INTRODUCTION

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The regulation of transcription initiation is mediated by the interplay between two classes of promoter elements: the basal promoter elements, which can be defined as those promoter elements sufficient to direct basal levels of transcription in vitro, and the regulatory elements, which modulate the levels of transcription. The basal elements are recognized by basal transcription factors, whereas the regulatory elements are recognized by either transcriptional activators or repressors. Eucaryotic activators are often modular, consisting of a DNA binding domain, which targets the activator to the correct promoter, and of activation domains, whose role is to enhance transcription (see references 21-23, 32, and 33 for reviews).

The human snRNA gene family contains both RNA polymerase II and RNA polymerase III genes. The RNA polymerase II snRNA promoters consist of a proximal sequence element (PSE), which is sufficient to direct basal levels of transcription in vitro, and a distal sequence element, which activates basal transcription. The RNA polymerase III snRNA promoters are similar, except that basal transcription is directed by the combination of a PSE and a TATA box (reviewed in reference 9). The PSE is recognized by a multisubunit complex called the SNAP complex (SNAP_c) (7) or PTF (34). Since SNAP_c can bind to the PSE on its own, it corresponds to a sequence-specific DNA binding basal transcription factor. SNAP_c contains at least four subunits, SNAP43, SNAP45, SNAP50, and SNAP190, and cDNAs encoding the SNAP43 (7) or PTF  (35), SNAP45 (24) or PTF  (35), and SNAP50 (6) or PTF  (2) subunits have been isolated. Cross-linking studies suggest that SNAP50 (6) and the largest subunit of the complex, SNAP190 (PTF ) (34), are in close contact with DNA. Recombinant SNAP50, however, does not bind to the PSE (6); thus, how SNAP_c recognizes its target sequence is not yet understood.

Although SNAP_c is capable of binding to the PSE on its own, its binding is strongly enhanced by the concomitant binding of at least two factors. On a basal RNA polymerase III promoter, containing both a PSE and a TATA box, SNAP_c binds cooperatively with TBP, and this effect is dependent on the amino-terminal domain of TBP (16). And on DNAs containing an octamer site and a PSE, SNAP_c binds cooperatively with the Oct-1 or Oct-2 POU domain (17) but not with the Pit-1 POU domain (15). Of all the amino acid differences between the Oct-1 and Pit-1 POU domains, a single one is the key determinant for the differential abilities of these two proteins to recruit SNAP_c to the PSE (15). This effect contributes to efficient transcription in vitro and is largely independent of the Oct-1 activation domains, indicating that a function that is the hallmark of activation domains, namely, recruitment of a basal transcription complex resulting in activation of transcription, can be performed by a DNA-binding domain (3, 15). However, it remains to be determined whether cooperative binding results from a direct Oct-1 POU-SNAP_c interaction and which subunit in SNAP_c is contacted by the Oct-1 POU domain.

Here, we report the isolation of a cDNA encoding a 1,469-amino-acid protein that corresponds to full-length SNAP190. SNAP190 is an unusual Myb domain protein. Unlike most Myb domains from animal cells and plant cells, which contain three and two repeats, respectively, (see references 11 and 14 for reviews), SNAP190 contains a half repeat (Rh) followed by four complete repeats, Ra, Rb, Rc, and Rd. We show that just the Rc and Rd repeats are capable of recognizing the PSE, suggesting that the SNAP190 Myb domain contributes to specific binding of the SNAP complex to the PSE. SNAP190 interacts with the SNAP45 subunit of SNAP_c and with activator Oct-1. Our observations suggest that the SNAP complexes required for RNA polymerase II and III snRNA gene transcription contain at least four common subunits, provide further insight into the architecture of the complex, and identify SNAP190 as a target for activation by Oct-1.

	MATERIALS AND METHODS

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Purification of SNAP190 and isolation of corresponding cDNAs. The biochemical purification of SNAP_c has been described previously (7). We also purified SNAP_c by immunoprecipitation. An S100 extract from HeLa cells was first fractionated by ammonium sulfate precipitation. The material precipitating between 18 and 32% ammonium sulfate was resuspended in buffer D (20 mM HEPES [pH 7.9], 0.2 mM EDTA, 15% glycerol, 3 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride) containing 100 mM KCl (D₁₀₀), dialyzed against the same buffer, and loaded on a P11 (Whatman) phosphocellulose column (8 mg of protein/ml of packed resin). The column was washed with 3 column volumes of buffer D₁₀₀ and then step eluted with 5 column volumes of buffer D₃₅₀, followed by five column volumes of buffer D₅₀₀. Then, 80 ml of the 350 to 500 mM KCl elution step mixture (P11-C fraction) was directly used in nondenaturing immunoprecipitations with 4 ml of protein A-agarose beads coupled to an anti-SNAP43 antibody (-CSH375 antibody) (7). The beads were washed extensively with HTMG₁₀₀ (20 mM HEPES [pH 7.9], 15% glycerol, 0.1% Tween 20, 5 mM MgCl₂, 100 mM KCl), and the bound material was then eluted twice in 12 ml of HTMG₆₀ containing 0.5-mg/ml specific peptide, CSH375, against which the antibody was raised. The eluate was further fractionated on a 200-µl Q-Sepharose column, and the fractions were tested in an electrophoretic mobility shift assay (EMSA) with a probe containing the mouse U6 PSE. The proteins in the fractions containing DNA binding activity were precipitated with trichloroacetic acid, redissolved in Laemmli buffer, and fractionated on a 5% sodium dodecyl sulfate (SDS)-polyacrylamide gel. The protein band migrating with an apparent molecular mass of 190 kDa was excised from the gel and subjected to amino acid sequencing as described previously (7). Amino acid sequence information obtained both from protein purified by immunoaffinity and from protein purified by conventional chromatography (7) was used to design degenerate oligonucleotide primers for use in PCRs, with cDNA prepared from total HeLa cell RNA as the template. A p190-specific 156-nucleotide (nt) fragment (encoding amino acids [aa] E242 to K294) was obtained.

Generation of antipeptide antibodies. Synthetic peptides derived from the predicted amino acid sequence for SNAP190 (see Fig. 1) were coupled to keyhole limpet hemocyanin (Pierce) and injected into rabbits to generate polyclonal antipeptide antibodies. Rabbit antisera were tested in an EMSA as previously described (25).

Immunodepletions and in vitro transcription assays. Rabbit preimmune or anti-SNAP190 antibodies were covalently cross-linked to protein A-agarose beads (5). All immunodepletions were performed for 30 min at room temperature in 1.5-ml Eppendorf tubes. For U6 snRNA, VAI, and AdML transcription experiments, immunodepletions were done with 100 µl of whole-cell extracts (13) depleted with an equal volume of preimmune antibody beads, preimmune and anti-SNAP190 antibody beads mixed at ratios of 2:1 and 1:2, or only anti-SNAP190 antibody beads. Depletions were also performed with the largest amount of anti-SNAP190 antibody beads, which had been preincubated with 10 µg of a nonspecific peptide (CSH483) (6) or a specific peptide (190-3 peptide). For U1 snRNA transcription experiments, 40 µl of whole-cell extract was depleted with 20 µl of antibody beads while maintaining the same bead ratios as those described above. Immunodepletion reaction mixtures were centrifuged for 1 min at room temperature, and the supernatants were transferred to fresh 1.5-ml Eppendorf tubes. The supernatants were immediately tested in in vitro transcription experiments. Eight, 4, 7, and 18 µl of extract were used for U6 snRNA, VAI, AdML, and U1 snRNA transcription reactions, respectively. To test for the effects of dilution of the extract by antibody beads, whole-cell extract or whole-cell extract diluted two- and fourfold in buffer D was tested. Reconstitution of transcription was tested by the addition of 2, 4, and 8 µl of a SNAP_c-enriched fraction (mono-Q fraction; approximately 0.3 mg of protein/ml) (7) to transcription reaction mixtures; the reactions were performed with extract treated with the highest level of anti-SNAP190 antibody beads.

Expression of recombinant proteins and coimmunoprecipitations. The SNAP190 coding sequence was subcloned into the pCITE-2a(+) vector (Novagen) to generate construct pCITE-SNAP190. Three micrograms of pCITE-SNAP190 and 1 µg of pCITE-SNAP45 (25) were used as the template for coupled in vitro transcription and translation (Promega) in a final volume of 50 µl containing 4 µl of L-[³⁵S]methionine (1,233 Ci/mmol; New England Nuclear), either in separate reactions or in cotranslation reactions. For immunoprecipitation control experiments, 10 µl of each individually labeled protein was combined with 190 µl of 20 mM HEPES (pH 7.9)-15% glycerol-0.1% Tween 20-5 mM MgCl₂-1 mM dithiothreitol-0.5 mM phenylmethylsulfonyl fluoride containing 100 mM KCl (HMGT₁₀₀) and incubated at 4°C for 2 h. Then, 10 µl of protein A-Sepharose beads coupled to either anti-SNAP190 antibody (antibody 402) or anti-SNAP45 antibody (-CSH467; rabbit 234) was added, and the reaction mixtures were incubated at 4°C for 1 h. The antibody beads were then washed extensively with HMGT₁₀₀, and the bound proteins were eluted by boiling the beads in Laemmli buffer. For coimmunoprecipitation experiments, 3, 10, or 30 µl of cotranslated labeled proteins was used and the reaction mixtures were adjusted to a 200-µl final volume with HMGT₁₀₀. The samples were then processed exactly as described above. The eluted proteins were fractionated by SDS-12.5% polyacrylamide gel electrophoresis and visualized by autoradiography. Similar experiments were performed with in vitro-translated SNAP43 (7) and SNAP50 (6), but no interactions with SNAP190 could be detected.

Expression of SNAP190 Myb repeats in E. coli. Fragments corresponding to aa 390 to 518 (RcRd), 283 to 414 (RaRb), 283 to 518 (RaRbRcRd), 283 to 572 (RaRbRcRd and Arg-rich and Ser-rich regions), 330 to 518 (RbRcRd), 238 to 518 (RhRaRbRcRd), and 238 to 572 (RhRaRbRcRd and Arg-rich and Ser-rich regions) of SNAP190 were generated by PCR amplification with Pfu polymerase (Stratagene). PCR fragments corresponding to aa 390 to 518, 283 to 414, 283 to 518, and 283 to 572 were ligated into a pET-GST vector (8) to generate constructs pET-GST-190RcRd, pET-GST-190RaRb, pET-GST-190RaRbRcRd, and pET-GST-190RaRbRcRdSer, respectively. PCR fragments corresponding to aa 330 to 518, 238 to 518, and 238 to 572 were ligated into a modified pSBET vector (27) containing a glutathione S-transferase (GST)-coding sequence to generate constructs pSBET-GST-190RbRcRd, pSBET-GST-190RhRaRbRcRd, and pSBET-GST-190RhRaRbRcRdSer, respectively. GST fusion proteins were expressed in Escherichia coli BL21-DE3 (30), and lysates were prepared as described previously (31). Fusion proteins were bound to glutathione-agarose (Sigma), the beads were washed with phosphate-buffered saline containing 0.1% Nonidet P-40 and 10% glycerol, and the bound proteins were eluted with 10 mM glutathione in 50 mM Tris, pH 8.8. Where indicated, the GST moiety was cleaved by treatment with thrombin. All the proteins were analyzed by SDS-polyacrylamide gel electrophoresis. The various proteins were then tested for binding to wild-type and mutated PSEs in an EMSA as described previously (25). The EMSA whose results are shown in in Fig. 7C was performed as described in reference 29.

Nucleotide sequence accession number. The nucleotide sequence obtained in this study has been assigned GenBank accession no. AF032387.

	RESULTS

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Isolation of cDNAs encoding p190. In our original purified SNAP_c preparation, three prominent polypeptides with apparent molecular masses of 43, 45, and 50 kDa were readily visible. These polypeptides have all been cloned and are indeed part of the SNAP complex (6, 7, 24). In addition, however, a polypeptide with an apparent molecular mass of about 190 kDa was clearly visible (7). We used an antibody raised against SNAP43 (7) to confirm by coimmunoprecipitation assays that the 190-kDa polypeptide (p190) is associated with SNAP_c (data not shown) and to purify the protein. We obtained p190 peptide sequences from both SNAP_c purified by conventional chromatography (7) and immunopurified SNAP_c. With this information, we designed degenerate oligonucleotides for PCR from cDNA prepared from total cellular RNA and obtained a p190-specific PCR fragment. We also identified a partial p190-encoding cDNA in a one-hybrid screen performed with Saccharomyces cerevisiae (29) (see below).

View larger version (52K): [in this window] [in a new window]   View larger version (44K): [in this window] [in a new window]   FIG. 1.   Amino acid sequence and schematic structure of SNAP190. (A) Amino acid sequence of SNAP190. The shaded regions correspond to peptide sequences obtained from the purified protein. The dashed arrow corresponds to the Myb half repeat, Rh, and the other arrows indicate Myb repeats Ra, Rb, Rc, and Rd, with the conserved tryptophan (or phenylalanine in the Ra repeat and tyrosines in Rb and Rc repeats) indicated in boldface. The arginine-rich and serine-rich regions are boxed. The leucines spaced as in a leucine zipper are indicated by asterisks. The synthetic peptides used to generate antibodies in rabbits are underlined: peptide 190-1 (aa 1452 to 1469) gave antibodies 398 and 399a; peptide 190-2 (aa 1309 to 1328) gave antibodies 400 and 401; peptide 190-3 (aa 843 to 865) gave antibodies 402 and 403; and peptide 190-4 (aa 712 to 735) gave antibodies 439 and 440. (B) Schematic structure of p190. The location of the Myb domain with the half repeat (Rh) and the four complete repeats (Ra, Rb, Rc, and Rd), as well as the locations of the arginine-rich, serine-rich, and leucine zipper-like regions, are indicated. The region of the protein that interacts with Oct-1 in a yeast one-hybrid assay is indicated by a bracket. (C) Alignment of the p190 half Myb repeat, Rh, and repeats Ra, Rb, Rc, and Rd and Myb repeats R1, R2, and R3 from the human A-Myb, B-Myb (19), and c-Myb (12, 28) proteins. The conserved tryptophans (replaced in some cases by tyrosines or phenylalanines) are indicated in boldface. In this alignment, the p190 Ra repeat is 17, 25, and 22% identical to the Myb R1, R2, and R3 repeats, respectively; the p190 Rb repeat is 14, 23, and 23% identical to the Myb R1, R2, and R3 repeats, respectively; the p190 Rc repeat is 27, 38, and 23% identical to the Myb R1, R2, and R3 repeats, respectively; and the p190 Rd repeat is 21, 38, and 30% identical to the Myb R1, R2, and R3 repeats, respectively.

p190 contains a Myb DNA binding domain. As shown in Fig. 1A and B, the p190 sequence contains several striking features. The protein contains an unusual Myb domain, immediately followed by an arginine-rich region and a serine-rich region, and in the carboxy-terminal region of the protein is a leucine zipper-like motif. The Myb domain similarity extends from the tryptophan at position 263 to the glutamine at position 503.

p190 corresponds to the largest subunit of SNAP_c. To characterize the function of p190, and in particular to determine whether p190 is indeed part of the SNAP complex, we raised antibodies against four peptides (Fig. 1A) and used the antibody raised against amino acids 843 to 865 (antibody 402) for the experiments described in this work. We first performed an immunoblot analysis. Recombinant p190 translated in vitro was fractionated alongside a SNAP_c-containing phosphocellulose P11 column fraction on an SDS-polyacrylamide gel, the proteins were transferred to nitrocellulose, and p190 was visualized with the anti-p190 antibody (antibody 402). As shown in Fig. 2, the recombinant protein comigrated with endogenous p190, even though its calculated molecular mass is only 159 kDa. This is consistent with the cDNA encoding a full-length p190 protein and indicates that p190 migrates anomalously on an SDS-polyacrylamide gel.

View larger version (46K): [in this window] [in a new window]   FIG. 2.   Recombinant p190 comigrates with SNAP190. Three (lane 1), 10 (lane 2), and 30 (lane 3) µl of a SNAPc-containing P11C fraction (7) and 5 (lane 4), 10 (lane 5), and 30 (lane 6) µl of reticulocyte lysate programmed with the p190 cDNA or a mixture of 10 µl of the P11C fraction and 10 µl of p190 cDNA-programmed reticulocyte lysate (lane 7) were fractionated on an SDS-5% polyacrylamide gel. The proteins were transferred to nitrocellulose, and the filter was immunoblotted with the anti-p190 402 antibody. Unprogrammed reticulocyte lysate gave no signal (data not shown). The positions of molecular weight markers and of SNAP190 in the P11C fraction are indicated.

View larger version (46K): [in this window] [in a new window]   FIG. 3.   p190 corresponds to the SNAP190 subunit of SNAPc. (A) EMSA performed with a probe containing the wild-type mouse U6 PSE (lanes 1 and 3 to 8) or a mutated mouse U6 PSE (ABC mutation [25];lane 2), a fraction highly enriched in SNAPc (mono-Q fraction) (7), and either no antibodies (lanes 1 and 2), 1 µl of preimmune antibodies (lane 3), or 1 µl of anti-p190 (402) antibodies (lanes 4 to 8). Lanes 5 and 6 also contained 1 and 3 µg, respectively, of specific peptide (peptide 190-3), and lanes 7 and 8 contained 1 and 3 µg, respectively, of nonspecific peptide (peptide CSH375). The locations of the free probe and the complexes containing SNAPc or SNAPc and anti-p190 antibodies (SNAPc + Ab) bound to the PSE are indicated. (B) Beads coated with either preimmune (lanes 3 to 6) or anti-p190 (402) (lanes 7 to 10) antibodies were incubated with the SNAPc-enriched mono-Q fraction. The bound material was then eluted either without (lanes 3, 4, 7, and 8) or with (lanes 5, 6, 9, and 10) a specific peptide (peptide 190-3) and used in an EMSA with a probe containing the wild-type mouse U6 PSE. For lanes 1 and 2, the EMSA was performed with a probe containing the wild-type (lane 1) or mutated (lane 2) mouse U6 PSE and the SNAPc-enriched mono-Q fraction.

SNAP190 is required for RNA polymerase II and III transcription of snRNA genes. Fractions enriched in the SNAP complex are required for RNA polymerase II and III transcription of snRNA genes, suggesting that SNAP_c is part of the initiation complexes assembled on both the RNA polymerase II and III snRNA promoters. It is possible, however, that the RNA polymerase II and III snRNA promoters recruit different versions of the SNAP complex containing, for example, different sets of subunits. We tested the involvement of SNAP190 in RNA polymerase II and III transcription from the four types of promoters depicted in Fig. 4A: the adenovirus 2 (Ad2) major-late promoter exemplifies a typical TATA box-containing RNA polymerase II mRNA promoter, the U1 and U6 promoters exemplify RNA polymerase II and III PSE-containing snRNA promoters, respectively, and the Ad2 VAI promoter exemplifies an RNA polymerase III promoter with gene-internal elements.

View larger version (19K): [in this window] [in a new window]   FIG. 4.   SNAP190 is required for RNA polymerase II and III transcription from snRNA promoters. (A) Schematic representation of the four promoters used for in vitro transcription. (B) SNAP190 is required for human snRNA gene transcription. For the AdML, VAI, and U6 snRNA gene transcription experiments, 100 µl of HeLa whole-cell extract was depleted with either 100 µl of rabbit preimmune antibody beads (lane 4), 67 µl of preimmune antibody beads plus 33 µl of anti-SNAP190 antibody (antibody 402) beads (lane 5), 33 µl of preimmune antibody beads plus 67 µl of anti-SNAP190 antibody beads (lane 6), or 100 µl of anti-SNAP190 antibody beads (lanes 7 to 12). In lanes 8 and 9, the anti-SNAP190 antibody beads were first incubated with an excess of a nonspecific (CSH483) and a specific (peptide 190-3) peptide, respectively. In lanes 10 to 12, 2, 4, and 8 µl, respectively, of the SNAPc-enriched mono-Q fraction were added to transcription reaction mixtures. Lanes 1 to 3 show transcription performed with undiluted extract (lane 1), extract diluted 1:2 with buffer D (lane 2), and extract diluted 1:4 with buffer D (lane 3). Lane 2 is directly comparable to lanes 4 to 12. The U1 snRNA gene transcription was sensitive to the effects of dilution (lanes 2 and 3); therefore, 40 µl of whole-cell extract was depleted with 20 µl of antibody beads by using the same ratio of preimmune to anti-SNAP190 antibody beads as that described above. The bands corresponding to correctly initiated RNA are labeled AdML for the Ad2 major-late promoter, U1 5' for the U1 snRNA promoter, U6 5' for the U6 snRNA promoter, and VAI for the Ad2 VAI promoter. The band labeled RT corresponds to transcripts derived from cryptic promoters located within vector sequences (25).

SNAP190 associates with SNAP45. To understand how SNAP190 fits into the architecture of the SNAP complex, we tested whether SNAP190 could associate with any of the previously cloned SNAP_c subunits. SNAP190 was cotranslated in a reticulocyte lysate with either SNAP43, SNAP45, SNAP50, or the three proteins together. We then performed immunoprecipitations with antibodies directed against the various subunits. Figure 5 shows the results for SNAP45. As shown in Fig. 5A, anti-SNAP190 antibodies were able to recognize in vitro-translated SNAP190 (lane 4) but not SNAP45 (lane 8), as expected. However, when increasing amounts of cotranslated SNAP45 and SNAP190 were incubated with the anti-SNAP190 antibody, increasing amounts of SNAP45 were coimmunoprecipitated with SNAP190 (lanes 5 to 7). Reciprocally, as shown in Fig. 5B, anti-SNAP45 antibodies recognized in vitro-translated SNAP45 (lane 4) but not SNAP190 (lane 8). However, when increasing amounts of cotranslated SNAP45 and SNAP190 were incubated with anti-SNAP45 antibodies, increasing amounts of SNAP190 were coimmunoprecipitated with SNAP45 (lanes 5 to 7). Together, these data indicate that SNAP190 and SNAP45 interact strongly with each other. We could not detect, however, any strong interaction between SNAP190 and SNAP43 or SNAP50, and cotranslation of all four subunits did not result in coimmunoprecipitation of SNAP43 or SNAP50 alongside SNAP45 and SNAP190 (data not shown). This raises the question of how the SNAP complex is assembled; this question is discussed below.

View larger version (32K): [in this window] [in a new window]   FIG. 5.   SNAP190 interacts with SNAP45. (A) A cDNA encoding SNAP190 (lanes 1 and 4), SNAP45 (lanes 2 and 8), or a mixture of cDNAs encoding SNAP190 and SNAP45 (lanes 3 and 5 to 7) were expressed by coupled in vitro transcription and translation in reticulocyte lysates. In lanes 1 to 3, 1 µl of the products was loaded directly on the SDS-polyacrylamide gel. In lanes 4 to 8, 10, 3, 10, 30, and 30 µl, respectively, of the products were first incubated with beads coated with the anti-SNAP190 (402) antibody. The beads were washed, and the bound proteins were eluted by boiling the beads in Laemmli buffer and were fractionated on the SDS-polyacrylamide gel. (B) The same experiment as in panel A was performed, except that in lanes 4 to 8, the products were first incubated with beads coated with an anti-SNAP45 antibody (-CSH467; rabbit 234).

The SNAP190 Rc and Rd Myb domain repeats can bind the PSE. The presence of a DNA binding domain sequence in SNAP190 immediately suggested that within the SNAP complex, SNAP190 is involved in recognizing the PSE. To test this hypothesis, we expressed full-length SNAP190 in a baculovirus expression system; we also expressed a number of SNAP190 fragments corresponding to the entire Myb domain or parts of it in E. coli (see Materials and Methods). Of these proteins, only one, consisting of the Rc and Rd repeats, bound to DNA, as shown in Fig. 6A. To test DNA binding we used the two probes shown in Fig. 6B. A GST fusion protein containing the Rc and Rd repeats bound to a wild-type mouse U6 PSE probe but not to a mutated probe carrying six point mutations within the PSE (compare lanes 3 to 5 with lanes 6 to 8). Similarly, an RcRd protein lacking the GST moiety bound specifically to the PSE (compare lanes 10 to 13 with lanes 15 to 18). These results suggest that the Rc and Rd repeats of the SNAP190 Myb domain contribute to recognition of the PSE within the SNAP complex.

View larger version (63K): [in this window] [in a new window]   View larger version (8K): [in this window] [in a new window]   FIG. 6.   DNA binding of the SNAP190 Myb repeats. (A) An EMSA was performed with a probe containing a wild-type (lanes 1, 3 to 5, and 9 to 13) or mutated (lanes 2, 6 to 8, and 14 to 18) mouse U6 PSE and either 2 µl of a fraction enriched in SNAPc (mono-Q fraction; 0.3 mg of protein/ml [7]) (lanes 1 and 2) or 0.1 (lanes 3, 6, 10, and 15), 0.3 (lanes 4, 7, 11, and 16), 1 (lanes 5, 8, 12, and 17) or 3 (lanes 13 and 18) µl of the bacterially expressed proteins indicated above the lanes. In lanes 9 and 14, no proteins were added to the probes. The locations of free probes, complexes containing SNAPc, GST-190RcRd, and 190RcRd are indicated. (B) The sequences of the PSEs present in the wild-type and mutant probes are shown. Uppercase letters correspond to sequences derived from the mouse U6 promoter, with underlined characters corresponding to mutations. Flanking sequences are in lowercase characters. The c-Myb consensus binding site (c-Myb BS) is also indicated.

SNAP190 interacts with Oct-1. SNAP_c and Oct-1 can bind cooperatively to DNA, suggesting protein-protein interactions between these two factors on DNA (15, 17). We therefore checked whether proteins identified in an Oct-1 interaction screen in yeast (29) corresponded to subunits of SNAP_c. In this screen, summarized in Fig. 7A, the parental yeast strain carried, as a selectable marker, an integrated copy of a HIS3 allele with six octamer sites upstream of the TATA element, as well as a plasmid that directs constitutive expression of full-length Oct-1. Because Oct-1 does not activate transcription in yeast, this strain transcribes the HIS3 gene at basal levels and thus does not grow in the presence of aminotriazole (AT), a competitive inhibitor of the HIS3 gene product. This tester strain was transformed with a library of cDNAs fused to the VP16 transcriptional activation domain, and AT-resistant colonies were selected. Cells that grow under these selective conditions may express VP16 hybrid proteins that stimulate HIS3 transcription by being recruited to the HIS3 promoter through their interaction with DNA-bound Oct-1 (29). Indeed, such a screen resulted in the isolation of cDNAs encoding OBF-1 (also called OCA-B [10] and Bob1 [4]), a protein that interacts with octamer-bound Oct-1 (29). As shown below, this type of screen also resulted in the isolation of a cDNA encoding part of SNAP190.

View larger version (26K): [in this window] [in a new window]   FIG. 7.   The carboxy-terminal half of SNAP190 interacts with DNA-bound Oct-1 both in vivo and in vitro. (A) The strategy for isolating proteins interacting with DNA-bound Oct-1 (29) is depicted. An interaction between Oct-1, which is transcriptionally inactive in yeast, and a fusion protein containing the VP16 activation domain results in induction of HIS3 transcription, which in turn allows cells to grow on AT-containing medium. The Oct-1 protein and the VP16 cDNA library were expressed from single-copy plasmids marked with the URA3 and TRP1 genes, respectively. Expression of the VP16 hybrid proteins was placed under control of the galactose-inducible GAL1, 10 regulatory sequences (UASG). (B) Yeast cells expressing Oct-1, SNAP190C, and OBF-1 as indicated above the lanes were plated on medium without AT or containing 40 mM AT. Only cells expressing induced levels of the HIS3 gene product can grow in the presence of AT. (C) EMSA performed with a probe containing an octamer motif and the in vitro-translated proteins indicated above the lanes. The total amount of reticulocyte lysate was kept constant in all lanes. Lane 1 contains unprogrammed reticulocyte lysate only, lanes 2 to 5 contain 1 µl of Oct-1 POU, lane 3 contains in addition 1 µl of OBF-1, and lanes 4 and 5 contain 1 and 2 µl, respectively, of SNAP190C.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

We describe the characterization of a cDNA encoding the largest subunit of the SNAP complex, SNAP190. That SNAP190 is part of SNAP_c was determined by the observations that antibodies directed against SNAP190 retard the migration of the SNAP_c-PSE complex in an EMSA and can immunoprecipitate a protein complex that binds to the PSE and retards the probe indistinguishably from SNAP_c. In addition, depletion of extracts with anti-SNAP190 antibodies inhibits both RNA polymerase II and III transcription from snRNA promoters, as expected for a subunit of SNAP_c. This result, together with the observations that depletions of transcription extracts with anti-SNAP43 (7), anti-SNAP45 (24), and anti-SNAP50 (6) antibodies also inhibit both RNA polymerase II and III transcription from snRNA promoters, indicates that the SNAP complexes involved in RNA polymerase II and III snRNA gene transcription contain at least four common subunits.

Architecture of the SNAP complex. SNAP190 interacts with SNAP45 in a coimmunoprecipitation assay. We have shown before that SNAP43 and SNAP50 can also be coimmunoprecipitated (6). We interpret these observations as reflecting protein-protein contacts that occur within the SNAP complex. Intriguingly, however, we have been unable to show protein-protein contacts between the SNAP190-SNAP45 pair and the SNAP43-SNAP50 pair of proteins by coimmunoprecipitation experiments in either the presence or absence of TBP (unpublished results). Our recent results indicate that this is due to the lack of a small, previously undetected, subunit. In the presence of this additional subunit, a SNAP complex capable of binding specifically to the PSE can be assembled (6a).

SNAP190 contains a Myb DNA binding domain. SNAP190 contains a region with strong similarity to the c-Myb DNA binding domain. However, unlike the c-Myb DNA binding domain, which contains three repeats, the SNAP190 Myb domain contains four repeats and one half repeat. A number of proteins, including the yeast SWI3 and ADA2 proteins, the transcriptional corepressor N-Cor, and the yeast TFC5 subunit of TFIIIB, contain one or two copies of a domain, which has been called the SANT domain, that resembles a Myb repeat (1). Although the SANT domain has been hypothesized to be involved in DNA binding, this has not been shown experimentally. The SNAP190 repeats are, however, much more related to the Myb R1 to R3 repeats than to the SANT domain, consistent with the ability of the SNAP190 Rc and Rd repeats to bind to the PSE.

SNAP190 interacts with Oct-1. One of the mechanisms by which transcriptional activators can activate transcription is by recruiting members of the basal machinery to a promoter. For example, the Drosophila melanogaster activators Bicoid and Hunchback can activate transcription in vitro by recruitment of basal transcription factor TFIID. Recruitment involves protein-protein contacts with two of the TFIID subunits, TAF_II110 and TAF_II60 (26). In the case of snRNA promoters, the DNA binding POU domain of activator Oct-1 can recruit SNAP_c to the PSE (15, 17), and this effect contributes to efficient snRNA gene transcription in vitro (15). We have isolated the region encoding the carboxy-terminal half of SNAP190 in a yeast one-hybrid screen as a DNA-bound Oct-1-interacting protein, and we could show that this region of SNAP190 can form a complex with the Oct-1 POU domain bound to an octamer motif in vitro. Thus, the DNA binding domain of Oct-1 may contribute to transcription activation by contacting the carboxy-terminal half of SNAP190 and so recruiting basal transcription complex SNAP_c to the PSE.