Characterization of a Trimeric Complex Containing Oct-1, SNAPc, and DNA
1997; Elsevier BV; Volume: 272; Issue: 25 Linguagem: Inglês
10.1074/jbc.272.25.16048
ISSN1083-351X
Autores Tópico(s)RNA Interference and Gene Delivery
ResumoThe human small nuclear (sn) RNA promoters contain a proximal sequence element (PSE), which recruits the basal transcription factor SNAPc, and a distal sequence element characterized by an octamer sequence, which recruits the POU domain transcription factor Oct-1. The Oct-1 POU domain and SNAPc bind cooperatively to probes containing a PSE and an octamer sequence, and this effect contributes to efficient transcription in vitro. In vivo, however, Oct-1 regions outside of the POU domain can activate snRNA gene transcription. Here, we have examined whether the role of these regions is to contribute to cooperative binding with SNAPc. We find that they indeed improve cooperative binding, but most of the effect is nevertheless mediated by just the POU domain. This suggests that Oct-1 activates transcription of snRNA genes in at least two steps, recruitment of SNAPc mediated primarily by the POU domain, and a later step mediated by regions outside of the POU domain. We also show that a PSE-binding complex observed in nuclear extracts consists of Oct-1 and SNAPc. Although Oct-1 cannot bind effectively to the PSE probe on its own, in the complex it contacts DNA. Thus, in a nuclear extract, SNAPc can recruit Oct-1 to a probe to which Oct-1 cannot bind on its own. The human small nuclear (sn) RNA promoters contain a proximal sequence element (PSE), which recruits the basal transcription factor SNAPc, and a distal sequence element characterized by an octamer sequence, which recruits the POU domain transcription factor Oct-1. The Oct-1 POU domain and SNAPc bind cooperatively to probes containing a PSE and an octamer sequence, and this effect contributes to efficient transcription in vitro. In vivo, however, Oct-1 regions outside of the POU domain can activate snRNA gene transcription. Here, we have examined whether the role of these regions is to contribute to cooperative binding with SNAPc. We find that they indeed improve cooperative binding, but most of the effect is nevertheless mediated by just the POU domain. This suggests that Oct-1 activates transcription of snRNA genes in at least two steps, recruitment of SNAPc mediated primarily by the POU domain, and a later step mediated by regions outside of the POU domain. We also show that a PSE-binding complex observed in nuclear extracts consists of Oct-1 and SNAPc. Although Oct-1 cannot bind effectively to the PSE probe on its own, in the complex it contacts DNA. Thus, in a nuclear extract, SNAPc can recruit Oct-1 to a probe to which Oct-1 cannot bind on its own. Transcriptional activators are key regulators of RNA polymerase II transcription, but their mode of action is still poorly understood. Activators often consist of a DNA-binding domain, whose role is to target the activator to the correct promoter, and of activation domains, whose role is to enhance transcription (1Ptashne M. Nature. 1988; 335: 683-689Crossref PubMed Scopus (1167) Google Scholar). The activation domains may help recruit members of the basal transcription machinery to the promoter, enhance transcription elongation, or perhaps trigger modifications of the basal machinery that result in enhanced transcription initiation (2Lieberman P.M. Berk A.J. Genes Dev. 1991; 5: 2441-2454Crossref PubMed Scopus (162) Google Scholar, 3Lin Y.-S. Green M.R. Cell. 1991; 64: 971-981Abstract Full Text PDF PubMed Scopus (366) Google Scholar, 4Choy B. Green M.R. Nature. 1993; 366: 531-536Crossref PubMed Scopus (235) Google Scholar, 5Barberis A. Pearlberg J. Simkovich N. Farrell S. Reinagel P. Bamdad C. Sigal G. Ptashne M. Cell. 1995; 81: 359-368Abstract Full Text PDF PubMed Scopus (234) Google Scholar, 6Yankulov K. Blau J. Purton T. Roberts S. Bentley D.L. Cell. 1994; 77: 749-759Abstract Full Text PDF PubMed Scopus (207) Google Scholar) (see Refs. 7Triezenberg S.J. Curr. Opin. Genet. Dev. 1995; 5: 190-196Crossref PubMed Scopus (348) Google Scholar and 8Ranish J.A. Hahn S. Curr. Opin. Genet. Dev. 1991; 6: 151-158Crossref Scopus (62) Google Scholar, for reviews).The RNA polymerase II and III snRNA 1The abbreviations used are: snRNA, small nuclear RNA, PSE; proximal sequence element; PCR, polymerase chain reaction; GST, glutathione S-transferase; Op-Cu, orthophenanthroline-Cu. 1The abbreviations used are: snRNA, small nuclear RNA, PSE; proximal sequence element; PCR, polymerase chain reaction; GST, glutathione S-transferase; Op-Cu, orthophenanthroline-Cu. gene promoters both contain an essential proximal sequence element (PSE), which recruits the basal transcription factor SNAPc (also called PTF) (9Murphy S. Yoon J.-B. Gerster T. Roeder R.G. Mol. Cell. Biol. 1992; 12: 3247-3261Crossref PubMed Scopus (149) Google Scholar, 10Henry R.W. Sadowski C.L. Kobayashi R. Hernandez N. Nature. 1995; 374: 653-657Crossref PubMed Scopus (122) Google Scholar, 11Yoon J.-B. Murphy S. Bai L. Wang Z. Roeder R.G. Mol. Cell. Biol. 1995; 15: 2019-2027Crossref PubMed Scopus (116) Google Scholar), and a distal sequence element, which serves as a transcriptional enhancer and is characterized by the presence of an octamer sequence. The octamer constitutes a binding site for both the Oct-1 and Oct-2 POU domain transcription factors, but the distal sequence element is thought to recruit Oct-1. Indeed, like snRNA genes, Oct-1 is broadly expressed, whereas Oct-2 is a B cell-specific factor (see Ref. 12Cleary M.A. Herr W. Mol. Cell. Biol. 1995; 15: 2090-2100Crossref PubMed Scopus (56) Google Scholar, for a review). Moreover, in vivo, the Oct-1 and Oct-2 activation domains display promoter specific activities; the Oct-1 activation domains preferentially activate snRNA promoters, whereas the Oct-2 activation domains preferentially activate transcription from mRNA promoters (13Das G. Hinkley C.S. Herr W. Nature. 1995; 374: 657-660Crossref PubMed Scopus (100) Google Scholar, 14Tanaka M. Lai J.-S. Herr W. Cell. 1992; 68: 755-767Abstract Full Text PDF PubMed Scopus (188) Google Scholar). This differential activation results from differences in the mRNA and snRNA basal promoter elements, suggesting that the Oct-1 and Oct-2 activation domains interact differentially with promoter-specific basal transcription factors (13Das G. Hinkley C.S. Herr W. Nature. 1995; 374: 657-660Crossref PubMed Scopus (100) Google Scholar).Both the Oct-1 and Oct-2 POU domains bind cooperatively with SNAPc/PTF to a probe containing a PSE and an octamer sequence, and at least in the case of the Oct-1 POU domain, this cooperative binding promotes increased levels of transcription in vitro (9Murphy S. Yoon J.-B. Gerster T. Roeder R.G. Mol. Cell. Biol. 1992; 12: 3247-3261Crossref PubMed Scopus (149) Google Scholar, 15Mittal V. Cleary M.A. Herr W. Hernandez N. Mol. Cell. Biol. 1996; 16: 1955-1965Crossref PubMed Scopus (66) Google Scholar). The observation that in vivo, Oct-1 regions outside of the POU domain activate snRNA gene transcription, and do so much more efficiently than Oct-2 regions outside of the POU domain, suggests that the POU domain is not sufficient for transcription activation in vivo (16Tanaka M. Herr W. Cell. 1990; 60: 375-386Abstract Full Text PDF PubMed Scopus (517) Google Scholar). How do Oct-1 regions outside of the POU domain contribute, then, to transcription activation?Here we have tested whether Oct-1 regions outside of the POU DNA-binding domain play any role in cooperative binding with SNAPc to probes containing a PSE and an octamer sequence. We find that they do contribute to cooperative binding but most of the effect is mediated by the POU domain, suggesting that the Oct-1 activation domains play their primary role at a later step in the activation process. We also show that in crude nuclear extracts, a complex consisting of Oct-1 and SNAPc forms on a probe containing a PSE-binding site but lacking an octamer site. Formation of the complex is dependent on the ability of Oct-1 to bind DNA, and indeed Oct-1 contacts DNA in the complex. Thus, in the very complex mixture of proteins that constitutes a nuclear extract, SNAPc can recruit Oct-1 to a probe to which Oct-1 cannot bind on its own.EXPERIMENTAL PROCEDURESConstructsConstructs for PCR ProbesThe plasmids containing the H2B octamer site and human U6 PSE were previously described (15Mittal V. Cleary M.A. Herr W. Hernandez N. Mol. Cell. Biol. 1996; 16: 1955-1965Crossref PubMed Scopus (66) Google Scholar). The plasmid containing the H2B octamer site was described previously (17Cleary M.A. Stern S. Tanaka M. Herr W. Genes Dev. 1993; 7: 72-83Crossref PubMed Scopus (82) Google Scholar). The plasmids AD (also referred to in the text as mouse U6 PSE probe) and the mouse U6 PSE probe with the ABC mutation were generated by annealing two oligos, filling in with the Klenow fragment of DNA polymerase, cutting with BamHI and HindIII, and inserting into pUC118. This resulted in plasmids containing inserts with the sequences GGATCCGAAACTCACCCTAACTGTAAAGTAATTGTGTTTCTTGGCTTCTCGAGCCTTGTGGAAGCTTAAG and GGATCCGAAACTCCCACTACCGGTCCAGTAATTGTGTTTCTTGGCTTCTCGAGCCTT-GTGGAAGCTTAAG for the AD plasmid and the plasmid containing the mouse U6 PSE with the ABC mutation, respectively. The N7 plasmid has been previously described (18Sadowski C.L. Henry R.W. Lobo S.M. Hernandez N. Genes Dev. 1993; 7: 1535-1548Crossref PubMed Scopus (141) Google Scholar). Probes were generated by PCR amplification of these constructs using the universal sequencing primer end-labeled with [γ-32P]ATP and T4 polynucleotide kinase and the reverse sequencing primer. The probes AD-mutHD and AD-short were generated by PCR with the plasmid AD as a template and primers with the sequences TCACACAGGAAACAGCTATGACCATGACCACGAATTCG and AGCTCGGTACCCGGGGATCC, respectively, substituted for the reverse sequencing primer. All the probes were generated with the same radiolabeled primer and had, therefore, the same specific activity.Expression ConstructsThe pET11c.G.POU-1 and pET11c.G.Pit-1 POU constructs, which contain the Oct-1 POU and Pit-1 POU domains fused to the glutathione S-transferase (GST) gene, were previously described (19Lai J.-S. Cleary M.A. Herr W. Genes Dev. 1992; 6: 2058-2065Crossref PubMed Scopus (116) Google Scholar, 20Aurora R. Herr W. Mol. Cell. Biol. 1992; 12: 455-467Crossref PubMed Scopus (83) Google Scholar). The constructs pET11c.H.Oct-1 and pET11c.H.1.P.1 were generated by PCR amplification of a plasmid containing the Oct-1 coding sequence and pBSoct-1(H)P(Pf)1 (20Aurora R. Herr W. Mol. Cell. Biol. 1992; 12: 455-467Crossref PubMed Scopus (83) Google Scholar), respectively, using oligonucleotides with the sequences GGGAATTCCATATGCATCACCATCACCATCACAACAATCCGTCAGAAACCAG and ACGCGGATCCTCACTGTGCCTTGGAGGC. The PCR products were cleaved withNdeI and BamHI and ligated into pET11c cleaved with NdeI and BamHI.Sources of ProteinsSNAPcThe SNAPc used in these experiments was derived from a Mono Q peak fraction, which corresponds to the fourth step in the purification of SNAPc and is purified approximately 2,500-fold (10Henry R.W. Sadowski C.L. Kobayashi R. Hernandez N. Nature. 1995; 374: 653-657Crossref PubMed Scopus (122) Google Scholar). The total protein concentration in the fraction is approximately 0.3 mg/ml.Expression and Purification of Oct Proteins in Escherichia coliAll proteins were expressed in E. coli BL21 (DE3) cells using the T7 expression system (21Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (5987) Google Scholar) as described previously (20Aurora R. Herr W. Mol. Cell. Biol. 1992; 12: 455-467Crossref PubMed Scopus (83) Google Scholar). The Oct-1 POU, Oct-1 POU R49A, and Pit-1 POU domains were expressed as GST fusion proteins and were purified with glutathione-agarose beads (Sigma). In some cases the GST moiety was removed by cleavage with thrombin and dialyzed against buffer D (20 mm HEPES, pH 7.9, 100 mm KCl, 0.5 mm EDTA, 20% glycerol, 1 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride). Proteins were analyzed by SDS-polyacrylamide gel electrophoresis and visualized by Coomassie Blue staining. In all cases the proteins appeared to be greater than 90% pure. Protein concentrations were measured by the Bio-Rad protein assay (Bio-Rad).Histidine-tagged proteins were produced by growing 1-liter cultures ofE. coli BL21 (DE3) cells expressing histidine-tagged Oct-1 (H.Oct-1) or histidine tagged Oct-1.P.1 (H.Oct-1.P.1) as described above. The cells were lysed by sonication in OctQ buffer (20 mm Tris-HCl, pH 8.0, 100 mm NaCl, 0.1% Tween 20, 5% glycerol, 5 mm 2-mercaptoethanol, 0.5 mm phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin A, 1 mm benzamidine, 2 μg/ml aprotonin, 1 μg/ml leupeptin). Cell lysate was centrifuged at 40,000 × g for 30 min. Supernatant was collected and passed over a Mono Q 10/10 column (Pharmacia). The flow-through fractions were kept and dialyzed against OctQ buffer containing 1 m NaCl. Protein was applied to a 1.5-ml Ni-NTA column (Qiagen) and eluted with a gradient from 0 to 40 mm imidazole. Fractions containing octamer binding activity were then dialyzed against OctQ buffer containing 100 mmNaCl and applied to a Mono S 5/5 column (Pharmacia) and eluted with a salt gradient from 0.1 to 1 m NaCl. Fractions containing octamer binding activity were pooled and dialyzed against buffer D. Protein purity and concentration were assessed by the same method as with the POU domains above except that the protein gels were stained with silver. For both Oct-1 and Oct-1.P.1, a number of truncated proteins were visible below the full-length products. Oct-2 and the Oct-2 POU domain were a generous gift of Dr. Masafumi Tanaka (Cold Spring Harbor Laboratory).Nuclear ExtractsHeLa cell nuclear extracts were prepared as described (22Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9143) Google Scholar).EMSAThe binding reactions were performed in a total volume of 20 μl containing final concentrations of 100 mm KCl, 20 mm HEPES, pH 7.9, 5 mm MgCl2, 0.2 mm EDTA, 10% glycerol, 20 μg of fetal calf serum as a protein carrier, 2 mm dithiothreitol, 0.5 mmphenylmethylsulfonyl fluoride, 0.4 μg each of poly(dI-dC) and pUC118. The amounts of SNAPc and Oct proteins added are indicated in the figure legends. The reactions were incubated at room temperature for 20 min before addition of 25,000 cpm (50–100 pg) of radiolabeled DNA probe followed by a 30-min incubation at room temperature. The reactions were electrophoresed through 5% nondenaturing polyacrylamide gels (acrylamide/bisacrylamide ratio, 39:1) in 1 × TGE running buffer (50 mm Tris base, 380 mm glycine, 2 mm EDTA) at 150 V for 4.5 h at room temperature. The gels were dried and autoradiographed. The intensities of the signals were measured with a Fuji BAS1000 PhosphorImager.OP-Cu FootprintingEMSA reactions containing 16 μl of SNAPc, O.4 μg of His-Oct-1, and 100,000 cpm of DNA probe in a total volume of 80 μl were performed as described above. In-gel footprinting reactions were performed essentially as described (23Sigman D.S. Kuwabara M.D. Chen C.-H.B. Bruice T.W. Methods Enzymol. 1991; 208: 414-433Crossref PubMed Scopus (134) Google Scholar, 24Kristie T.M. Sharp P.A. Genes Dev. 1990; 4: 2383-2396Crossref PubMed Scopus (156) Google Scholar) except that the reactions were performed at 4 °C. The gel was washed with 600 ml of 50 mm Tris, pH 8.0. The wash solution was removed and the gel was immersed in 400 ml of 50 mm Tris, pH 8.0, 40 ml of solution A (2 mm 1,10-phenanthroline, 0.45 mmCuSO4), and 40 ml of solution B (58 mm3-mercaptopropionic acid). The cleavage reaction was allowed to proceed for 13 min and stopped by the addition of 40 ml of 28 mm2,9-dimethyl-1,10-phenanthroline. The gel was incubated for an additional 15 min at 4 °C and 10 min at room temperature. The gel was subject to autoradiography and the bands corresponding to specific protein-DNA complexes, as well as free probe, were excised. Gel slices were eluted overnight in 0.4 ml of 0.1% SDS, 5 mm EDTA, 20 mm Tris, pH 8.0. Eluted DNA was extracted with phenol:chloroform (1:1), precipitated with ethanol, and analyzed on a 6% polyacrylamide (19:1), 8 m urea, 0.5 × TBE sequencing gel. The gel was dried and autoradiographed.DISCUSSIONThe snRNA promoters contain an enhancer, the distal sequence element, which is nearly always characterized by the presence of an octamer sequence as well as, in some cases, an Sp1-binding site (28Hernandez N. Cold Spring Harbor Monogr. Ser. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992: 281-313Google Scholar). This contrasts with the enhancers of mRNA promoters, which differ from one promoter to the next and can consist of a wide variety of protein-binding sites. Consistent with the uniformity of the distal sequence element, basal transcription from snRNA promoters is activated by Oct-1 activation domains and a glutamine-rich activation domain derived from Sp1 (13Das G. Hinkley C.S. Herr W. Nature. 1995; 374: 657-660Crossref PubMed Scopus (100) Google Scholar), but not, in general, by activation domains derived from other activators. For example, the VP16 or Oct-2 activation domains do not activate snRNA gene transcription (13Das G. Hinkley C.S. Herr W. Nature. 1995; 374: 657-660Crossref PubMed Scopus (100) Google Scholar, 14Tanaka M. Lai J.-S. Herr W. Cell. 1992; 68: 755-767Abstract Full Text PDF PubMed Scopus (188) Google Scholar,16Tanaka M. Herr W. Cell. 1990; 60: 375-386Abstract Full Text PDF PubMed Scopus (517) Google Scholar, 29Tanaka M. Grossniklaus U. Herr W. Hernandez N. Genes Dev. 1988; 2: 1764-1778Crossref PubMed Scopus (104) Google Scholar). This selectivity suggests that the Oct-1 activation domains exert their action on a snRNA promoter-specific transcription factor, such as SNAPc.The Oct-1 DNA-binding domain (POU domain) has been shown before to bind cooperatively with SNAPc to a probe containing a PSE and an octamer motif (9Murphy S. Yoon J.-B. Gerster T. Roeder R.G. Mol. Cell. Biol. 1992; 12: 3247-3261Crossref PubMed Scopus (149) Google Scholar, 15Mittal V. Cleary M.A. Herr W. Hernandez N. Mol. Cell. Biol. 1996; 16: 1955-1965Crossref PubMed Scopus (66) Google Scholar). Thus, a possibility is that the role of the Oct-1 activation domains is to reinforce this effect. Indeed, we find that the Oct-1 regions outside of the POU domain contribute to cooperative binding with SNAPc, and the effect is significantly larger than that observed with Oct-2 regions outside of the POU domain. There is, therefore, a correlation between the ability of the Oct-1 and Oct-2 regions outside of the POU domain to recruit SNAPc to the PSE and to enhance snRNA gene transcriptionin vivo (14Tanaka M. Lai J.-S. Herr W. Cell. 1992; 68: 755-767Abstract Full Text PDF PubMed Scopus (188) Google Scholar). Nevertheless, most of the SNAPcrecruitment effect is contributed by the POU domain, suggesting that the main function of the Oct-1 activation domains is different. They may be involved either in recruiting other members of the basal machinery, perhaps other snRNA-promoter specific factors, or, for example, in inducing conformational changes in the basal machinery that result in more efficient transcription.While characterizing a PSE-binding complex observed in crude nuclear extracts, we found that it consists of SNAPc and Oct-1. This was unexpected, because Oct-1 on its own did not bind effectively to the probe, and our previous results had indicated that cooperative binding of Oct-1 and SNAPc to DNA requires the presence of both a PSE and an octamer motif (15Mittal V. Cleary M.A. Herr W. Hernandez N. Mol. Cell. Biol. 1996; 16: 1955-1965Crossref PubMed Scopus (66) Google Scholar). However, we find that within the complex, Oct-1 contacts DNA in a sequence-specific manner, and that this contact is required for formation of the complex. Thus, as we had observed previously, cooperative binding of SNAPc and Oct-1 to DNA requires Oct-1-DNA contacts. Interestingly, the location of these contacts relative to the location of the PSE is flexible and changes on different probes. This is consistent with the observation that neither the distance between the octamer sequence and the PSE (9Murphy S. Yoon J.-B. Gerster T. Roeder R.G. Mol. Cell. Biol. 1992; 12: 3247-3261Crossref PubMed Scopus (149) Google Scholar) nor the orientation of the octamer (15Mittal V. Cleary M.A. Herr W. Hernandez N. Mol. Cell. Biol. 1996; 16: 1955-1965Crossref PubMed Scopus (66) Google Scholar), are critical for cooperative binding.The POU domain consists of two helix-turn-helix-containing DNA-binding structures, the POU homeodomain (POUH) and the POU-specific domain (POUS), joined together by a flexible linker (30Herr W. Sturm R.A. Clerc R.G. Corcoran L.M. Baltimore D. Sharp P.A. Ingraham H.A. Rosenfeld M.G. Finney M. Ruvkun G. Horvitz H.R. Genes Dev. 1988; 2: 1513-1516Crossref PubMed Scopus (599) Google Scholar, 31Assa-Munt N. Mortishire-Smith R.J. Aurora R. Herr W. Wright P.E. Cell. 1993; 73: 193-205Abstract Full Text PDF PubMed Scopus (121) Google Scholar, 32Dekker N. Cox M. Boelens R. Verrijzer C. van der Vliet P.C. Kaptein R. Nature. 1993; 362: 852-855Crossref PubMed Scopus (134) Google Scholar, 33Klemm J.D. Rould M.A. Aurora R. Herr W. Pabo C.O. Cell. 1994; 77: 21-32Abstract Full Text PDF PubMed Scopus (454) Google Scholar, 34Herr W. Cleary M.A. Genes Dev. 1995; 9: 1679-1693Crossref PubMed Scopus (345) Google Scholar). Cooperative binding of the Oct-1 POU domain and SNAPc can be disrupted by a single amino acid change within the POUS domain, which maps to the surface of helix 1 away from the DNA and has no effect on DNA binding (15Mittal V. Cleary M.A. Herr W. Hernandez N. Mol. Cell. Biol. 1996; 16: 1955-1965Crossref PubMed Scopus (66) Google Scholar). This suggests that the POUS domain is involved in direct protein-protein interactions with SNAPc, and that its position may, therefore, be fixed relative to SNAPc and the PSE. We show here that cooperative binding can also be disrupted by a single amino acid mutation within the POUS domain that maps to the surface of helix 3 pointing toward the DNA and that affects DNA binding (12Cleary M.A. Herr W. Mol. Cell. Biol. 1995; 15: 2090-2100Crossref PubMed Scopus (56) Google Scholar). This suggests that the POUS domain also contacts the DNA in the complex. How, then, can there be so much flexibility in the spacing between the PSE and the sequences contacted by Oct-1 POU? Perhaps the POUS domain-DNA contact is transient, occurring only during formation of the trimeric complex, whereas the POUH domain remains bound to DNA in the formed complex. Or perhaps the location of the POUS domain-DNA contact is dictated more by protein-protein interactions with SNAPcthan by specific DNA sequence. In contrast, the position of the POUH may be much more dependent on local DNA sequences than on the position of SNAPc and the PSE. Indeed, the sequence ATT (or AAT on the other strand) constitutes part of the AAAT sequence recognized by the POUH domain on a histone H2B-octamer site (33Klemm J.D. Rould M.A. Aurora R. Herr W. Pabo C.O. Cell. 1994; 77: 21-32Abstract Full Text PDF PubMed Scopus (454) Google Scholar). Thus, perhaps on different probes, the relative locations of the Oct-1 POUS and POUH domains changes, the first being dictated mainly by the location of SNAPc, and the second by the local DNA sequence. Alternatively, SNAPc may itself be flexible, allowing different positionings of Oct-1 on the DNA while maintaining protein-protein contacts. Transcriptional activators are key regulators of RNA polymerase II transcription, but their mode of action is still poorly understood. Activators often consist of a DNA-binding domain, whose role is to target the activator to the correct promoter, and of activation domains, whose role is to enhance transcription (1Ptashne M. Nature. 1988; 335: 683-689Crossref PubMed Scopus (1167) Google Scholar). The activation domains may help recruit members of the basal transcription machinery to the promoter, enhance transcription elongation, or perhaps trigger modifications of the basal machinery that result in enhanced transcription initiation (2Lieberman P.M. Berk A.J. Genes Dev. 1991; 5: 2441-2454Crossref PubMed Scopus (162) Google Scholar, 3Lin Y.-S. Green M.R. Cell. 1991; 64: 971-981Abstract Full Text PDF PubMed Scopus (366) Google Scholar, 4Choy B. Green M.R. Nature. 1993; 366: 531-536Crossref PubMed Scopus (235) Google Scholar, 5Barberis A. Pearlberg J. Simkovich N. Farrell S. Reinagel P. Bamdad C. Sigal G. Ptashne M. Cell. 1995; 81: 359-368Abstract Full Text PDF PubMed Scopus (234) Google Scholar, 6Yankulov K. Blau J. Purton T. Roberts S. Bentley D.L. Cell. 1994; 77: 749-759Abstract Full Text PDF PubMed Scopus (207) Google Scholar) (see Refs. 7Triezenberg S.J. Curr. Opin. Genet. Dev. 1995; 5: 190-196Crossref PubMed Scopus (348) Google Scholar and 8Ranish J.A. Hahn S. Curr. Opin. Genet. Dev. 1991; 6: 151-158Crossref Scopus (62) Google Scholar, for reviews). The RNA polymerase II and III snRNA 1The abbreviations used are: snRNA, small nuclear RNA, PSE; proximal sequence element; PCR, polymerase chain reaction; GST, glutathione S-transferase; Op-Cu, orthophenanthroline-Cu. 1The abbreviations used are: snRNA, small nuclear RNA, PSE; proximal sequence element; PCR, polymerase chain reaction; GST, glutathione S-transferase; Op-Cu, orthophenanthroline-Cu. gene promoters both contain an essential proximal sequence element (PSE), which recruits the basal transcription factor SNAPc (also called PTF) (9Murphy S. Yoon J.-B. Gerster T. Roeder R.G. Mol. Cell. Biol. 1992; 12: 3247-3261Crossref PubMed Scopus (149) Google Scholar, 10Henry R.W. Sadowski C.L. Kobayashi R. Hernandez N. Nature. 1995; 374: 653-657Crossref PubMed Scopus (122) Google Scholar, 11Yoon J.-B. Murphy S. Bai L. Wang Z. Roeder R.G. Mol. Cell. Biol. 1995; 15: 2019-2027Crossref PubMed Scopus (116) Google Scholar), and a distal sequence element, which serves as a transcriptional enhancer and is characterized by the presence of an octamer sequence. The octamer constitutes a binding site for both the Oct-1 and Oct-2 POU domain transcription factors, but the distal sequence element is thought to recruit Oct-1. Indeed, like snRNA genes, Oct-1 is broadly expressed, whereas Oct-2 is a B cell-specific factor (see Ref. 12Cleary M.A. Herr W. Mol. Cell. Biol. 1995; 15: 2090-2100Crossref PubMed Scopus (56) Google Scholar, for a review). Moreover, in vivo, the Oct-1 and Oct-2 activation domains display promoter specific activities; the Oct-1 activation domains preferentially activate snRNA promoters, whereas the Oct-2 activation domains preferentially activate transcription from mRNA promoters (13Das G. Hinkley C.S. Herr W. Nature. 1995; 374: 657-660Crossref PubMed Scopus (100) Google Scholar, 14Tanaka M. Lai J.-S. Herr W. Cell. 1992; 68: 755-767Abstract Full Text PDF PubMed Scopus (188) Google Scholar). This differential activation results from differences in the mRNA and snRNA basal promoter elements, suggesting that the Oct-1 and Oct-2 activation domains interact differentially with promoter-specific basal transcription factors (13Das G. Hinkley C.S. Herr W. Nature. 1995; 374: 657-660Crossref PubMed Scopus (100) Google Scholar). Both the Oct-1 and Oct-2 POU domains bind cooperatively with SNAPc/PTF to a probe containing a PSE and an octamer sequence, and at least in the case of the Oct-1 POU domain, this cooperative binding promotes increased levels of transcription in vitro (9Murphy S. Yoon J.-B. Gerster T. Roeder R.G. Mol. Cell. Biol. 1992; 12: 3247-3261Crossref PubMed Scopus (149) Google Scholar, 15Mittal V. Cleary M.A. Herr W. Hernandez N. Mol. Cell. Biol. 1996; 16: 1955-1965Crossref PubMed Scopus (66) Google Scholar). The observation that in vivo, Oct-1 regions outside of the POU domain activate snRNA gene transcription, and do so much more efficiently than Oct-2 regions outside of the POU domain, suggests that the POU domain is not sufficient for transcription activation in vivo (16Tanaka M. Herr W. Cell. 1990; 60: 375-386Abstract Full Text PDF PubMed Scopus (517) Google Scholar). How do Oct-1 regions outside of the POU domain contribute, then, to transcription activation? Here we have tested whether Oct-1 regions outside of the POU DNA-binding domain play any role in cooperative binding with SNAPc to probes containing a PSE and an octamer sequence. We find that they do contribute to cooperative binding but most of the effect is mediated by the POU domain, suggesting that the Oct-1 activation domains play their primary role at a later step in the activation process. We also show that in crude nuclear extracts, a complex consisting of Oct-1 and SNAPc forms on a probe containing a PSE-binding site but lacking an octamer site. Formation of the complex is dependent on the ability of Oct-1 to bind DNA, and indeed Oct-1 contacts DNA in the complex. Thus, in the very complex mixture of proteins that constitutes a nuclear extract, SNAPc can recruit Oct-1 to a probe to which Oct-1 cannot bind on its own. EXPERIMENTAL PROCEDURESConstructsConstructs for PCR ProbesThe plasmids containing the H2B octamer site and human U6 PSE were previously described (15Mittal V. Cleary M.A. Herr W. Hernandez N. Mol. Cell. Biol. 1996; 16: 1955-1965Crossref PubMed Scopus (66) Google Scholar). The plasmid containing the H2B octamer site was described previously (17Cleary M.A. Stern S. Tanaka M. Herr W. Genes Dev. 1993; 7: 72-83Crossref PubMed Scopus (82) Google Scholar). The plasmids AD (also referred to in the text as mouse U6 PSE probe) and the mouse U6 PSE probe with the ABC mutation were generated by annealing two oligos, filling in with the Klenow fragment of DNA polymerase, cutting with BamHI and HindIII, and inserting into pUC118. This resulted in plasmids containing inserts with the sequences GGATCCGAAACTCACCCTAACTGTAAAGTAATTGTGTTTCTTGGCTTCTCGAGCCTTGTGGAAGCTTAAG and GGATCCGAAACTCCCACTACCGGTCCAGTAATTGTGTTTCTTGGCTTCTCGAGCCTT-GTGGAAGCTTAAG for the AD plasmid and th
Referência(s)