lanes 1 to 4), the 1.0 M potassium acetate fraction of HeLa nuclear extract eluted from the phosphocellulose (PC) column alone (lanes 5 and 6), or the 1.0 M potassium acetate fraction in the presence of affinity-purified SPT4 and SPT5 proteins (lanes 7 and 8), SPT5 alone (lane 9 and 10), or SPT4 and affinity-purified SPT5 mutants (lanes 11 to 24) as indicated. Tat (25 ng) was added to the odd-numbered lanes and GST (25 ng) was added to the even-numbered lanes in the in vitro transcription assays. Following the in vitro transcription analysis with these templates containing G-less cassettes, the labeled RNAs were digested with RNase T1 and gel electrophoresis and autoradiography were performed.

The CTR1 domain of SPT5 is critical for mediating Tat activation. The 1.0 M potassium acetate phosphocellulose fraction was next assayed for its ability to support Tat activation. First, we demonstrated, using unfractionated HeLa nuclear extract, that Tat strongly activated in vitro transcription from the wild-type HIV-1 LTR template (Fig. 4D, lanes 1 to 4, upper panel) but not from an HIV-1 template with mutations in the TAR RNA loop sequences (lanes 1 to 4, lower panel) (58). In contrast, there was no Tat activation when the 1.0 M potassium acetate phosphocellulose fraction of HeLa nuclear extract was used (lanes 5 and 6). The addition of the affinity-purified SPT4 and SPT5 resulted in the restoration of low levels of Tat activation for the wild type (lanes 7 and 8, upper panel) but not the HIV-1 loop mutant template (lanes 7 and 8, lower panel). Similar results were seen with the 1.0 M potassium acetate phosphocellulose fraction following the addition of baculovirus-produced SPT4 and SPT5 (data not shown). The addition of SPT5 alone did not markedly stimulate Tat activation in the absence of transfected SPT4 (lanes 9 and 10).

P-TEFb phosphorylation of SPT5. Previous data suggest that P-TEFb is critical for SPT5 function (54). For example, immunodepletion of CDK9 from HeLa nuclear extract prevented SPT5 from mediating DRB repression. Thus, one possible mechanism to explain DRB-mediated repression is that this compound inhibits P-TEFb phosphorylation of RNA polymerase II, which alters its interaction with SPT5 and results in transcriptional inhibition (31). Alternatively, it is possible that DRB could inhibit P-TEFb phosphorylation of SPT5, which may lead to enhanced SPT5 inhibitory effects on transcription. Thus, the CTR1 domain of SPT5 may be a substrate for P-TEFb phosphorylation which is important in the regulation of both DRB-mediated repression and Tat activation.

View larger version (31K): [in this window] [in a new window]   FIG. 5.   P-TEFb phosphorylation of SPT5. (A) Baculovirus-produced and purified proteins including CDK9 (lanes 1 and 6), CDK9 and cyclin T1 (lanes 2 and 7), CDK9 and cyclin T2a (lanes 3 and 8), and CDK9 and cyclin T2b (lanes 4 and 8) were assayed in in vitro kinase assays in the absence of the GST-SPT5 substrate (lanes 1 to 4), the GST-SPT5 substrate was assayed alone (lane 5), or the GST-SPT5 substrate was added along with the different CDK9 preparations (lanes 6 to 9). (B) The baculovirus-produced and purified preparations of CDK9 (lane 1), CDK9 and cyclin T1 (lane 2), CDK9 and cyclin T2a (lane 3), and CDK9 and cyclin T2b (lane 4) were assayed in Western blot analysis using antibodies directed against CDK9 (top panel), cyclin T1 (middle panel), or cyclin T2 (lower panel).

View larger version (28K): [in this window] [in a new window]   FIG. 6.   Comparison of P-TEFb and CAK phosphorylation of SPT5 constructs. (A) Purified RNA polymerase II (Pol II) was assayed in in vitro kinase assays using RNA polymerase II alone (lane 1) or in the presence of baculovirus-produced and purified CAK including CDK7, cyclin H, and MAT1 (lane 2) or CDK9 and cyclin T1 (lane 3). MW, molecular weight in thousands. (B) In vitro kinase assays were performed using baculovirus-produced preparations of CDK9/cyclin T1 (lanes 1 to 6) or CAK (lanes 7 to 12) with either no added substrate (lanes 1 and 7), GST-SPT5 (lanes 2 and 8), GST-SPT5 (aa 1 to 754) (lanes 3 and 9), GST-SPT5 (aa 760 to 852) (lanes 4 and 10), GST-SPT5 (aa 814 to 1087) (lanes 5 and 11), or GST-SPT5 (aa 760 to 1087) (lanes 6 and 12). Phosphorylation was assayed following SDS-PAGE and autoradiography. (C) A Coomassie blue-stained gel of the various GST-SPT5 fusion proteins used in the in vitro kinase assays in part B is shown.

CTR1 amino acid residues phosphorylated by P-TEFb. Next we determined which amino acids within the CTR1 repeat (Fig. 7A) are phosphorylated by CDK9-cyclin T1. The consensus repeat in CTR1, GS(R/Q)TPXY, contains serine and threonine residues that can potentially be phosphorylated by P-TEFb. We constructed a GST fusion protein containing two CTR1 repeats spanning aa 772 to 787 and then either left this region intact or created mutations of serine or threonine residues in both repeats to alanine (Fig. 7B). The wild-type and mutant GST fusion proteins were then used as substrates in in vitro kinase reactions with baculovirus-produced CDK9-cyclin T1 (Fig. 7C). The wild-type and serine mutant GST-CTR1 fusion proteins were readily phosphorylated by CDK9-cyclin T1 (Fig. 7C, lanes 4 and 5), while the GST-CTR1 threonine mutant was not significantly phosphorylated (lane 6). This result indicated that threonine residues in the CTR1 repeat are probably the preferred amino acids that are phosphorylated by P-TEFb.

View larger version (27K): [in this window] [in a new window]   FIG. 7.   Comparison of P-TEFb and CAK phosphorylation of SPT5 CTR1 repeats. (A) Sequences of CTR1 and CTR2 repeats. The positions of the C-terminal repeat motifs in the CTR1 and CTR2 domains of SPT5 are indicated, as is the consensus sequence for each domain, as previously described (49). (B) The sequences of the two CTR1 repeats that were fused to GST and used as substrates in in vitro kinase reactions are shown. The boxes indicate the positions of the repeats, and the mutated amino acids are shown in boldface type. (C) In vitro kinase reactions were performed using baculovirus-produced and purified CDK9-cyclin T1 (lanes 1 to 6) or the baculovirus-produced and purified CAK components including CDK7, cyclin H, and MAT1 (lanes 7 to 12). The kinase reactions were performed without the addition of substrate (lanes 1 and 7) or following the addition of GST (lanes 2 and 8), a GST-CTD fusion protein containing two repeats of the RNA polymerase II CTD (lanes 3 and 9), or GST fusion proteins containing two CTR1 repeats with either the wild-type (WT) sequences (lanes 4 and 10), serine residues substituted for alanine (lanes 5 and 11), or threonine residues substituted for alanine (lanes 6 and 12). (D) A Coomassie blue-stained SDS-polyacrylamide gel of the GST fusion proteins that were used as substrates for kinase assays in panel B is shown.

Role of P-TEFb phosphorylation on SPT5 function. Finally, we tested whether P-TEFb phosphorylation of SPT5 alters its functional properties. The recombinant SPT4-SPT5 complex purified following baculovirus production was incubated with P-TEFb in either the presence or absence of ATP. SPT4-SPT5 was then added to in vitro transcription reaction mixtures with the pTF3-6C₂AT template and HeLa nuclear extract depleted of endogenous SPT5. First, we demonstrated that DRB inhibited in vitro transcription of this template by using both unfractionated HeLa nuclear extract and HeLa nuclear extract immunodepleted with GST antibody (Fig. 8A, lanes 1 to 4). In contrast, HeLa nuclear extract immunodepleted of SPT5 exhibited only minimal DRB sensitivity (lanes 5 and 6), which was restored by the addition of recombinant SPT4-SPT5 complex (lanes 7 and 8). Preincubation of the SPT4-SPT5 complex with P-TEFb, in the absence of ATP, did not change its ability to confer DRB sensitivity to in vitro transcription reactions with HeLa extract depleted of SPT5 (lanes 9 to 12). However, when ATP was present during the preincubation of SPT4-SPT5 complex with P-TEFb, little DRB sensitivity was observed (lanes 15 and 16). When SPT4-SPT5 was preincubated with ATP in the absence of P-TEFb, it was still able to confer DRB sensitivity, although to a somewhat lesser extent than was the control lacking ATP (lanes 17 and 18). This result may be explained by the fact that the preparation of recombinant SPT4-SPT5 contains trace amounts of kinases that are capable of phosphorylating SPT5. Thus, phosphorylation of SPT5 by P-TEFb before beginning the in vitro transcription reaction alters SPT5 function and abrogates its ability to mediate DRB inhibition of transcription.

View larger version (37K): [in this window] [in a new window]   FIG. 8.   Role of P-TEFb phosphorylation on SPT4-SPT5 function in mediating DRB repression. (A) In vitro transcription assays were performed with the pTF3-6C2AT template in the presence (even-numbered lanes) or absence (odd-numbered lanes) of DRB. Untreated HeLa nuclear extract (lanes 1 and 2), HeLa nuclear extract immunodepleted with GST antibody (lanes 3 and 4), or HeLa nuclear extract immunodepleted with SPT5 antibody (lanes 6 to 20) were used in the in vitro transcription assays. In vitro transcription assay mixtures with the SPT5-immunodepleted HeLa nuclear extracts were supplemented with the SPT4-SPT5 complex purified following baculovirus expression in the absence of other treatment (lanes 7 and 8), the SPT4-SPT5 complex preincubated with P-TEFb (lanes 9 and 10 and lanes 15 and 16), the SPT4-SPT5 complex preincubated without P-TEFb (lanes 11 and 12 and lanes 17 and 18), or P-TEFb alone preincubated in the absence of SPT4-SPT5 (lanes 13 and 14 and lanes 19 and 20). The preincubation procedure was performed in the absence of ATP (lanes 9 to 14) or in the presence of 5 mM ATP (lanes 15 to 20), which was subsequently removed by dialysis. (B) A schematic of the functional domains in the SPT5 protein that were analyzed in this study is shown.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

The SPT4 and SPT5 proteins are involved in the control of transcriptional elongation (23, 53, 58, 61). Depending on the conditions, such as the concentrations of nucleotides used in the in vitro transcription assay, SPT4 and SPT5 function in both inhibiting and enhancing transcriptional elongation (53). Although SPT5 directly interacts with RNA polymerase II (53, 61), the mechanism by which it alters the processivity of polymerase remains to be determined. One study suggests that SPT5 associates better with the unphosphorylated (IIa) form of RNA polymerase II than with the phosphorylated (IIo) form (54). Therefore, it is possible that P-TEFb may stimulate transcriptional elongation both by increasing the phosphorylation of SPT5 bound to RNA polymerase II and by increasing the phosphorylation of the polymerase CTD. This could result in the release of unphosphorylated SPT5 from the IIo form of the polymerase, while the phosphorylated form of SPT5 stimulates its polymerase processivity. Further studies are needed to test this model. In addition to P-TEFb (54), multiprotein complexes including NELF (59) and Tat-SF1 (29, 35) modulate the SPT4-SPT5, function suggesting that a number of positive and negative factors may regulate its activity.

In the present study, we analyzed domains in SPT5 that are involved in in vivo interactions with SPT4, RNA polymerase II, and CDK9. Furthermore, we characterized domains in SPT5 that are responsible for DRB-mediated repression and Tat activation (Fig. 8B). This analysis defines domains in SPT5 that bind SPT4 and RNA polymerase II that are in agreement with a previous study (61). In contrast to the results of that study, our analysis demonstrates that a CTD of SPT5 with nine conserved heptad motifs, designated CTR1, is critical for DRB-mediated repression. Furthermore, CTR1 is involved in SPT5 modulation of Tat activation and is also a target for P-TEFb phosphorylation. One possible explanation for these results is that the recombinant SPT5 and SPT4 used in the previous study was produced in bacteria and subjected to a denaturation and renaturation protocol that may have altered its activity compared to the SPT4 and SPT5 proteins used in our study.

Our results demonstrate that both DRB inhibition and Tat activation can be restored by the addition of purified SPT4 and SPT5 to fractionated HeLa nuclear extract that is not competent for either DRB inhibition or Tat activation. Our previous work (58) suggested that SPT5 but not SPT4 was critical for Tat activation. However, this result was due to the low affinity of the antibody used to immunodeplete SPT4. The effects of SPT4 and SPT5 are due to both increases in transcriptional repression in the presence of DRB (1.5- to 2-fold) and increases in basal transcription in the absence of DRB (2.5- to 3.5-fold). The effects of SPT4 and SPT5 on Tat activation of the HIV LTR are primarily due to decreases in basal transcription (1.5- to 2-fold), although increases in Tat-induced transcription (1.5-fold) are noted. Similar effects on DRB inhibition and Tat activation are observed with the SPT5 deletion mutants and SPT4 when they are added to HeLa nuclear extract immunodepleted of SPT5 rather than when they are added to the 1.0 M potassium acetate fraction of HeLa nuclear extract (D. Ivanov and R. Gaynor, unpublished observation). However, there are differences in the ability of SPT4 and SPT5 to activate HIV-1 transcription in the SPT5-immunodepleted HeLa extract compared to the 1.0 M potassium acetate fraction. When SPT4 and SPT5 are added to the 1.0 M potassium acetate phosphocellulose fraction, these proteins activate HIV-1 transcription in the presence of Tat and repress basal transcription. However when SPT4 and SPT5 are added to the SPT5-immunodepleted extract, they result primarily in repression of basal transcription rather than stimulation of transcription in the presence of Tat.

The yeast SPT5 homologue, like human SPT5, contains a repetitive structure in its C-terminal portion (50). It is composed of 15 copies of a 6-aa repeat with a consensus consequence S(T/A)WGG(A/G) that is very different from the repeats found in mammalian SPT5 protein. Since no homologue of P-TEFb has been identified in yeast, it is possible that the serine residue in this repeat is phosphorylated by another kinase which recognizes a different consensus sequence. In support of this possibility, partial deletion of these repeats in the yeast SPT5 protein impairs its function while deletion of all 15 of these repeats virtually eliminates SPT5 function (50). Therefore, it is likely that the C-terminal repeats found in both yeast and mammalian SPT5 proteins function to promote protein-protein interactions and regulate SPT5 activity. It is likely that other domains in SPT5, including acidic residues in its amino terminus, function in other regulatory properties such as modulating chromatin structure, which would not be detected in the in vitro transcription assays used in this study.

Our model would suggest that it is probably the unphosphorylated form of SPT5 bound to RNA polymerase II that is initially involved in modulating its transcriptional elongation properties. P-TEFb phosphorylation of SPT5 may alter its interactions with RNA polymerase II or facilitate the interaction with other factors that associate with polymerase. In the absence of the CTR1 domain, SPT5 can bind to RNA polymerase II but is not able to modulate the transcriptional elongation properties of polymerase. SPT5 that is phosphorylated prior to the onset of transcription is unable to mediate DRB inhibition. This result is consistent with the idea that the timing of SPT5 phosphorylation during transcription is critical in regulating its ability to modulate polymerase processivity. Further work is needed to address how phosphorylation of SPT5 leads to changes in its functional properties.

Interestingly, SPT5 protein from mitotic HeLa cells appears to migrate more slowly in SDS-polyacrylamide gels than does SPT5 isolated from interphase cells, and this effect is probably the result of enhanced SPT5 phosphorylation (49). We find that SPT5 phosphorylation by P-TEFb also results in a slower-migrating form of SPT5, probably reflecting the phosphorylation of multiple C-terminal repeats. In this regard, it is remarkable that during mitosis RNA polymerase II aborts transcription and that nascent transcripts dissociate from the template (46). This suggests the existence of mechanisms controlling the elongation phase of transcription during mitosis, which might involve inactivation of specific transcription elongation factors such as SPT5 by enhancing their phosphorylation prior to preventing the transcription of specific genes. In addition, a number of transcription factors such as Sp1 and Oct-1 are phosphorylated during mitosis and demonstrate decreased DNA binding properties (43).

Among the known elongation factors, TFIIF increases the rate of RNA chain synthesis relatively uniformly (2, 16, 38) while TFIIS releases polymerase stalled at intrinsic pause sites (40, 42). Elongin was suggested to facilitate the proper positioning of the 3'-hydroxyl terminus of the nascent transcript with respect to the catalytic site of RNA polymerase II to increase the rate of RNA chain elongation (52). We speculate that the SPT4-SPT5 complex works through a different mechanism by actually decreasing the rate of transcriptional elongation while at the same time preventing the collapse of the RNA polymerase II into a "dead-end" configuration that might happen during the "leap" coinciding with the polymerase leaving an intrinsic pause site (6). This would explain why SPT4-SPT5 can function as both positive and negative transcription factors depending on the experimental conditions. Since the rate of transcriptional elongation is usually assessed by the amount of transcript that reaches a certain point, it is difficult to distinguish between an elongation factor that causes polymerase to increase RNA synthesis and a factor that allows polymerase to move more slowly but extend past intrinsic pause sites such as nucleosomes (1, 5, 50, 51, 57). This model would agree with genetic data indicating that mutations in the two largest subunits of the RNA polymerase II complex, which reduce the efficiency of elongation, can suppress conditional mutations in SPT5.

The existing model of SPT5 action (54) suggests that the sole function of P-TEFb is to relieve the repression caused by SPT4-SPT5. P-TEFb-mediated CTD phosphorylation may prevent SPT4-SPT5 from interacting with RNA polymerase II. Our results indicate that P-TEFb also phosphorylates SPT5 directly and that this phosphorylation is critical for SPT5 function. Thus, P-TEFb, like another CTD kinase, TFIIH, is able to stimulate the activity of other transcriptional regulators (44). The ability of SPT4-SPT5 to stimulate transcription in the absence of DRB suggests that phosphorylation by P-TEFb not only overcomes its negative effect on transcription but also converts it into a positive factor. We propose that phosphorylation of the CTR1 domain in SPT5 may result in recruitment of additional positive regulators of transcriptional elongation. This hypothesis is also supported by the observation that DRB does not function in transcription assay mixtures containing purified RNA polymerase II, recombinant SPT4-SPT5, and P-TEFb (54). In addition, our preliminary results suggest the existence of additional proteins that specifically interact with the phosphorylated CTR1 and function to stimulate transcriptional elongation in the absence of DRB. Further studies are necessary to better understand the mechanism by which SPT5 modulates transcriptional elongation.