The Nuclear Factor SPBP Contains Different Functional Domains and Stimulates the Activity of Various Transcriptional Activators
2000; Elsevier BV; Volume: 275; Issue: 51 Linguagem: Inglês
10.1074/jbc.m006978200
ISSN1083-351X
AutoresCecilie Rekdal, Eva Sjøttem, Terje Johansen,
Tópico(s)Epigenetics and DNA Methylation
ResumoSPBP (stromelysin-1platelet-derived growth factor-responsive elementbinding protein) was originally cloned from a cDNA expression library by virtue of its ability to bind to a platelet-derived growth factor-responsive element in the human stromelysin-1 promoter. A 937-amino acid-long protein was deduced from a 3995-nucleotide murine cDNA sequence. By analyses of both human and murine cDNAs, we now show that SPBP is twice as large as originally found. The human SPBP gene contains six exons and is located on chromosome 22q13.1–13.3. Two isoforms differing in their C termini are expressed due to alternative splicing. PCR analyses of multitissue cDNA panels showed that SPBP is expressed in most tissues except for ovary and prostate. Functional mapping revealed that SPBP is a nuclear, multidomain protein containing an N-terminal region with transactivating ability, a novel type of DNA-binding domain containing an AT hook motif, and a bipartite nuclear localization signal as well as a C-terminal zinc finger domain. This type of zinc finger domain is also found in the trithorax family of chromatin-based transcriptional regulator proteins. Using cotransfection experiments, we find that SPBP enhances the transcriptional activity of various transcription factors such as c-Jun, Ets1, Sp1, and Pax6. Hence, SPBP seems to act as a transcriptional coactivator. SPBP (stromelysin-1platelet-derived growth factor-responsive elementbinding protein) was originally cloned from a cDNA expression library by virtue of its ability to bind to a platelet-derived growth factor-responsive element in the human stromelysin-1 promoter. A 937-amino acid-long protein was deduced from a 3995-nucleotide murine cDNA sequence. By analyses of both human and murine cDNAs, we now show that SPBP is twice as large as originally found. The human SPBP gene contains six exons and is located on chromosome 22q13.1–13.3. Two isoforms differing in their C termini are expressed due to alternative splicing. PCR analyses of multitissue cDNA panels showed that SPBP is expressed in most tissues except for ovary and prostate. Functional mapping revealed that SPBP is a nuclear, multidomain protein containing an N-terminal region with transactivating ability, a novel type of DNA-binding domain containing an AT hook motif, and a bipartite nuclear localization signal as well as a C-terminal zinc finger domain. This type of zinc finger domain is also found in the trithorax family of chromatin-based transcriptional regulator proteins. Using cotransfection experiments, we find that SPBP enhances the transcriptional activity of various transcription factors such as c-Jun, Ets1, Sp1, and Pax6. Hence, SPBP seems to act as a transcriptional coactivator. platelet-derived growth factor glutathioneS-transferase gel mobility shift assays DNA-binding domain minimal DBD maximal DBD green fluorescent protein phosphate-buffered saline polymerase chain reaction rapid amplification of cDNA ends base pair(s) kilobase pair(s) stromelysin-1 PDGF-responsive element open reading frame hemagglutinin fluorescence in situ hybridization expressed sequence tag cAMP-response element-binding protein 4′,6′-diamidino-2-phenylindole SPBP (stromelysin-1PDGF1-responsive element-binding protein) was originally isolated from a murine λgt11 cDNA library as a protein that bound to the stromelysin-1 PDGF-responsive element (SPRE) of the human stromelysin-1 promoter (1Sanz L. Moscat J. Diaz-Meco M.T. Mol. Cell. Biol. 1995; 15: 3164-3170Crossref PubMed Scopus (28) Google Scholar). The published cDNA sequence of SPBP is 3995 base pairs (bp) long with an open reading frame (ORF) of 2822 bp encoding a protein of 937 amino acids (1Sanz L. Moscat J. Diaz-Meco M.T. Mol. Cell. Biol. 1995; 15: 3164-3170Crossref PubMed Scopus (28) Google Scholar). SPBP has also been called AR1 or TCF20 (2Rajadhyaksha A. Riviere M. Van Vooren P. Szpirer J. Szpirer C. Babin J. Bina M. Cytogenet. Cell. Genet. 1998; 81: 176-177Crossref PubMed Google Scholar). Stromelysin-1, also known as MMP3, is an extracellular matrix-degrading metalloproteinase that is active against a broad range of substrates. These proteases are invariably up-regulated in epithelial cancers, recognized as targets of oncogenic signal transduction pathways and shown to contribute to tumor invasion and metastasis (3Kahari V.M. Saarialho-Kere U. Ann. Med. 1999; 31: 34-45Crossref PubMed Scopus (349) Google Scholar, 4Matrisian L.M. Curr. Biol. 1999; 9: 776-778Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Recently, it was reported that stromelysin-1 actually also promoted mammary carcinogenesis in a mouse model system. Overexpression of stromelysin-1 in the mammary gland of transgenic mice induced all stages of tumor progression from hyperplasia to malignant carcinomas (5Sternlicht M.D. Lochter A. Sympson C.J. Huey B. Rougier J.P. Gray J.W. Pinkel D. Bissell M.J. Werb Z. Cell. 1999; 98: 137-146Abstract Full Text Full Text PDF PubMed Scopus (764) Google Scholar). In such experiments, stromelysin-1 seems to act as a natural tumor promoter enhancing cancer susceptibility (6Sternlicht M.D. Bissell M.J. Werb Z. Oncogene. 2000; 19: 1102-1113Crossref PubMed Scopus (232) Google Scholar).The transcriptional activity of the stromelysin-1 promoter is stimulated by PDGF, and SPBP is reported to contribute to this up-regulation (7Kirstein M. Sanz L. Quinones S. Moscat J. Diaz-Meco M.T. Saus J. J. Biol. Chem. 1996; 271: 18231-18236Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 8Sanz L. Berra E. Municio M.M. Dominguez I. Lozano J. Johansen T. Moscat J. Diaz Meco M.T. J. Biol. Chem. 1994; 269: 10044-10049Abstract Full Text PDF PubMed Google Scholar). Interestingly, the expression of SPBP itself was also found to be induced by PDGF or serum (1Sanz L. Moscat J. Diaz-Meco M.T. Mol. Cell. Biol. 1995; 15: 3164-3170Crossref PubMed Scopus (28) Google Scholar). The stromelysin-1 promoter contains three elements that are important for induction by mitogenic stimuli. These are an AP-1 element binding the c-Fos/c-Jun transcription factors, two head-to-head PEA3 elements binding Ets family transcription factors, and the SPRE element that binds SPBP (see Ref. 7Kirstein M. Sanz L. Quinones S. Moscat J. Diaz-Meco M.T. Saus J. J. Biol. Chem. 1996; 271: 18231-18236Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar and references therein). The signal from the PDGF receptor is dependent on active Ras protein and bifurcates into one pathway that involves Raf-1 and activates the stromelysin-1 promoter via the AP-1 and PEA3 elements. The other pathway involves the atypical protein kinase C isoforms λ and ζ and acts via the AP-1 and SPRE elements (7Kirstein M. Sanz L. Quinones S. Moscat J. Diaz-Meco M.T. Saus J. J. Biol. Chem. 1996; 271: 18231-18236Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 8Sanz L. Berra E. Municio M.M. Dominguez I. Lozano J. Johansen T. Moscat J. Diaz Meco M.T. J. Biol. Chem. 1994; 269: 10044-10049Abstract Full Text PDF PubMed Google Scholar). It has been proposed that SPBP cooperates with c-Jun to transactivate via the SPRE site, and these two proteins are found to interact in vitro (7Kirstein M. Sanz L. Quinones S. Moscat J. Diaz-Meco M.T. Saus J. J. Biol. Chem. 1996; 271: 18231-18236Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar).Transcriptional regulation requires the concerted action of a large number of proteins, many of which act in multiprotein complexes (reviewed in Refs. 9Hampsey M. Reinberg D. Curr. Opin. Genet. Dev. 1999; 9: 132-139Crossref PubMed Scopus (137) Google Scholar and 10Lemon B.D. Freedman L.P. Curr. Opin. Genet. Dev. 1999; 9: 499-504Crossref PubMed Scopus (88) Google Scholar). These factors, termed transcription factors, transcriptional cofactors, or mediators, seem to be involved at two levels: 1) modulation of chromatin structure making DNA more or less accessible for other transcription factors to bind and 2) recruitment of the general transcriptional machinery to the promoter.Here we show that SPBP is twice as large as originally described. Due to alternative splicing, two isoforms with different C termini are produced (1983 and 1965 amino acids long for the murine protein). The human and murine proteins show a sequence identity of 92%. The murine gene is located on chromosome 15, while the human is found on chromosome 22 in positions q13.1 to q13.3. SPBP is expressed in most tissues except ovary and prostate. Functional mapping revealed SPBP to be a nuclear multidomain protein. It contains three nuclear localization signals, a novel type of DNA-binding domain with a single AT-hook, a transactivation domain in the N-terminal end, and in the very C-terminal end an evolutionary conserved zinc finger domain also found in the trithorax family of chromatin-based transcriptional regulator proteins. Interestingly, SPBP has the ability to enhance the transcriptional activity of various transcription factors, such as c-Jun, Ets, Sp1, and Pax6, suggesting that SPBP may be a novel transcriptional coactivator.DISCUSSIONWe show that SPBP, originally described as a 110-kDa novel transcription factor acting to regulate stromelysin-1 transcription, is actually a 220-kDa nuclear factor that is also able to function as a transcriptional coactivator. SPBP is expressed in most tissues, except for prostate and ovary. Two different isoforms, differing in their C termini, are produced by alternative splicing. The splicing pattern varies between different tissues and during embryonic development. The significance of the different isoforms is currently unknown. However, the differential expression of the two splice forms in the various tissues analyzed suggests that they may serve different roles or functions. Since SPBP is expressed in most tissues while stromelysin-1 expression is more restricted, SPBP may have a more general role in transcriptional regulation than only to control stromelysin-1 expression.Except for the evolutionary conserved ZNF2 zinc finger domain, we have been unable to detect sequences in the sequenced genomes ofSaccharomyces cerevisiae, Caenorhabditis elegans, or Drosophila that could represent direct homologues of SPBP. Thus, SPBP may be a vertebrate-specific protein. By FISH analysis, the human gene encoding SPBP was located at 22q13.1–3, and the murine gene was localized to the syntenic chromosome 15E region. Consistently, while this work was in progress Rajadhyaksha et al. (2Rajadhyaksha A. Riviere M. Van Vooren P. Szpirer J. Szpirer C. Babin J. Bina M. Cytogenet. Cell. Genet. 1998; 81: 176-177Crossref PubMed Google Scholar) reported that AR1 or TCF20 (other names for SPBP) is located at 22q13.3. We findSPBP to be located at position 26 Mb by alignment to the sequence data from chromosome 22q (21Dunham I. Hunt A.R. Collins J.E. Bruskiewich R. Beare D.M. Clamp M. Smink L.J. Ainscough R. Almeida J.P. Babbage A. Bagguley C. Bailey J. Barlow K. Bates K.N. Beasley O. et al.Nature. 1999; 402: 489-495Crossref PubMed Scopus (911) Google Scholar). Several tumor forms have been associated with deletions and other lesions in this part of chromosome 22 including gliomas, meningiomas, and ovarian and colon cancers. However, the mapping data are too imprecise to say anything about a possible involvement of SPBP.SPBP was originally isolated by virtue of its ability to bind specifically to a concatenated SPRE probe (1Sanz L. Moscat J. Diaz-Meco M.T. Mol. Cell. Biol. 1995; 15: 3164-3170Crossref PubMed Scopus (28) Google Scholar). We found the region from amino acid 1535 to 1584 to contain a minimal DNA binding domain (Min-DBD), but both the affinity and the specificity of the binding increased when the region was extended C-terminally to amino acid 1731 to give the maximal DNA binding domain (Max-DBD). The SPRE consists of an AT-rich palindrome (Pal-1), which partially overlaps with another palindrome (Pal-2). These two palindromes may constitute half-sites. Our results from GMSA with the Min- and Max-DBDs are compatible with the notion that SPBP binds as a dimer or multimer to the SPRE motif when the complete binding site is present and as a monomer when Pal-1 or Pal-2 is mutated. This notion is supported by the occurrence of a differently migrating complex when the Min- and Max-DBDs were mixed compared with the complexes observed when these two domains were assayed separately. Interestingly, the affinity of DNA binding increased significantly when the protein concentration was raised above a certain level. This is indicative of cooperativity in the DNA binding or dimerization. A cooperative, dimeric/multimeric DNA binding by SPBP is also supported by the hyperbolic activation kinetics of a SPRE reporter upon cotransfection of increasing amounts of SPBP-(1016–1965) (7Kirstein M. Sanz L. Quinones S. Moscat J. Diaz-Meco M.T. Saus J. J. Biol. Chem. 1996; 271: 18231-18236Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The Min-DBD contains a single AT-hook motif. Deletion of the AT-hook from the Min-DBD abolished DNA binding. The region C-terminal to the AT-hook, constituting the rest of the Max-DBD, does not show any sequence homology to any known classes of DBDs. By itself, this region is unable to bind to the SPRE motif, suggesting that the Min-DBD with an intact single AT-hook motif is essential for DNA binding. Multiple and single AT-hooks motifs are found in several multidomain proteins that associate with chromatin (27Aravind L. Landsman D. Nucleic Acids Res. 1998; 26: 4413-4421Crossref PubMed Scopus (378) Google Scholar). Interestingly, among these are members of the trithorax group of regulatory genes that also contain zinc finger domains (ZNF and PHD) with homology to the zinc finger domain of SPBP. Trithorax proteins are involved in chromatin decondensation (31Pirrotta V. Cell. 1998; 93: 333-336Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar), and their AT-hooks have been implicated in several chromosomal translocations resulting in different lymphoid leukemias (32Slany R.K. Lavau C. Cleary M.L. Mol. Cell. Biol. 1998; 18: 122-129Crossref PubMed Scopus (204) Google Scholar, 33Tkachuk D.C. Kohler S. Cleary M.L. Cell. 1992; 71: 691-700Abstract Full Text PDF PubMed Scopus (856) Google Scholar, 34Zeleznik-Le N.J. Harden A.M. Rowley J.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10610-10614Crossref PubMed Scopus (211) Google Scholar). The type 1 AT-hook motifs such as the one found in SPBP seem to be auxiliary elements necessary for cooperation with other DNA binding activities in the same or different proteins (27Aravind L. Landsman D. Nucleic Acids Res. 1998; 26: 4413-4421Crossref PubMed Scopus (378) Google Scholar). This is consistent with our finding that SPBP has a Min-DBD with the AT-hook and a larger domain C-terminal to it that together with the Min-DBD constitutes the Max-DBD. AT-hooks are known to bind to AT-rich sequences in the minor groove of DNA. In doing so they often change the DNA conformation, making DNA more or less accessible for other DNA binding factors, depending on the sequence context. This suggests that the Min-DBD of SPBP recognizes the AT-rich Pal-1 of the SPRE motif, binds in the minor groove, and thereby makes the Pal-2 sequence more accessible for binding by another SPBP molecule.A dual picture of the function(s) of SPBP is emerging. On one hand, two previous reports suggest that SPBP acts as a classical sequence-specific transcriptional activator. SPBP-(1016–1965) and c-Jun were found to cooperate to transactivate a promoter under SPRE control (1Sanz L. Moscat J. Diaz-Meco M.T. Mol. Cell. Biol. 1995; 15: 3164-3170Crossref PubMed Scopus (28) Google Scholar, 7Kirstein M. Sanz L. Quinones S. Moscat J. Diaz-Meco M.T. Saus J. J. Biol. Chem. 1996; 271: 18231-18236Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). On the other hand, we find that SPBP may act as a coactivator for structurally and functionally rather different transcription factors binding to distinct target sequences in promoters/enhancers. However, this is not inconsistent with the previously reported collaboration between c-Jun and SPBP-(1016–1965). In fact, it was found that c-Jun and SPBP bound to each other in vitro and that c-Jun could activate the reporter alone but that this activation was markedly increased upon coexpression of SPBP (7Kirstein M. Sanz L. Quinones S. Moscat J. Diaz-Meco M.T. Saus J. J. Biol. Chem. 1996; 271: 18231-18236Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). This is in complete agreement with our finding of SPBP as a potent coactivator of c-Jun. Since SPBP coactivated such diverse transcription factors as Sp1, Pax6, c-Jun, and Ets-1, the mechanism(s) involved may be relatively general targeting common components. The finding that the zinc finger domain was necessary for stimulation of the c-Jun and Sp1 activity but not for the stimulation of the Ets-1 and Pax6 activity indicates that different parts of SPBP are involved, depending on the specific transcription factor. At what level does SPBP act to stimulate the transcription mediated by these factors? We found that SPBP does not act by increasing translation or increasing protein stability. The fact that SPBP did not enhance the transcriptional activation potential of E2F-1, together with the nonstimulatory effect of SPBP alone, suggests that SPBP does not act at the levels of pre-mRNA processing or mRNA stability of the luciferase reporter gene transcript. Thus, most likely, SPBP acts on the transcriptional initiation step, either at the chromatin/DNA level or in the recruitment of necessary factors to the initiation site. Several AT-hook-containing proteins play important roles in modulating chromatin structure and act as transcriptional cofactors (see Refs. 35Chin M.T. Pellacani A. Wang H. Lin S.S. Jain M.K. Perrella M.A. Lee M.E. J. Biol. Chem. 1998; 273: 9755-9760Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 36Falvo J.V. Thanos D. Maniatis T. Cell. 1995; 83: 1101-1111Abstract Full Text PDF PubMed Scopus (276) Google Scholar, 37Girard F. Bello B. Laemmli U.K. Gehring W.J. EMBO J. 1998; 17: 2079-2085Crossref PubMed Scopus (63) Google Scholar, 38Onate S.A. Prendergast P. Wagner J.P. Nissen M. Reeves R. Pettijohn D.E. Edwards D.P. Mol. Cell. Biol. 1994; 14: 3376-3391Crossref PubMed Google Scholar and references therein). AT-hook proteins may act both as coactivators or corepressors. In addition to changing the DNA conformation, the stimulatory effect of some AT-hook-containing proteins is also dependent on protein-protein contacts (35Chin M.T. Pellacani A. Wang H. Lin S.S. Jain M.K. Perrella M.A. Lee M.E. J. Biol. Chem. 1998; 273: 9755-9760Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Thus, SPBP may stimulate the activity of other transcription factors both by changing the DNA conformation, making it more accessible, and by protein-protein contacts recruiting the transcription factors to DNA. In this context, it is noteworthy that the ZNF2 or extended PHD domain that SPBP shares with the trithorax family of chromatin-based transcriptional regulatory proteins is most likely a protein-protein interaction domain. We have found that a GST fusion of the ZNF2 domain of SPBP binds to in vitro translated full-length SPBP. Furthermore, in a yeast two-hybrid screen with the ZNF2 domain as bait, we isolated a part of murine GT1 corresponding to the GT1-F homology region of SPBP. 4E. Sjøttem and T. Johansen, unpublished results.In future studies, it will be important to interfere with or knock out the endogenous SPBP to determine what physiological processes may depend on active SPBP. Our attempts to inhibit the expression of endogenous SPBP using antisense RNA expression were unsuccessful. Thus, other strategies like microinjection of neutralizing antibodies (not presently available) or overexpression of dominant negative mutants and the production of knock-out mice are needed to study the effects on transcription and phenotypic changes associated with blockade of endogenous SPBP. SPBP (stromelysin-1PDGF1-responsive element-binding protein) was originally isolated from a murine λgt11 cDNA library as a protein that bound to the stromelysin-1 PDGF-responsive element (SPRE) of the human stromelysin-1 promoter (1Sanz L. Moscat J. Diaz-Meco M.T. Mol. Cell. Biol. 1995; 15: 3164-3170Crossref PubMed Scopus (28) Google Scholar). The published cDNA sequence of SPBP is 3995 base pairs (bp) long with an open reading frame (ORF) of 2822 bp encoding a protein of 937 amino acids (1Sanz L. Moscat J. Diaz-Meco M.T. Mol. Cell. Biol. 1995; 15: 3164-3170Crossref PubMed Scopus (28) Google Scholar). SPBP has also been called AR1 or TCF20 (2Rajadhyaksha A. Riviere M. Van Vooren P. Szpirer J. Szpirer C. Babin J. Bina M. Cytogenet. Cell. Genet. 1998; 81: 176-177Crossref PubMed Google Scholar). Stromelysin-1, also known as MMP3, is an extracellular matrix-degrading metalloproteinase that is active against a broad range of substrates. These proteases are invariably up-regulated in epithelial cancers, recognized as targets of oncogenic signal transduction pathways and shown to contribute to tumor invasion and metastasis (3Kahari V.M. Saarialho-Kere U. Ann. Med. 1999; 31: 34-45Crossref PubMed Scopus (349) Google Scholar, 4Matrisian L.M. Curr. Biol. 1999; 9: 776-778Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Recently, it was reported that stromelysin-1 actually also promoted mammary carcinogenesis in a mouse model system. Overexpression of stromelysin-1 in the mammary gland of transgenic mice induced all stages of tumor progression from hyperplasia to malignant carcinomas (5Sternlicht M.D. Lochter A. Sympson C.J. Huey B. Rougier J.P. Gray J.W. Pinkel D. Bissell M.J. Werb Z. Cell. 1999; 98: 137-146Abstract Full Text Full Text PDF PubMed Scopus (764) Google Scholar). In such experiments, stromelysin-1 seems to act as a natural tumor promoter enhancing cancer susceptibility (6Sternlicht M.D. Bissell M.J. Werb Z. Oncogene. 2000; 19: 1102-1113Crossref PubMed Scopus (232) Google Scholar). The transcriptional activity of the stromelysin-1 promoter is stimulated by PDGF, and SPBP is reported to contribute to this up-regulation (7Kirstein M. Sanz L. Quinones S. Moscat J. Diaz-Meco M.T. Saus J. J. Biol. Chem. 1996; 271: 18231-18236Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 8Sanz L. Berra E. Municio M.M. Dominguez I. Lozano J. Johansen T. Moscat J. Diaz Meco M.T. J. Biol. Chem. 1994; 269: 10044-10049Abstract Full Text PDF PubMed Google Scholar). Interestingly, the expression of SPBP itself was also found to be induced by PDGF or serum (1Sanz L. Moscat J. Diaz-Meco M.T. Mol. Cell. Biol. 1995; 15: 3164-3170Crossref PubMed Scopus (28) Google Scholar). The stromelysin-1 promoter contains three elements that are important for induction by mitogenic stimuli. These are an AP-1 element binding the c-Fos/c-Jun transcription factors, two head-to-head PEA3 elements binding Ets family transcription factors, and the SPRE element that binds SPBP (see Ref. 7Kirstein M. Sanz L. Quinones S. Moscat J. Diaz-Meco M.T. Saus J. J. Biol. Chem. 1996; 271: 18231-18236Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar and references therein). The signal from the PDGF receptor is dependent on active Ras protein and bifurcates into one pathway that involves Raf-1 and activates the stromelysin-1 promoter via the AP-1 and PEA3 elements. The other pathway involves the atypical protein kinase C isoforms λ and ζ and acts via the AP-1 and SPRE elements (7Kirstein M. Sanz L. Quinones S. Moscat J. Diaz-Meco M.T. Saus J. J. Biol. Chem. 1996; 271: 18231-18236Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 8Sanz L. Berra E. Municio M.M. Dominguez I. Lozano J. Johansen T. Moscat J. Diaz Meco M.T. J. Biol. Chem. 1994; 269: 10044-10049Abstract Full Text PDF PubMed Google Scholar). It has been proposed that SPBP cooperates with c-Jun to transactivate via the SPRE site, and these two proteins are found to interact in vitro (7Kirstein M. Sanz L. Quinones S. Moscat J. Diaz-Meco M.T. Saus J. J. Biol. Chem. 1996; 271: 18231-18236Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Transcriptional regulation requires the concerted action of a large number of proteins, many of which act in multiprotein complexes (reviewed in Refs. 9Hampsey M. Reinberg D. Curr. Opin. Genet. Dev. 1999; 9: 132-139Crossref PubMed Scopus (137) Google Scholar and 10Lemon B.D. Freedman L.P. Curr. Opin. Genet. Dev. 1999; 9: 499-504Crossref PubMed Scopus (88) Google Scholar). These factors, termed transcription factors, transcriptional cofactors, or mediators, seem to be involved at two levels: 1) modulation of chromatin structure making DNA more or less accessible for other transcription factors to bind and 2) recruitment of the general transcriptional machinery to the promoter. Here we show that SPBP is twice as large as originally described. Due to alternative splicing, two isoforms with different C termini are produced (1983 and 1965 amino acids long for the murine protein). The human and murine proteins show a sequence identity of 92%. The murine gene is located on chromosome 15, while the human is found on chromosome 22 in positions q13.1 to q13.3. SPBP is expressed in most tissues except ovary and prostate. Functional mapping revealed SPBP to be a nuclear multidomain protein. It contains three nuclear localization signals, a novel type of DNA-binding domain with a single AT-hook, a transactivation domain in the N-terminal end, and in the very C-terminal end an evolutionary conserved zinc finger domain also found in the trithorax family of chromatin-based transcriptional regulator proteins. Interestingly, SPBP has the ability to enhance the transcriptional activity of various transcription factors, such as c-Jun, Ets, Sp1, and Pax6, suggesting that SPBP may be a novel transcriptional coactivator. DISCUSSIONWe show that SPBP, originally described as a 110-kDa novel transcription factor acting to regulate stromelysin-1 transcription, is actually a 220-kDa nuclear factor that is also able to function as a transcriptional coactivator. SPBP is expressed in most tissues, except for prostate and ovary. Two different isoforms, differing in their C termini, are produced by alternative splicing. The splicing pattern varies between different tissues and during embryonic development. The significance of the different isoforms is currently unknown. However, the differential expression of the two splice forms in the various tissues analyzed suggests that they may serve different roles or functions. Since SPBP is expressed in most tissues while stromelysin-1 expression is more restricted, SPBP may have a more general role in transcriptional regulation than only to control stromelysin-1 expression.Except for the evolutionary conserved ZNF2 zinc finger domain, we have been unable to detect sequences in the sequenced genomes ofSaccharomyces cerevisiae, Caenorhabditis elegans, or Drosophila that could represent direct homologues of SPBP. Thus, SPBP may be a vertebrate-specific protein. By FISH analysis, the human gene encoding SPBP was located at 22q13.1–3, and the murine gene was localized to the syntenic chromosome 15E region. Consistently, while this work was in progress Rajadhyaksha et al. (2Rajadhyaksha A. Riviere M. Van Vooren P. Szpirer J. Szpirer C. Babin J. Bina M. Cytogenet. Cell. Genet. 1998; 81: 176-177Crossref PubMed Google Scholar) reported that AR1 or TCF20 (other names for SPBP) is located at 22q13.3. We findSPBP to be located at position 26 Mb by alignment to the sequence data from chromosome 22q (21Dunham I. Hunt A.R. Collins J.E. Bruskiewich R. Beare D.M. Clamp M. Smink L.J. Ainscough R. Almeida J.P. Babbage A. Bagguley C. Bailey J. Barlow K. Bates K.N. Beasley O. et al.Nature. 1999; 402: 489-495Crossref PubMed Scopus (911) Google Scholar). Several tumor forms have been associated with deletions and other lesions in this part of chromosome 22 including gliomas, meningiomas, and ovarian and colon cancers. However, the mapping data are too imprecise to say anything about a possible involvement of SPBP.SPBP was originally isolated by virtue of its ability to bind specifically to a concatenated SPRE probe (1Sanz L. Moscat J. Diaz-Meco M.T. Mol. Cell. Biol. 1995; 15: 3164-3170Crossref PubMed Scopus (28) Google Scholar). We found the region from amino acid 1535 to 1584 to contain a minimal DNA binding domain (Min-DBD), but both the affinity and the specificity of the binding increased when the region was extended C-terminally to amino acid 1731 to give the maximal DNA binding domain (Max-DBD). The SPRE consists of an AT-rich palindrome (Pal-1), which partially overlaps with another palindrome (Pal-2). These two palindromes may constitute half-sites. Our results from GMSA with the Min- and Max-DBDs are compatible with the notion that SPBP binds as a dimer or multimer to the SPRE motif when the complete binding site is present and as a monomer when Pal-1 or Pal-2 is mutated. This notion is supported by the occurrence of a differently migrating complex when the Min- and Max-DBDs were mixed compared with the complexes observed when these two domains were assayed separately. Interestingly, the affinity of DNA binding increased significantly when the protein concentration was raised above a certain level. This is indicative of cooperativity in the DNA binding or dimerization. A cooperative, dimeric/multimeric DNA binding by SPBP is also supported by the hyperbolic activation kinetics of a SPRE reporter upon cotransfection of increasing amounts of SPBP-(1016–1965) (7Kirstein M. Sanz L. Quinones S. Moscat J. Diaz-Meco M.T. Saus J. J. Biol. Chem. 1996; 271: 18231-18236Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The Min-DBD contains a single AT-hook motif. Deletion of the AT-hook from the Min-DBD abolished DNA binding. The region C-terminal to the AT-hook, constituting the rest of the Max-DBD, does not show any sequence homology to any known classes of DBDs. By itself, this region is unable to bind to the SPRE motif, suggesting that the Min-DBD with an intact single AT-hook motif is essential for DNA binding. Multiple and single AT-hooks motifs are found in several multidomain proteins that associate with chromatin (27Aravind L. Landsman D. Nucleic Acids Res. 1998; 26: 4413-4421Crossref PubMed Scopus (378) Google Scholar). Interestingly, among these are members of the trithorax group of regulatory genes that also contain zinc finger domains (ZNF and PHD) with homology to the zinc finger domain of SPBP. Trithorax proteins are involved in chromatin decondensation (31Pirrotta V. Cell. 1998; 93: 333-336Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar), and their AT-hooks have been implicated in several chromosomal translocations resulting in different lymphoid leukemias (32Slany R.K. Lavau C. Cleary M.L. Mol. Cell. Biol. 1998; 18: 122-129Crossref PubMed Scopus (204) Google Scholar, 33Tkachuk D.C. Kohler S. Cleary M.L. Cell. 1992; 71: 691-700Abstract Full Text PDF PubMed Scopus (856) Google Scholar, 34Zeleznik-Le N.J. Harden A.M. Rowley J.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10610-10614Crossref PubMed Scopus (211) Google Scholar). The type 1 AT-hook motifs such as the one found in SPBP seem to be auxiliary elements necessary for cooperation with other DNA binding activities in the same or different proteins (27Aravind L. Landsman D. Nucleic Acids Res. 1998; 26: 4413-4421Crossref PubMed Scopus (378) Google Scholar). This is consistent with our finding that SPBP has a Min-DBD with the AT-hook and a larger domain C-terminal to it that together with the Min-DBD constitutes the Max-DBD. AT-hooks are known to bind to AT-rich sequences in the minor groove of DNA. In doing so they often change the DNA conformation, making DNA more or less accessible for other DNA binding factors, depending on the sequence context. This suggests that the Min-DBD of SPBP recognizes the AT-rich Pal-1 of the SPRE motif, binds in the minor groove, and thereby makes the Pal-2 sequence more accessible for binding by another SPBP molecule.A dual picture of the function(s) of SPBP is emerging. On one hand, two previous reports suggest that SPBP acts as a classical sequence-specific transcriptional activator. SPBP-(1016–1965) and c-Jun were found to cooperate to transactivate a promoter under SPRE control (1Sanz L. Moscat J. Diaz-Meco M.T. Mol. Cell. Biol. 1995; 15: 3164-3170Crossref PubMed Scopus (28) Google Scholar, 7Kirstein M. Sanz L. Quinones S. Moscat J. Diaz-Meco M.T. Saus J. J. Biol. Chem. 1996; 271: 18231-18236Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). On the other hand, we find that SPBP may act as a coactivator for structurally and functionally rather different transcription factors binding to distinct target sequences in promoters/enhancers. However, this is not inconsistent with the previously reported collaboration between c-Jun and SPBP-(1016–1965). In fact, it was found that c-Jun and SPBP bound to each other in vitro and that c-Jun could activate the reporter alone but that this activation was markedly increased upon coexpression of SPBP (7Kirstein M. Sanz L. Quinones S. Moscat J. Diaz-Meco M.T. Saus J. J. Biol. Chem. 1996; 271: 18231-18236Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). This is in complete agreement with our finding of SPBP as a potent coactivator of c-Jun. Since SPBP coactivated such diverse transcription factors as Sp1, Pax6, c-Jun, and Ets-1, the mechanism(s) involved may be relatively general targeting common components. The finding that the zinc finger domain was necessary for stimulation of the c-Jun and Sp1 activity but not for the stimulation of the Ets-1 and Pax6 activity indicates that different parts of SPBP are involved, depending on the specific transcription factor. At what level does SPBP act to stimulate the transcription mediated by these factors? We found that SPBP does not act by increasing translation or increasing protein stability. The fact that SPBP did not enhance the transcriptional activation potential of E2F-1, together with the nonstimulatory effect of SPBP alone, suggests that SPBP does not act at the levels of pre-mRNA processing or mRNA stability of the luciferase reporter gene transcript. Thus, most likely, SPBP acts on the transcriptional initiation step, either at the chromatin/DNA level or in the recruitment of necessary factors to the initiation site. Several AT-hook-containing proteins play important roles in modulating chromatin structure and act as transcriptional cofactors (see Refs. 35Chin M.T. Pellacani A. Wang H. Lin S.S. Jain M.K. Perrella M.A. Lee M.E. J. Biol. Chem. 1998; 273: 9755-9760Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 36Falvo J.V. Thanos D. Maniatis T. Cell. 1995; 83: 1101-1111Abstract Full Text PDF PubMed Scopus (276) Google Scholar, 37Girard F. Bello B. Laemmli U.K. Gehring W.J. EMBO J. 1998; 17: 2079-2085Crossref PubMed Scopus (63) Google Scholar, 38Onate S.A. Prendergast P. Wagner J.P. Nissen M. Reeves R. Pettijohn D.E. Edwards D.P. Mol. Cell. Biol. 1994; 14: 3376-3391Crossref PubMed Google Scholar and references therein). AT-hook proteins may act both as coactivators or corepressors. In addition to changing the DNA conformation, the stimulatory effect of some AT-hook-containing proteins is also dependent on protein-protein contacts (35Chin M.T. Pellacani A. Wang H. Lin S.S. Jain M.K. Perrella M.A. Lee M.E. J. Biol. Chem. 1998; 273: 9755-9760Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Thus, SPBP may stimulate the activity of other transcription factors both by changing the DNA conformation, making it more accessible, and by protein-protein contacts recruiting the transcription factors to DNA. In this context, it is noteworthy that the ZNF2 or extended PHD domain that SPBP shares with the trithorax family of chromatin-based transcriptional regulatory proteins is most likely a protein-protein interaction domain. We have found that a GST fusion of the ZNF2 domain of SPBP binds to in vitro translated full-length SPBP. Furthermore, in a yeast two-hybrid screen with the ZNF2 domain as bait, we isolated a part of murine GT1 corresponding to the GT1-F homology region of SPBP. 4E. Sjøttem and T. Johansen, unpublished results.In future studies, it will be important to interfere with or knock out the endogenous SPBP to determine what physiological processes may depend on active SPBP. Our attempts to inhibit the expression of endogenous SPBP using antisense RNA expression were unsuccessful. Thus, other strategies like microinjection of neutralizing antibodies (not presently available) or overexpression of dominant negative mutants and the production of knock-out mice are needed to study the effects on transcription and phenotypic changes associated with blockade of endogenous SPBP. We show that SPBP, originally described as a 110-kDa novel transcription factor acting to regulate stromelysin-1 transcription, is actually a 220-kDa nuclear factor that is also able to function as a transcriptional coactivator. SPBP is expressed in most tissues, except for prostate and ovary. Two different isoforms, differing in their C termini, are produced by alternative splicing. The splicing pattern varies between different tissues and during embryonic development. The significance of the different isoforms is currently unknown. However, the differential expression of the two splice forms in the various tissues analyzed suggests that they may serve different roles or functions. Since SPBP is expressed in most tissues while stromelysin-1 expression is more restricted, SPBP may have a more general role in transcriptional regulation than only to control stromelysin-1 expression. Except for the evolutionary conserved ZNF2 zinc finger domain, we have been unable to detect sequences in the sequenced genomes ofSaccharomyces cerevisiae, Caenorhabditis elegans, or Drosophila that could represent direct homologues of SPBP. Thus, SPBP may be a vertebrate-specific protein. By FISH analysis, the human gene encoding SPBP was located at 22q13.1–3, and the murine gene was localized to the syntenic chromosome 15E region. Consistently, while this work was in progress Rajadhyaksha et al. (2Rajadhyaksha A. Riviere M. Van Vooren P. Szpirer J. Szpirer C. Babin J. Bina M. Cytogenet. Cell. Genet. 1998; 81: 176-177Crossref PubMed Google Scholar) reported that AR1 or TCF20 (other names for SPBP) is located at 22q13.3. We findSPBP to be located at position 26 Mb by alignment to the sequence data from chromosome 22q (21Dunham I. Hunt A.R. Collins J.E. Bruskiewich R. Beare D.M. Clamp M. Smink L.J. Ainscough R. Almeida J.P. Babbage A. Bagguley C. Bailey J. Barlow K. Bates K.N. Beasley O. et al.Nature. 1999; 402: 489-495Crossref PubMed Scopus (911) Google Scholar). Several tumor forms have been associated with deletions and other lesions in this part of chromosome 22 including gliomas, meningiomas, and ovarian and colon cancers. However, the mapping data are too imprecise to say anything about a possible involvement of SPBP. SPBP was originally isolated by virtue of its ability to bind specifically to a concatenated SPRE probe (1Sanz L. Moscat J. Diaz-Meco M.T. Mol. Cell. Biol. 1995; 15: 3164-3170Crossref PubMed Scopus (28) Google Scholar). We found the region from amino acid 1535 to 1584 to contain a minimal DNA binding domain (Min-DBD), but both the affinity and the specificity of the binding increased when the region was extended C-terminally to amino acid 1731 to give the maximal DNA binding domain (Max-DBD). The SPRE consists of an AT-rich palindrome (Pal-1), which partially overlaps with another palindrome (Pal-2). These two palindromes may constitute half-sites. Our results from GMSA with the Min- and Max-DBDs are compatible with the notion that SPBP binds as a dimer or multimer to the SPRE motif when the complete binding site is present and as a monomer when Pal-1 or Pal-2 is mutated. This notion is supported by the occurrence of a differently migrating complex when the Min- and Max-DBDs were mixed compared with the complexes observed when these two domains were assayed separately. Interestingly, the affinity of DNA binding increased significantly when the protein concentration was raised above a certain level. This is indicative of cooperativity in the DNA binding or dimerization. A cooperative, dimeric/multimeric DNA binding by SPBP is also supported by the hyperbolic activation kinetics of a SPRE reporter upon cotransfection of increasing amounts of SPBP-(1016–1965) (7Kirstein M. Sanz L. Quinones S. Moscat J. Diaz-Meco M.T. Saus J. J. Biol. Chem. 1996; 271: 18231-18236Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The Min-DBD contains a single AT-hook motif. Deletion of the AT-hook from the Min-DBD abolished DNA binding. The region C-terminal to the AT-hook, constituting the rest of the Max-DBD, does not show any sequence homology to any known classes of DBDs. By itself, this region is unable to bind to the SPRE motif, suggesting that the Min-DBD with an intact single AT-hook motif is essential for DNA binding. Multiple and single AT-hooks motifs are found in several multidomain proteins that associate with chromatin (27Aravind L. Landsman D. Nucleic Acids Res. 1998; 26: 4413-4421Crossref PubMed Scopus (378) Google Scholar). Interestingly, among these are members of the trithorax group of regulatory genes that also contain zinc finger domains (ZNF and PHD) with homology to the zinc finger domain of SPBP. Trithorax proteins are involved in chromatin decondensation (31Pirrotta V. Cell. 1998; 93: 333-336Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar), and their AT-hooks have been implicated in several chromosomal translocations resulting in different lymphoid leukemias (32Slany R.K. Lavau C. Cleary M.L. Mol. Cell. Biol. 1998; 18: 122-129Crossref PubMed Scopus (204) Google Scholar, 33Tkachuk D.C. Kohler S. Cleary M.L. Cell. 1992; 71: 691-700Abstract Full Text PDF PubMed Scopus (856) Google Scholar, 34Zeleznik-Le N.J. Harden A.M. Rowley J.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10610-10614Crossref PubMed Scopus (211) Google Scholar). The type 1 AT-hook motifs such as the one found in SPBP seem to be auxiliary elements necessary for cooperation with other DNA binding activities in the same or different proteins (27Aravind L. Landsman D. Nucleic Acids Res. 1998; 26: 4413-4421Crossref PubMed Scopus (378) Google Scholar). This is consistent with our finding that SPBP has a Min-DBD with the AT-hook and a larger domain C-terminal to it that together with the Min-DBD constitutes the Max-DBD. AT-hooks are known to bind to AT-rich sequences in the minor groove of DNA. In doing so they often change the DNA conformation, making DNA more or less accessible for other DNA binding factors, depending on the sequence context. This suggests that the Min-DBD of SPBP recognizes the AT-rich Pal-1 of the SPRE motif, binds in the minor groove, and thereby makes the Pal-2 sequence more accessible for binding by another SPBP molecule. A dual picture of the function(s) of SPBP is emerging. On one hand, two previous reports suggest that SPBP acts as a classical sequence-specific transcriptional activator. SPBP-(1016–1965) and c-Jun were found to cooperate to transactivate a promoter under SPRE control (1Sanz L. Moscat J. Diaz-Meco M.T. Mol. Cell. Biol. 1995; 15: 3164-3170Crossref PubMed Scopus (28) Google Scholar, 7Kirstein M. Sanz L. Quinones S. Moscat J. Diaz-Meco M.T. Saus J. J. Biol. Chem. 1996; 271: 18231-18236Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). On the other hand, we find that SPBP may act as a coactivator for structurally and functionally rather different transcription factors binding to distinct target sequences in promoters/enhancers. However, this is not inconsistent with the previously reported collaboration between c-Jun and SPBP-(1016–1965). In fact, it was found that c-Jun and SPBP bound to each other in vitro and that c-Jun could activate the reporter alone but that this activation was markedly increased upon coexpression of SPBP (7Kirstein M. Sanz L. Quinones S. Moscat J. Diaz-Meco M.T. Saus J. J. Biol. Chem. 1996; 271: 18231-18236Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). This is in complete agreement with our finding of SPBP as a potent coactivator of c-Jun. Since SPBP coactivated such diverse transcription factors as Sp1, Pax6, c-Jun, and Ets-1, the mechanism(s) involved may be relatively general targeting common components. The finding that the zinc finger domain was necessary for stimulation of the c-Jun and Sp1 activity but not for the stimulation of the Ets-1 and Pax6 activity indicates that different parts of SPBP are involved, depending on the specific transcription factor. At what level does SPBP act to stimulate the transcription mediated by these factors? We found that SPBP does not act by increasing translation or increasing protein stability. The fact that SPBP did not enhance the transcriptional activation potential of E2F-1, together with the nonstimulatory effect of SPBP alone, suggests that SPBP does not act at the levels of pre-mRNA processing or mRNA stability of the luciferase reporter gene transcript. Thus, most likely, SPBP acts on the transcriptional initiation step, either at the chromatin/DNA level or in the recruitment of necessary factors to the initiation site. Several AT-hook-containing proteins play important roles in modulating chromatin structure and act as transcriptional cofactors (see Refs. 35Chin M.T. Pellacani A. Wang H. Lin S.S. Jain M.K. Perrella M.A. Lee M.E. J. Biol. Chem. 1998; 273: 9755-9760Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 36Falvo J.V. Thanos D. Maniatis T. Cell. 1995; 83: 1101-1111Abstract Full Text PDF PubMed Scopus (276) Google Scholar, 37Girard F. Bello B. Laemmli U.K. Gehring W.J. EMBO J. 1998; 17: 2079-2085Crossref PubMed Scopus (63) Google Scholar, 38Onate S.A. Prendergast P. Wagner J.P. Nissen M. Reeves R. Pettijohn D.E. Edwards D.P. Mol. Cell. Biol. 1994; 14: 3376-3391Crossref PubMed Google Scholar and references therein). AT-hook proteins may act both as coactivators or corepressors. In addition to changing the DNA conformation, the stimulatory effect of some AT-hook-containing proteins is also dependent on protein-protein contacts (35Chin M.T. Pellacani A. Wang H. Lin S.S. Jain M.K. Perrella M.A. Lee M.E. J. Biol. Chem. 1998; 273: 9755-9760Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Thus, SPBP may stimulate the activity of other transcription factors both by changing the DNA conformation, making it more accessible, and by protein-protein contacts recruiting the transcription factors to DNA. In this context, it is noteworthy that the ZNF2 or extended PHD domain that SPBP shares with the trithorax family of chromatin-based transcriptional regulatory proteins is most likely a protein-protein interaction domain. We have found that a GST fusion of the ZNF2 domain of SPBP binds to in vitro translated full-length SPBP. Furthermore, in a yeast two-hybrid screen with the ZNF2 domain as bait, we isolated a part of murine GT1 corresponding to the GT1-F homology region of SPBP. 4E. Sjøttem and T. Johansen, unpublished results. In future studies, it will be important to interfere with or knock out the endogenous SPBP to determine what physiological processes may depend on active SPBP. Our attempts to inhibit the expression of endogenous SPBP using antisense RNA expression were unsuccessful. Thus, other strategies like microinjection of neutralizing antibodies (not presently available) or overexpression of dominant negative mutants and the production of knock-out mice are needed to study the effects on transcription and phenotypic changes associated with blockade of endogenous SPBP. We are very grateful to Jorge Moscat for gifts of the plasmid 5′KS-SPBP, pcDNA3-HA-SPBP-(1016–1965), and pcDNA3-HA as well as the rabbit polyclonal antibody to SPBP. Takahiro Nagase is acknowledged for the generous gift of the cDNA clone for KIAA0292. We thank Joseph R. Nevins, Guntram Suske, and Michael Sieweke for gifts of pcDNA3-E2F-1, pCMV-Sp1, and pRc-Ets68, respectively.
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