INTRODUCTION

The octamer element (ATGCAAAT) has been shown to be one of the major determinants for B cell-specific promoter and enhancer function both in vivo (1, 2) and in vitro (3, 4). Surprisingly, the same element has been implicated as a critical motif in a number of other genes that include ubiquitously expressed small nuclear RNA genes, the cell cycle-regulated histone H2B gene, and VP16-dependent herpes simplex virus (HSV)¹ immediate early genes (reviewed in Refs. 5 and 6). Cognate octamer binding factors include the ubiquitous Oct-1 and a B cell-restricted Oct-2, which both contain a conserved DNA-binding domain designated the POU domain (7, 8). However, activation of octamer-containing promoters through Oct-1 requires additional promoter-specific cofactors. For example, the B cell-specific coactivator OCA-B, the ubiquitous PTF, and the HSV-encoded VP16 are necessary for the maximum activation of immunoglobulin promoters, small nuclear RNA genes, and HSV immediate early genes, respectively. These additional factors interact with the POU domains of Oct-1 and Oct-2 (9-13).

To identify other factors that modulate Oct-1 activity, we cloned additional POU domain-interacting proteins using a yeast two-hybrid screen. One of several isolates was identified as MAT1, a subunit of CDK-activating kinase (CAK). CAK was originally identified as a kinase that activates CDKs in vitro by phosphorylating a conserved threonine residue in the T-loop of CDKs (14). CAK consists of MAT1 along with CDK7 and cyclin H; MAT1 acts to assemble the latter two subunits to form a stable active kinase containing all three subunits (15-17). Some populations of CAK exist as part of the general transcription factor TFIIH (18-20). CAK-containing TFIIH phosphorylates the C-terminal domain (CTD) of the largest subunit of RNA polymerase II, and this phosphorylation has been implicated in promoter clearance and transcriptional elongation (Refs. 18-21; Ref. 22 and references therein; reviewed in Ref. 23). Here, we show that MAT1 directly binds to the POU domains of several Oct factors and, consequently, enhances their phosphorylation by CAK.

EXPERIMENTAL PROCEDURES

The expression vector for 6HisT7MAT1 was constructed as follows. First, 6HisT-pET21b was constructed by ligating the larger fragment of NdeI-PstI-digested 6HisT-pET11d (24) and the smaller fragment of NdeI-PstI-digested pET21b (Novagen). The coding region of MAT1 was amplified by polymerase chain reaction with primers (5-CCTTGAATTCCATGGACGATCAGGGTTG-3 (upstream) and 5-CCGGGTCGACTTATAAATGGTTAACTGGGCT-3 (downstream)) from the I.M.A.G.E. Consortium cDNA clone (identification number 230976), which contains the whole coding region of MAT1. The amplified fragment was digested with EcoRI and SalI and cloned into the EcoRI and SalI sites of 6HisT-pET21b. The resulting vector (pHT7MAT1) was used to express MAT1 containing N-terminal hexahistidine and T7 epitope tags. A hybrid gene encoding the GAL4 DNA-binding domain (amino acids 1-147), the hemagglutinin epitope tag, and the POU domain of Oct-1 was constructed in the yeast bait vector pCENCYH as follows. First the larger PvuI fragment of the yeast multiple copy vector pAS2, which contains the GAL4 DNA-binding domain (25), was ligated with the larger PvuI fragment of pRS314, which contains CEN6/ARSH4 and TRP1 (26), to regenerate the -lactamase gene. The resulting plasmid, pCENCYH is a low copy number plasmid in yeast. The POU domain (amino acids 279-438) of Oct-1 was amplified by polymerase chain reaction from pBSoct1 (27) with an NdeI site in the N terminus and a termination codon followed by a BamHI site in the C terminus. This NdeI-BamHI fragment was cloned into the NdeI-BamHI sites of pCENCYH to obtain pAI11.

The expression vector for FLAG-Oct-1 (pFOct-1) was constructed as follows. Oligonucleotide-directed mutagenesis (28) was carried out to introduce an NdeI site in the N terminus (nucleotides 54 to 49) and a BglII site after the termination codon (nucleotides 2232-2237) of Oct-1 in pBSOct-1 (27). The NdeI-BglII fragment containing the full-length Oct-1 was swapped with the NdeI-BamHI fragment of TFIIE in pF:TFIIE-11d (29) to generate pFOct-1.

pAI11 was transformed into the Y190 yeast strain (25). The expression of the GAL4-(1-147)-POU-1 hybrid protein was verified by Western blotting with anti-hemagglutinin monoclonal antibody 12CA5 (Babco) (data not shown). This yeast was transformed by a B cell cDNA library in pACT (30) and plated on SC medium lacking tryptophan, leucine, and histidine (containing 25 mM 3-aminotriazole). His⁺ colonies exhibiting -galactosidase activity using the filter lift assay (31) were restreaked onto SC medium plates lacking tryptophan and leucine. 39 colonies that exhibited -galactosidase activity reproducibly were further characterized. Library plasmids were recovered by transforming leucine-deficient Escherichia coli MH4 (leuB, (lac)X74, galU, galK, hsr, hsm⁺, strA) with total DNA prepared from these colonies. Transformants were identified on minimal medium lacking leucine and containing ampicillin. To ensure that the correct cDNAs were identified, isolated library plasmids were individually cotransformed with pAI11 into Y190, and only four library plasmids were found to reproduce -galactosidase activity. To check the specificity of interaction of these clones with the POU domain, Y190 was cotransformed with the library plasmids and pLAM5, which encodes a human lamin C fused to a GAL4 DNA-binding domain (CLONTECH). Since the four positive plasmids failed to give -galactosidase activity, the sequences of the inserts were determined by dideoxynucleotide sequencing using Sequenase 2.0 according to the manufacturer's instructions (U.S. Biochemical Corp.).

GST and GST-POU domain fusion proteins (10) were expressed in E. coli and purified by glutathione-Sepharose (Pharmacia Biotech Inc.). The amount of proteins bound to the resin was determined by SDS-PAGE using BSA (Boehringer Mannheim) as a standard.

[³⁵S]Methionine-labeled 6HisT7MAT1 and luciferase were transcribed and translated in vitro from pHT7MAT1 and the luciferase control DNA, respectively, by using the TnT system (Promega) according to the manufacturer's instructions. Six micrograms of GST-POU proteins or 8 µg of GST protein bound to 10 µl of glutathione-Sepharose in 210 µl of BC100 buffer (20 mM Tris-HCl (pH 7.9), 20% (v/v) glycerol, 0.2 mM EDTA, 100 mM KCl, 0.25 mM PMSF, 0.1% (v/v) Nonidet P-40, 10 mM 2-mercaptoethanol) containing 0.2 mg/ml BSA were used for each binding experiment. One microliter (each) of reticulocyte lysate containing expressed proteins was added to initiate binding. After 1 h of incubation at room temperature, the resin was washed extensively with BC100 buffer at 4 °C. Proteins retained on the resin were analyzed by SDS-PAGE after elution with SDS-loading buffer.

Similarly, 3.7 µg of GST-POU-1 or GST-POU-2 proteins or 7.2 µg of GST protein bound to 3 µl of glutathione-Sepharose in 70 µl of BC100 buffer containing 0.2 mg/ml BSA were used for binding experiments with 30 µl of HeLa nuclear extract (8.4 mg of protein/ml) in BC100 buffer. After 17 h of incubation at 4 °C, resins were washed extensively with BC100 buffer. Retained proteins were eluted with SDS-loading buffer, separated by SDS-PAGE, blotted onto a polyvinylidene difluoride membrane, probed with appropriate antibodies, and visualized by the ECL system (Amersham Corp.).

6HisT7MAT1 was expressed in E. coli BL21(DE3)/pLysS after induction by isopropyl -D-thiogalactopyranoside. Cells were disrupted in binding buffer (20 mM Tris-HCl (pH 7.9 at 4 °C), 500 mM NaCl, 0.1% (v/v) Nonidet P-40, 20% (v/v) glycerol, 0.25 mM PMSF) by sonication, and the insoluble fraction containing most of 6HisT7MAT1 was collected by centrifugation. Proteins were solubilized in binding buffer containing 6 M guanidine hydrochloride and incubated at room temperature with nickel-nitrilotriacetate resin (Qiagen) preequilibrated with the same buffer. After extensive washes with binding buffer containing 6 M guanidine hydrochloride and 0.1 mM ZnSO₄, the protein on the resin was renatured by serial dilution of the guanidine hydrochloride with equal volumes of binding buffer containing 0.1 mM ZnSO₄, such that the final concentration of guanidine hydrochloride was 3, 1.5, 0.75, 0.375, and finally 0.1875 M. Resin was incubated in each diluted buffer for at least 10 min at room temperature and then transferred to 4 °C after the concentration of guanidine hydrochloride was 0.75 M. After washes with binding buffer containing 0.1 mM ZnSO₄ and then binding buffer with 20 mM imidazole and 0.1 mM ZnSO₄, the protein was eluted by adding binding buffer with 200 mM imidazole and 0.1 mM ZnSO₄. Dithiothreitol was added at a final concentration of 1 mM to the eluate.

A complex of CDK7 and cyclin H (bipartite CAK) was prepared as follows. 20 flasks (150 cm²) of monolayer high five cells (Invitrogen), maintained in Grace's medium supplemented with 10% fetal bovine serum with 70-80% confluency, were infected simultaneously with recombinant baculoviruses that produce CDK7 or cyclin H for 48 h at 27 °C. To harvest, the cells were centrifuged at 180 × g for 12 min followed by washing one time with ice-cold PBS. The cell pellet was then resuspended in 20 ml of hypotonic buffer (10 mM Tris-HCl (pH 7.4), 25 mM NaCl, 1 mM EDTA, 5 mM dithiothreitol, 0.1 mM PMSF, 1 µg/ml antipain, and 1 µg/ml leupeptin) and lysed with 20 strokes of a Dounce homogenizer. After centrifugation at 38,000 × g at 4 °C, the resulting supernatant (60 mg of protein) was directly loaded onto a Q-Sepharose column (1.0 × 1.3 cm, 1.5 ml) preequilibrated with buffer A (25 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.01% Nonidet P-40, 1 mM dithiothreitol, 10% glycerol, 0.1 mM PMSF, 0.2 µg/ml antipain, and 0.1 µg/ml leupeptin) plus 0.025 M NaCl. After washing the column with 30 ml of the equilibration buffer, the bound protein was eluted with 40 ml of a linear gradient from 0.025 to 0.4 M NaCl in buffer A. The peak of CDK7-cyclin H complex, monitored by silver staining following SDS-PAGE, was eluted around 0.18 M NaCl. The pooled CDK7-cyclin H fractions were then diluted 2-fold with buffer A prior to chromatography onto a heparin-Sepharose column (1.0 × 2.5 cm, 2 ml) preequilibrated with buffer A plus 0.1 M NaCl. After washing the column with 100 ml of the equilibration buffer, the bound protein was eluted with 40 ml of a linear gradient from 0.1 to 1 M NaCl in buffer A. The CDK7-cyclin H complex was eluted at around 0.35 M NaCl with a yield of ~2 mg with more than 90% purity as judged by silver staining following SDS-PAGE.

FLAG-Oct-1 was expressed in E. coli BL21(DE3)/pLysS harboring pFOct-1 after induction by isopropyl -D-thiogalactopyranoside. The expressed protein was purified as described by Chiang and Roeder (29). GST-CDK2 and cyclin A were expressed and purified from bacteria as described (32).

In addition to the buffer components introduced by the addition of kinase and substrates, the components of the kinase buffer were 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 1 mM dithiothreitol, 100 µg/ml BSA, 50 µM ATP, and 30 µCi [-³²P]ATP. Reactions were incubated for 1 h at 30 °C and stopped by adding EDTA. Phosphorylated proteins were precipitated with trichloroacetic acid, dissolved in SDS sample buffer, and analyzed by SDS-PAGE and autoradiography. For phosphopeptide analyses of 6HisPOU-1, reaction conditions were the same as above except for the use of 2.5 µM ATP and 150 µCi of [-³²P]ATP (1.25 µM ATP). 20 ng of 6HisPOU-1 was phosphorylated by 15 ng of the bipartite CAK with 10 ng of MAT1 or 0.05 units of protein kinase A (New England Biolabs) for 2 h at 30 °C. For phosphopeptide analyses of FLAG-Oct-1, 600 µCi of [-³²P]ATP (5 µM ATP) was used. 50 ng of FLAG-Oct-1 was phosphorylated by 15 ng of bipartite CAK with 10 ng of MAT1 or 2 units of Cdc2/cyclin B (New England Biolabs) for 2 h at 30 °C. Quantitation was performed with a PhosphorImager and ImageQuant version 1.1 software (Molecular Dynamics).

Phosphopeptide analysis was performed as described by Roberts et al. (33).

RESULTS

To isolate possible cofactors modulating Oct-1 activity, screening with the yeast two-hybrid system was employed (34). Since several cofactors are already known to interact with Oct-1 through the POU domain (POU-1), this domain was used as a bait for the screening. Yeast cells that express the GAL4-(1-147)-POU-1 fusion protein were transformed with a GAL4 activation domain-tagged human B cell cDNA library. Out of 11.3 × 10⁷ transformants, about 150 were His⁺ and lacZ⁺. Out of these, 38 colonies were selected, and plasmids containing cDNAs were recovered by transforming E. coli. Four plasmids could reproduce the -galactosidase activity by cotransforming the yeast with the GAL4-(1-147)-POU-1 plasmid. The inability of these plasmids to direct activation of the lacZ reporter gene by itself or with a GAL4-(1-147)-lamin plasmid (an unrelated control GAL4 plasmid) suggested that the interactions were specific. Therefore, these four plasmids were sequenced and were found to encode four different proteins (designated PIP-1, -2, -3, and -4 for POU-interacting proteins). The nucleotide sequence of PIP-4 revealed that this clone contains a sequence encoding approximately of the carboxyl terminus of the recently cloned CAK assembly factor, MAT1. MAT1 is also a subunit of the general transcription factor TFIIH (Fig. 1A; Refs. 15, 17, and 35).

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To show that the interaction detected in the yeast two-hybrid assay is due to a specific interaction between MAT1 and the Oct-1 POU domain (POU-1), in vitro binding studies were performed. The POU domains of Oct-1, Oct-2, and Oct-3 were fused to GST (GST-POU-1, GST-POU-2, and GST-POU-3, respectively), expressed in bacteria, and immobilized on glutathione-Sepharose beads for analysis of MAT1 interactions. A full-length MAT1 clone was obtained from an expressed sequence tag clone and subcloned for expression in the reticulocyte lysate system. MAT1 was synthesized in vitro in the presence of [³⁵S]methionine and incubated with the different POU domain affinity resins. After washing, the bound proteins were eluted with SDS and analyzed by SDS-PAGE. As shown in Fig. 2A, MAT1 was preferentially retained by the GST-POU-1, GST-POU-2, and GST-POU-3 affinity resins, while no detectable MAT1 was retained by the resin containing GST alone. It has been argued that inclusion of ethidium bromide in the binding assay allows a distinction between a bona fide protein-protein interaction and interactions due to contaminating nonspecific bridging DNA (36). Binding of MAT1 to GST-POU proteins was unaffected by the presence of up to 100 µg/ml ethidium bromide, strongly arguing in favor of bona fide direct protein-protein interactions between MAT1 and POU domains (Fig. 2B). Moreover, the inability of a nonrelated control protein, luciferase, to be retained by any of the resin-bound GST proteins further indicates the specificity of these interactions (Fig. 2A).

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Since MAT1 is a subunit of CAK, it was of interest to test whether CAK can phosphorylate interacting POU domains. For this purpose, bipartite CAK containing stoichiometric amounts of CDK7 and cyclin H was prepared from baculovirus-coinfected insect cells, and the influence of MAT1 on the phosphorylation of histidine-tagged POU-1 (6HisPOU-1) by bipartite CAK was examined. The addition of MAT1 to the bipartite CAK enhanced the kinase activity on 6HisPOU-1 in a concentration-dependent manner, up to 30-fold (Fig. 3A, lanes 7-10). Maximum phosphorylation was observed when an approximately stoichiometric amount (10 ng) of MAT1 was added to the bipartite CAK (Fig. 3A, lane 10), suggesting that recombinant MAT1 is fully active and incorporated into the bipartite CAK. When MAT1 levels were increased above the stoichiometric amount, the kinase activity decreased (data not shown); this presumably reflects binding of free MAT1 to 6HisPOU-1 and sequestration of the substrate from the tripartite CAK containing CDK7, cyclin H, and MAT1. Control assays showed that phosphorylation of 6HisPOU-1 was dependent upon bipartite CAK (Fig. 3A, lane 6 versus lane 10) as well as MAT1 and that autophosphorylation of CDK7 was only weakly stimulated by MAT1 (Fig. 3A, lanes 4, 5, and 7-10).

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To examine whether bipartite CAK can also phosphorylate full-length Oct-1 and whether this activity is also enhanced by the addition of MAT1, bacterially expressed FLAG-tagged full-length Oct-1 (FLAG-Oct-1) was used as a substrate. The addition of a stoichiometric amount of MAT1 to the bipartite CAK enhanced phosphorylation of FLAG-Oct-1 approximately 300-fold (Fig. 3B), suggesting that MAT1 is indispensable for CAK activity on full-length Oct-1. Consistent with the effect of MAT1 on phosphorylation of POU-1 and Oct-1 by CAK, as well as the physical interaction results in Fig. 2, A and B, MAT1 also enhanced phosphorylation of GST-POU-2 and GST-POU-3 by bipartite CAK (Fig. 3C). GST alone was not phosphorylated by CAK under the same conditions (data not shown).

Two other known substrates for CAK, the C-terminal domain of the large subunit of RNA polymerase II and CDK2, were also tested. Phosphorylation of GST-CTD by bipartite CAK was increased about 12-fold by MAT1 (Fig. 3D), while phosphorylation of GST-CDK2, the original substrate used to identify and purify CAK, was not further enhanced by the addition of MAT1 (Fig. 3E). Consistent with this, MAT1 did not affect the bipartite CAK-mediated activation of cyclin A-CDK2 to phosphorylate histone H1 (data not shown). The observed effects of MAT1 on the phosphorylation of CTD and CDK2 by bipartite CAK agree with recent results reported by Yankulov and Bentley (37). These results indicate that although CAK can phosphorylate a variety of substrates, MAT1 can further stimulate this phosphorylation on only a subset of these targets. This indicates that MAT1 is acting in a substrate-specific manner.

Earlier studies of Oct-1 during progression through the cell cycle revealed a complex temporal program of phosphorylation. In particular, Oct-1 is hyperphosphorylated during mitosis, and DNA binding of Oct-1 is blocked by mitosis-specific phosphorylation of Ser³⁸⁵ in the POU homeodomain by an unidentified cell cycle-regulated kinase (33, 38). Therefore, it was important to determine whether CAK can phosphorylate Ser³⁸⁵ in vitro. Since protein kinase A was shown to phosphorylate Ser³⁸⁵ in vitro (38), 6HisPOU-1 phosphorylated by protein kinase A was used as a marker in phosphopeptide analysis. As shown in Fig. 4A, thermolytic peptide maps from 6HisPOU-1 phosphorylated by CAK clearly differ from those generated with protein kinase A, indicating that CAK does not phosphorylate Ser³⁸⁵ in vitro.

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Somewhat unexpectedly, the tryptic peptide map of FLAG-Oct-1 phosphorylated by CAK (Fig. 4B, left panel) appeared very similar to the tryptic peptide map of FLAG-Oct-1 phosphorylated in vitro by a p13-associated Cdc2-related protein kinase (Fig. 4B, middle panel; Ref. 33). Moreover, analysis of a mixture of the two sets of tryptic phosphopeptides showed that six of the CAK-phosphorylated peptides comigrated with six of the Cdc2-phosphorylated peptides (Fig. 4B, right panel). The dependence on MAT1 of FLAG-Oct-1 phosphorylation by CAK (Fig. 3B) rules out the possibility that the overlapping CAK and Cdc2 phosphopeptide maps result from contaminating Cdc2 kinase present in our CAK preparation. Significantly, a prior analysis of the Cdc2-labeled phosphopeptides showed that one of the six common phosphopeptides (indicated by horizontal arrowheads in Fig. 4B) comigrates with an in vivo labeled mitosis-specific phosphopeptide (33). These results suggest that Oct-1 may be a physiological substrate of CAK or a CAK-related kinase. Since the reported CAK recognition sites in CDKs ((R/M) X (Y/L)(S/T) XXV), as well as the consensus Cdc2 recognition site ((S/T) PX(R/K); Ref. 39), differ significantly from the CAK recognition site in the CTD (YSPTSPS; Ref. 40), the results presented here raise the possibility that these kinases may nonetheless have an overlapping specificity, at least in vitro.

Since MAT1 is also a subunit of TFIIH and since the CTD of the large subunit of RNA polymerase II is a known substrate for the CAK within TFIIH, we tested whether POU domains can bind to TFIIH. As described above, GST and GST-POU-1 proteins were immobilized on glutathione-Sepharose beads and employed as affinity resins. After incubation of the resins with HeLa nuclear extract, followed by extensive washes, bound proteins were separated by SDS-PAGE and visualized by immunoblot with appropriate antibodies. As shown in Fig. 5, various subunits of TFIIH (MAT1, CDK7, cyclin H, p62, and ERCC3) were retained by immobilized GST-POU-1 (lanes 3) but not by immobilized GST (lanes 4). The failure to observe any significant retention of TFIIE, RAP74, or TFIIB on GST-POU-1 (lanes 3) indicates the specificity of the TFIIH interaction. Similar interactions were observed when nuclear extract was incubated with GST-POU-2 and when chromatographic fractions enriched in TFIIH were incubated with GST-POU-1 and GST (data not shown). Inclusion of 100 µg/ml ethidium bromide in the binding buffer did not affect the retention of TFIIH subunits, indicating that this retention is due to direct protein-protein interactions (data not shown).

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DISCUSSION

The present results demonstrate that MAT1 can interact directly with isolated POU domains, that it can stimulate phosphorylation by CDK7-cyclin H of Oct-1 and isolated POU domains (including, apparently, at least one physiological site) as well as isolated CTD repeats, and that the POU domain selectively interacts with TFIIH relative to other general initiation factors. These results have implications for the regulation of CAK substrate specificity and function, the modulation of Oct-1 functions, and the modulation of basic initiation and elongation mechanisms.

The previously reported substrates of CAK were restricted to the CDKs, the CTD of the large subunit of RNA polymerase II, and TBP (reviewed in Refs. 41 and 42). It is notable, therefore, that the DNA-binding Oct factors also fall within this group and are phosphorylated (minimally) in the POU domains in a MAT1-dependent manner. The much greater effect on CAK-mediated phosphorylation of Oct-1 (300-fold) than on CAK-mediated phosphorylation of the POU domain (30-fold) may reflect a conformational change that allows increased phosphorylation or it may reflect the increased number of phosphorylation sites available on the full-length molecule.

Bipartite CDK7-cyclin H and tripartite CDK7-cyclin H-MAT1 complexes have been purified from vertebrates (15-17). However, it is not clear whether both species exist in vivo or whether they arise artifactually during purification, although bipartite CAK can be reconstituted from recombinant proteins (43, 44). In addition, two groups have identified an essential yeast protein that functions as a CAK and lacks polypeptides homologous to vertebrate CDK7, cyclin H, and MAT1 (45, 46), while another group has reported that the yeast kinase KIN28, which is homologous to CDK7, functions only in transcription and not as a CAK (47). This has led to the proposal that the CDK7-cyclin H-MAT1 complex identified as the predominant CAK in vertebrate cell extracts may function primarily during transcription (as part of TFIIH) and not as a physiological CAK (45). Our finding of the Oct family of transcription factors as new substrates of CAK further supports this view and, minimally, suggests a novel MAT1-regulated function of CDK7-cyclin H in transcription regulation. Our finding that MAT1 greatly enhances phosphorylation by CDK7-cyclin H further supports the idea that MAT1 may provide a regulatory mechanism for selective substrate phosphorylation. A similar suggestion was made by Yankulov and Bentley (37) on the basis of their observation (also confirmed here) that MAT1 switches the substrate specificity of CDK7-cyclin H to favor the RNA polymerase II CTD over CDK2.

Since the POU domains of Oct factors comprise the DNA-binding domains, the observed MAT1-dependent POU domain phosphorylation events could regulate site-specific DNA binding on cognate target genes. However, while the DNA binding ability of Oct-1 is blocked during mitosis by phosphorylation (38), the site critical for this inhibition (Ser³⁸⁵ in the POU homeodomain of Oct-1) is not phosphorylated by CAK in vitro. On the other hand, the observed phosphorylation by CAK could modulate the function of coactivators known to interact with Oct-1 or Oct-2 through the POU domain. These include the HSV-encoded VP16 (6), the B cell-specific OCA-B implicated in immunoglobulin promoter function (5, 10), the ubiquitous PTF implicated in small nuclear RNA gene transcription (11), and a presumptive coactivator for histone H2B transcription (10). It also has been reported that Oct-1 and Oct-2 facilitate preinitiation complex formation and that Oct-2 may act at an early stage in this process (48, 49). Consistent with this conclusion, Zwilling et al. (50) showed that TBP can bind to the POU domains of Oct-1 and Oct-2. On the other hand, our finding that POU domains interact directly with MAT1 raises the possibility that Oct factors may also affect a late stage of preinitiation complex formation by recruiting TFIIH to octamer-containing promoters. This possibility is further supported by our observation that TFIIH, but not TFIIE or TFIIF, selectively binds Oct-1 and Oct-2 POU domains. This result is consistent with earlier reports of direct activator interactions with TFIIH (22, 51, 52).

Since a TFIIH helicase is essential for an early step in the initiation process (Refs. 53 and 54; reviewed in Ref. 55), an Oct-1-MAT1 interaction also could facilitate initiation per se. Significantly, however, some promoters may require CTD phosphorylation by the CAK component of TFIIH for promoter clearance (Refs. 56 and 57; reviewed in Ref. 55) and/or enhanced elongation events (22, 58). It has been speculated that such events might result in the release of constraining interactions of the CTD with TBP (59), TFIIE (60), or possibly other cofactors (reviewed in Refs. 23 and 61); TFIIH-mediated phosphorylation of TBP, TFIIE, and TFIIF has also been reported (37, 41, 62). By analogy, the phosphorylation of Oct factors by TFIIH might facilitate postinitiation events by releasing Oct-TFIIH interactions; alternatively, POU interactions with MAT1 could regulate other TFIIH interactions and functions.

A final important question (see above) is whether the CAK functions that result from MAT1 interactions with Oct-1 take place in the context of free CAK or (as observed for phosphorylation of TFIIE, RAP74, and the intact CTD within RNA polymerase II (see Ref. 41 and references therein)), in the context of TFIIH.