The Human Homologue of Drosophila TRF-proximal Protein Is Associated with an RNA Polymerase II-SRB Complex*

Mammalian RNA polymerase II holoenzymes are large complexes that have been reported to contain, in addition to RNA polymerase II, homologues of several yeast SRBs, various general transcription factors, and other polypeptides. On the basis of its copurification with an SRB-containing RNA polymerase II complex by conventional chromatography procedures, we have identified a human homologue of Drosophila TRF-proximal protein, designated hTRFP, and isolated its cognate cDNA. Antibody specific for SRB7 can immunoprecipitate hTRFP and RNA polymerase II and, reciprocally, antibody specific for hTRFP can immunoprecipitate RNA polymerase II and SRB7. These data indicate that hTRFP is an integral component of an RNA polymerase II-SRB complex. Whereas the precise function of hTRFP remains to be determined, the hTRFP-containing RNA polymerase II-SRB complex supports basal level transcription and, relative to RNA polymerase II alone, enhances transcriptional activation by Gal4-VP16 in the presence of cofactor PC4. Thus, hTRFP may regulate transcription of class II genes through association with the RNA polymerase II-SRB complex.

Mammalian RNA polymerase II holoenzymes are large complexes that have been reported to contain, in addition to RNA polymerase II, homologues of several yeast SRBs, various general transcription factors, and other polypeptides. On the basis of its copurification with an SRB-containing RNA polymerase II complex by conventional chromatography procedures, we have identified a human homologue of Drosophila TRF-proximal protein, designated hTRFP, and isolated its cognate cDNA. Antibody specific for SRB7 can immunoprecipitate hTRFP and RNA polymerase II and, reciprocally, antibody specific for hTRFP can immunoprecipitate RNA polymerase II and SRB7. These data indicate that hTRFP is an integral component of an RNA polymerase II-SRB complex. Whereas the precise function of hTRFP remains to be determined, the hTRFP-containing RNA polymerase II-SRB complex supports basal level transcription and, relative to RNA polymerase II alone, enhances transcriptional activation by Gal4-VP16 in the presence of cofactor PC4. Thus, hTRFP may regulate transcription of class II genes through association with the RNA polymerase II-SRB complex.
Eukaryotic RNA polymerase II holoenzymes have been purified from yeast and mammalian cells by a variety of procedures (reviewed in Refs. 1 and 2). The eukaryotic RNA polymerase II holoenzyme (3) was first isolated from yeast extract as a multicomponent complex containing RNA polymerase II, TFIIB, TFIIF, TFIIH, and a group of cofactors (SRBs) originally identified by genetic analysis (4). Another yeast holoenzyme lacking all general initiation factors except TFIIF was found to contain RNA polymerase II in association with a subset of SRBs, the regulatory factors GAL11, ROX3, SIN4, and RGR1, and a novel group of MED proteins (5)(6)(7)(8)(9)(10). These RNA polymerase II-associated proteins form a stable complex, designated mediator, that can be dissociated from RNA polymerase II by anti-CTD 1 antibodies and that supports transcriptional activation with RNA polymerase II (5). Two mediator subcomplexes have been described, one consisting of RGR1, GAL11, SIN4, SRB7, MED1, MED3, MED4, MED7, MED8, and MED9, and another consisting of MED6, ROX3, SRB2, SRB4, SRB5, and SRB6 (10,11).
The function of SRBs in transcription was initially indicated by the isolation, in yeast, of SRB mutants that suppressed an RNA polymerase II mutant containing only a portion of the C-terminal heptapeptide repeat domain of the largest subunit (4,12). SRB4 is required for expression of most class II gene in yeast, suggesting that it has a general function in regulating transcription (13,14). On the other hand, SRB2 and SRB5 are dispensable for cell viability, indicating that they have specific functions in the regulation of certain nonessential genes (13,15). Similarly, certain MED proteins are necessary for transcription of most genes whereas others are required only for specific genes (10,11). Therefore, the yeast SRB-mediator complex may play a number of important roles in transcriptional regulation.
Dependent upon the specific purification procedures, RNA polymerase II holoenzymes from mammalian cells vary in size from 2 to 4 MDa (reviewed in Refs. 1 and 2). In general, these holoenzyme preparations have been found to variably contain various SRBs and general transcription factors (16 -18), as well as a variety of other proteins that include elongation factor SII (19), RNA processing factors (20), RNA helicase (21), factors involved in DNA repair and recombination (18), histone acetyltransferases CBP and PCAF (22), Tat cofactors (23,24), components of the SWI-SNF complex (25), and the tumor suppressor BRCA1 (26). This has led to the proposal that the regulation of transcription during growth and differentiation of mammalian cells may involve multiple RNA polymerase II holoenzymes. Apart from the above mentioned proteins, a number of unidentified polypeptides also co-purified with mammalian holoenzymes. Here we describe the purification of a human RNA polymerase II holoenzyme by a combination of conventional chromatographic steps and affinity chromatography with an antibody specific for SRB7. We identified a component of the holoenzyme as a homologue of Drosophila TRF-proximal protein (27), indicating that TRF proximal protein (hTRFP) may be involved in the regulation of transcription via association with RNA polymerase II holoenzyme.

EXPERIMENTAL PROCEDURES
Protein Purification-100 ml of HeLa nuclear extract in BC100 were subjected to chromatography on phosphocellulose (P11) as described (29). 40 ml of the 0.5 M KCl fraction were loaded onto a hydroxylapatite column (15 ml), which was washed with 0.1 M phosphate buffer (pH 7.9) and step eluted first with 0.2, 0.3, and 1 M phosphate buffers (pH 7.9) and then with 0.5 M phosphate buffer (pH 6). The 0.5 M phosphate buffer (pH 6) fraction was then subjected to chromatography on a Sephacryl S-500 column (140 ml). The peak fraction was then immunoprecipitated with anti-SRB7 and anti-RPB6 antibodies coupled to Protein A beads (40 l) (33). * This work was supported by National Institutes of Health Grants AI37327 and CA42567 (to R. G. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AF097725.
‡ These two authors contributed equally to this work. § Supported in part by a fellowship from the National Institutes of Health.
For large scale purification for polypeptide sequence analysis, 100 ml of nuclear extract in BC300 were loaded directly onto an affinity column (2 ml) containing immobilized anti-SRB7 antibody. The column was washed with BC300 and BC1000 and subsequently eluted with 0.1 M glycine buffer (pH 2.5). The eluates were concentrated by trichloroacetic acid precipitation, and proteins were separated by SDS-PAGE and transferred onto a PVDF membrane. The proteins on the membrane were visualized by Ponceau S staining. The 28-kDa polypeptide was excised and digested with endoproteinase Lys-C. Peptides were isolated by high pressure liquid chromatography and subjected to amino acid sequence analysis. Derived sequences were used to identify an EST clone that was sequenced by the Rockefeller University core facility.
Antibody Preparation-Expression plasmids pRSET-his-hTRFP, pR-SET-his-SRB7, and pRSET-his-CDK8 were constructed by inserting the full-length cDNA (amplified by polymerase chain reaction) into the plasmid pREST-6his (33). This created, in each case, an NdeI site at the N terminus and a BamHI (BglII for CDK8) site at the C terminus. His-tagged hTRFP, His-tagged SRB7, and His-tagged CDK8 were used to prepare antibody as described previously (33). All antibody affinity columns were prepared with antigen-purified antibodies as described (33). Anti-RPB6 was provided by Dr. Zhengxin Wang.

RESULTS AND DISCUSSION
Purification of a Human RNA Polymerase II-SRB Complex-A HeLa nuclear extract was fractionated by conventional chromatographic steps according the scheme in Fig.  1A, with the holoenzyme being monitored by immunoblot with antibodies against RNA polymerase II and the previously described SRB7 component (17,18) of human holoenzyme. On the phosphocellulose column most of RNA polymerase II and SRB7 were detected in the 0.5 M KCl step fraction. On the subsequent hydroxylapatite column, RNA polymerase II was detected in the 0.2, 0.3, and 1 M phosphate buffer (pH 7.9) fractions and in the 0.5 M phosphate buffer (pH 6) fraction. SRB7 and CDK8 were detected mainly in the 1 M phosphate (pH 7.9) and the 0.5 M phosphate buffer (pH 6) fractions. Because RNA polymerase II and SRB7 were present in a more highly purified state in the 0.5 M phosphate buffer (pH 6) fraction, which contained only about 5% of total input protein, this fraction was subjected to chromatography on a gel filtration column. RNA polymerase II and SRB7 appeared to copurify in fractions corresponding to a size of about 2 MDa. The peak fractions of RNA polymerase II and SRB7 were pooled, and equal portions were subjected to independent immunoprecipitations with antibody specific for SRB7 and with antibody for the RPB6 subunit of RNA polymerase II. Each antibody immunoprecipitated a common population of about 30 polypeptides (Fig. 1B), and the presence of SRB7 and RNA polymerase II in both immunoprecipitates was verified by immunoblot analysis with anti-SRB7 and anti-RPB1 antibodies (data not shown). The fact that each antibody immunoprecipitated most of the major polypeptides in the input gel filtration fraction reflects the high degree of purification (about 400-fold) of SRB7 and RNA polymerase II at this step, whereas the few input proteins that were not precipitated indicate the specificity of the immunoprecipitation. A direct comparison of the anti-SRB7 immunoprecipitate with highly purified core RNA polymerase II revealed several polypeptides, specifically immunoprecipitated with anti-SRB7, with the same molecular weight as the subunits of RNA polymerase II (Fig. 1C). As a further control to show that the major group of polypeptides was specifically precipitated by anti-SRB7 antibody, control beads containing only protein A and beads containing protein A and bound anti-SRB7 antibody were used to immunoprecipitate proteins directly from HeLa nuclear extract. SRB7 and RNA polymerase II, as well as the 28-kDa protein (see below), were specifically detected in the anti-SRB7 immunoprecipitates by Western blot analysis and silver staining (data not shown).
Identification of Human TRF-proximal Protein-Because the antibody against SRB7 was previously reported to be able to immunoprecipitate the holoenzyme (17), an anti-SRB7 antibody affinity column was used to purify the holoenzyme on a   (Fig. 1B) was excised from the PVDF membrane and digested with protease. Three polypeptide sequences, SVQQTVELLTR, QGTFCVDCETYHTAAS, and XXQVPVA-GIR, were obtained. These peptide sequences were used as queries to search the NCBI data base of expressed sequence tags with the TBLASTN homology searching program. All three peptides were found in an open reading frame encoded by an EST cDNA clone (accession number 531746). This clone was obtained from I.M.A.G.E., and the DNA sequence of the 0.8kilobase pair insertion was determined. The complete open reading frame encodes a 209-amino acid protein that has 66% sequence similarity and 44% sequence identity with Drosophila TRF-proximal protein, whose cognate cDNA was found upstream of the TRF gene (Fig. 2) (27).
Association of Human TRF-proximal Protein with an RNA Polymerase II-SRB Complex-To investigate the functional role of hTRFP, the full-length protein was expressed in bacteria, purified, and used for polyclonal antibody production. An-tibody specific for hTRFP, antibody specific for CDK8 (a holoenzyme component equivalent to yeast SRB10) as a positive control, and purified rabbit antibody (from preimmune serum) as a negative control were used for immunoprecipitation. As shown in Fig. 3A, the anti-CDK8 (lane 4) and anti-hTRFP (lane 3) antibody, but not preimmune serum (lane 2), specifically precipitated both RNA polymerase II (RPB1 subunit) and SRB7. This result, together with a reciprocal experiment in which hTRFP was immunoprecipitated with anti-SRB7 antibody (as evident from Fig. 1B and from the purification protocol that generated the hTRFP employed for direct sequence analysis (see above)), indicates that hTRFP is associated with RNA polymerase II and SRBs in vivo.
To further determine whether hTRFP is present in a preassembled protein complex containing RNA polymerase II and SRBs, the hydroxylapatite fraction was subjected to gel filtration on Sephacryl S-500, and derived fractions were immunoblotted with antibodies directed against hTRFP, the RPB1 subunit of RNA polymerase II, SRB7, and MED7. As shown in Fig. 3B, four polypeptides are colocalized in fractions (peak fraction 33) corresponding to a size of about 2 MDa, consistent with the idea that they are present in a large complex.
Effect of the hTRFP-and SRB-containing RNA Polymerase II Complex on in Vitro Transcription-Because it was previously shown that SRB-containing yeast holoenzyme and mediator complexes are able to support transactivation (5) (reviewed in Ref. 1), we tested whether the hTRFP-containing RNA polymerase II-SRB complex could support both basal and activated transcription. Because our purified hTRFP-and SRB-containing RNA polymerase II complex appears not to contain any general transcription factors as judged by Western blot (data not shown), an in vitro system reconstituted with ectopic general transcription factors was used to assay transcription. To compare the activity of the hTRFP-containing RNA polymerase II complex with that of the 12-subunit core RNA polymerase II, the amount of the complex assayed was adjusted to contain the same amount of RNA polymerase II as the core RNA polymerase II preparation that was assayed (based on quantitative immunoblot assays with anti-RPB1 antibody; data not shown). As shown in Fig. 4, the assay system has little or no basal transcriptional activity without RNA polymerase II (lanes 1  and 2), and the hTRFP-containing RNA polymerase II-SRB complex can substitute for core RNA polymerase II in effecting basal transcription from a minimal promoter (lanes 3 and 4). This complex could not support transcriptional activation by Gal4-VP16 in the absence of the USA cofactor fraction (28) or the recombinant PC4 (29) (lanes 3 and 4). However, addition of either the USA cofactor fraction (lanes 7 and 8)  Each reaction contained all basal transcription factors and two DNA templates (30). As judged by Western blot analysis with anti-RPB1 antibodies, the hTRFP-and SRB7-containing RNA polymerase II and core RNA polymerase II preparations added to the reaction contained equivalent amounts of RNA polymerase II. Reactions were carried out in the presence or absence of activator Gal4-VP16 and in the presence of PC4 or USA as indicated. Arrows indicate the RNA transcripts from the specific DNA templates. New Component of RNA Polymerase II Holoenzyme 3939 and 6) to the reactions enhanced activated transcription by Gal4-VP16. In contrast, at the equivalent concentration tested, the core RNA polymerase supported a low level of basal transcription (lanes 9 and 10) but was not able to support transcriptional activation in the presence of recombinant PC4 (lanes 11 and 12). Consistent with previous data (28,29), the core RNA polymerase II did support transcriptional activation in the presence of the USA cofactor fraction (lanes 13 and 14). This latter result may reflect the presence in the partially purified USA fraction not only of other co-activators, including PC2 (30), but also substantial amounts of SRB7, CDK8, and hTRFP (data not shown). Thus, SRB and mediator components in USA may synergize with other endogenous coactivators (including PC4 and PC2), as observed for purified PC4 and holoenzyme components, to effect high level activation in conjunction with core RNA polymerase II. As observed previously (28,29), The present results demonstrate that hTRFP is a component of an RNA polymerase II-SRB complex that can synergize with coactivators derived from the USA fraction to enhance transcriptional activation. Our results are also consistent with a recent report by Kornberg and colleagues (31) of a mouse cell extract-derived mediator complex that contains SRBs, MED proteins, and other polypeptides. The amino acid sequence of a 28-kDa polypeptide in the mouse complex matched the amino acid sequence of hTRFP, indicating that a mouse TRFP is present in the mediator complex. Additionally, a human SRBand MED-containing cofactor complex that mediates activated transcription by RNA polymerase II has recently been purified in our laboratory by affinity methods (32), and this complex has also been found to contain hTRFP. 2 The conservation of TRFP among Drosophila, mouse, and man indicates that TRFP may have a specific role in regulation of transcription. Because of our inability to significantly deplete hTRFP from nuclear extract with anti-TRFP antibody, the role of hTRFP in transcription remains unknown. However, because deletion of the gene encoding TRFP in Drosophila results in larval (but not embryonic) lethality (27), it is unlikely that TRFP is a general transcriptional cofactor for transcriptional activation in Drosophila. Thus, it is possible that hTRFP is involved in regulation of transcription of specific gene(s) and that it functions via the RNA polymerase II-SRB complex.