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• W2067229881 abstract "Nuclear DEAF-1-related (NUDR) protein is a novel transcriptional regulator with sequence similarity to developmental and oncogenic proteins. NUDR protein deletions were used to localize the DNA binding domain between amino acids 167 and 368, and site-specific DNA photocross-linking indicated at least two sites of protein-DNA contact within this domain. The DNA binding domain contains a proline-rich region and a region with similarity to a Myc-type helix-loop-helix domain but does not include the zinc finger motif at the C terminus. Deoxyribonuclease I protection assays confirmed the presence of multiple NUDR binding motifs (TTC(C/G)G) in the heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) promoter and also in the 5′-untranslated region (UTR) of hNUDR cDNA. NUDR produced a 65–70% repression of the hnRNP A2/B1 promoter activity, and NUDR binding motifs in the 5′-UTR were found to mediate this repression. NUDR-dependent repression was also observed when the 5′-UTR of NUDR was placed onto a heterologous thymidine kinase promoter in an analogous 5′-UTR position but not when placed upstream of transcription initiation. These results suggest that NUDR may regulate the in vivo expression of hnRNP A2/B1 and NUDR genes and imply that inactivation of NUDR could contribute to the overexpression of hnRNP A2/B1 observed in some human cancers. Nuclear DEAF-1-related (NUDR) protein is a novel transcriptional regulator with sequence similarity to developmental and oncogenic proteins. NUDR protein deletions were used to localize the DNA binding domain between amino acids 167 and 368, and site-specific DNA photocross-linking indicated at least two sites of protein-DNA contact within this domain. The DNA binding domain contains a proline-rich region and a region with similarity to a Myc-type helix-loop-helix domain but does not include the zinc finger motif at the C terminus. Deoxyribonuclease I protection assays confirmed the presence of multiple NUDR binding motifs (TTC(C/G)G) in the heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) promoter and also in the 5′-untranslated region (UTR) of hNUDR cDNA. NUDR produced a 65–70% repression of the hnRNP A2/B1 promoter activity, and NUDR binding motifs in the 5′-UTR were found to mediate this repression. NUDR-dependent repression was also observed when the 5′-UTR of NUDR was placed onto a heterologous thymidine kinase promoter in an analogous 5′-UTR position but not when placed upstream of transcription initiation. These results suggest that NUDR may regulate the in vivo expression of hnRNP A2/B1 and NUDR genes and imply that inactivation of NUDR could contribute to the overexpression of hnRNP A2/B1 observed in some human cancers. nuclear DEAF-1-related human NUDR monkey NUDR Deformed epidermal autoregulatory factor-1 heterogeneous nuclear ribonucleoprotein A2/B1 cDNA region corresponding to the 5′-untranslated region retinoic acid response element LIM-only polymerase chain reaction nuclear localization signal thymidine kinase chloramphenicol acetyltransferase electrophoretic mobility shift assay helix-loop-helix glutathione S-transferase polyacrylamide gel electrophoresis base pair(s) cytomegalovirus 5-[N-(4-azidobenzoyl)-3-aminoallyl]-deoxyuridine triphosphate NUDR1 is a transcriptional regulatory protein that was initially identified in a monkey kidney cell (CV-1) cDNA library through protein expression and binding to a radiolabeled retinoic acid response element (RARE) based on the sequence in human retinoic acid receptor β2 gene (1Huggenvik J.I. Michelson R.J. Collard M.W. Ziemba A.J. Gurley P. Mowen K.A. Mol. Endocrinol. 1998; 12: 1619-1639Crossref PubMed Scopus (68) Google Scholar). The encoded protein had 46% overall amino acid similarity toDrosophila Deformed epidermal autoregulatory factor-1 (DEAF-1) (2Gross C.T. McGinnis W. EMBO J. 1996; 15: 1961-1970Crossref PubMed Scopus (129) Google Scholar) and was therefore named nuclearDEAF-1 related (NUDR) (1Huggenvik J.I. Michelson R.J. Collard M.W. Ziemba A.J. Gurley P. Mowen K.A. Mol. Endocrinol. 1998; 12: 1619-1639Crossref PubMed Scopus (68) Google Scholar). DEAF-1 has been shown to bind to TTCG-containing motifs located adjacent to DNA binding sites for the Deformed homeodomain protein that occur in the promoter regions of Deformed and other Deformed-regulated genes, indicating that DEAF-1 may act as a transcriptional cofactor of Deformed (2Gross C.T. McGinnis W. EMBO J. 1996; 15: 1961-1970Crossref PubMed Scopus (129) Google Scholar). NUDR was also shown to recognize TTCG-containing motifs (1Huggenvik J.I. Michelson R.J. Collard M.W. Ziemba A.J. Gurley P. Mowen K.A. Mol. Endocrinol. 1998; 12: 1619-1639Crossref PubMed Scopus (68) Google Scholar), and the combination of sequence and functional similarities suggests that NUDR may be the mammalian homolog of DEAF-1. In our previous report, NUDR was shown to transcriptionally activate a minimal proenkephalin promoter, and activation was increased by the addition of synthetic RAREs placed 5′ of the promoter (1Huggenvik J.I. Michelson R.J. Collard M.W. Ziemba A.J. Gurley P. Mowen K.A. Mol. Endocrinol. 1998; 12: 1619-1639Crossref PubMed Scopus (68) Google Scholar). Because we were unable to demonstrate NUDR binding to proenkephalin sequences in either DNase I protection assays or mobility shift assays, we concluded that the activation of the proenkephalin promoter was likely to occur through protein-protein interactions (1Huggenvik J.I. Michelson R.J. Collard M.W. Ziemba A.J. Gurley P. Mowen K.A. Mol. Endocrinol. 1998; 12: 1619-1639Crossref PubMed Scopus (68) Google Scholar). Using a yeast two hybrid system, Sugihara et al. (3Sugihara T.M. Bach I. Kioussi C. Rosenfeld M.G. Andersen B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15418-15423Crossref PubMed Scopus (84) Google Scholar) identified the mouse homolog of NUDR (called mDEAF-1) through interaction with LMO-4, a new member of the LIM-only (LMO) family. LMOs contain two tandem repeats of the LIM zinc finger domain, which can associate tightly with another family of cofactors called Clims (also referred to as Ldb or NLI) to activate (4Bach I. Carriere C. Ostendorff H.P. Andersen B. Rosenfeld M.G. Genes Dev. 1997; 11: 1370-1380Crossref PubMed Scopus (268) Google Scholar) or inhibit (5Jurata L.W. Gill G.N. Mol. Cell. Biol. 1997; 17: 5688-5698Crossref PubMed Scopus (160) Google Scholar) transcription. Since LMO and Clim complexes have not been demonstrated to bind directly to DNA, they have been postulated to regulate transcription through the recruitment of DNA-binding proteins and the assembly of transcriptional complexes (3Sugihara T.M. Bach I. Kioussi C. Rosenfeld M.G. Andersen B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15418-15423Crossref PubMed Scopus (84) Google Scholar, 6Breen J.J. Agulnick A.D. Westphal H. Dawid I.B. J. Biol. Chem. 1998; 273: 4712-4717Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 7Jurata L.W. Pfaff S.L. Gill G.N. J. Biol. Chem. 1998; 273: 3152-3157Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Mouse NUDR was shown to interact with LMO-4, LMO-2, and Clim-2 in both in vitroand in vivo assays, and it was proposed that NUDR could provide the critical DNA binding function to LMO-Clim complexes (3Sugihara T.M. Bach I. Kioussi C. Rosenfeld M.G. Andersen B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15418-15423Crossref PubMed Scopus (84) Google Scholar). Because NUDR showed only moderate affinity for the RARE sequence, higher affinity sequences were selected from a library of random oligonucleotides through binding to recombinant NUDR protein and amplification by PCR (1Huggenvik J.I. Michelson R.J. Collard M.W. Ziemba A.J. Gurley P. Mowen K.A. Mol. Endocrinol. 1998; 12: 1619-1639Crossref PubMed Scopus (68) Google Scholar). Analysis of the selected sequences revealed the presence of one or more copies of TTCG and/or TTTCCG, and multiple sequence alignment suggested a NUDR binding consensus sequence of TTCGGGNNTTTCCGG (1Huggenvik J.I. Michelson R.J. Collard M.W. Ziemba A.J. Gurley P. Mowen K.A. Mol. Endocrinol. 1998; 12: 1619-1639Crossref PubMed Scopus (68) Google Scholar). Comparison of the NUDR binding motifs and the RARE sequence suggested that the original identification of NUDR was likely through the fortuitous binding of NUDR protein to the TTCGG sequence found between the RARE half-sites. The similarity in DNA recognition sequences between NUDR and Drosophila DEAF-1 implied that the DNA binding domain may be in a region of greater amino acid homology between the proteins, namely, the zinc finger homology region at the C terminus (56% similarity) and/or the nuclear domain (1Huggenvik J.I. Michelson R.J. Collard M.W. Ziemba A.J. Gurley P. Mowen K.A. Mol. Endocrinol. 1998; 12: 1619-1639Crossref PubMed Scopus (68) Google Scholar)/KDWK domain (2Gross C.T. McGinnis W. EMBO J. 1996; 15: 1961-1970Crossref PubMed Scopus (129) Google Scholar) located in the central region of the proteins (70% similarity). The distantly related zinc finger region of the progesterone receptor has been shown to be involved directly in the DNA binding domain, whereas the more homologous zinc finger region of MTG8 (ETO) has recently been shown to be involved in protein-protein interaction and the recruitment of nuclear corepressors and histone deacetylases (8Lutterbach B. Westendorf J.J. Linggi B. Patten A. Moniwa M. Davie J.R. Huynh K.D. Bardwell V.J. Lavinsky R.M. Rosenfeld M.G. Glass C. Seto E. Hiebert S.W. Mol. Cell. Biol. 1998; 18: 7176-7184Crossref PubMed Scopus (370) Google Scholar, 9Gelmetti V. Zhang J. Fanelli M. Minucci S. Pelicci P.G. Lazar M.A. Mol. Cell. Biol. 1998; 18: 7185-7191Crossref PubMed Scopus (430) Google Scholar). The nuclear/KDWK domains of NUDR and DEAF-1 have similarity with proteins from the SP100 family (1Huggenvik J.I. Michelson R.J. Collard M.W. Ziemba A.J. Gurley P. Mowen K.A. Mol. Endocrinol. 1998; 12: 1619-1639Crossref PubMed Scopus (68) Google Scholar, 10Dent A.L. Yewdell J. Puvion-Dutilleul F. Koken M.H.M. de The H. Staudt L.M. Blood. 1996; 88: 1423-1436Crossref PubMed Google Scholar). SP100 proteins are localized to subnuclear structures termed “nuclear bodies” and are thought to play a role in the etiology of acute promyelocytic leukemia (reviewed in Ref. 11Sternsdorf T. Grotzinger T. Jensen K. Will H. Immunobiology. 1997; 198: 307-331Crossref PubMed Scopus (133) Google Scholar). Recently it was demonstrated that SP100B associates with non-histone chromatin components that behave as transcriptional silencers, and when fused to a GAL4 DNA binding domain, SP100B can repress transcription (12Seeler J.S. Marchio A. Sitterlin D. Transy C. Dejean A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7316-7321Crossref PubMed Scopus (229) Google Scholar, 13Lehming N. Le Saux A. Schuller J. Ptashne M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7322-7326Crossref PubMed Scopus (149) Google Scholar). In this report, we identify the DNA binding domain in the central region of NUDR that includes the nuclear/KDWK domain and a Myc-type helix-loop-helix structure, and we demonstrate that there are at least two sites of protein contact with the DNA. The hnRNP A2/B1gene, a potential early biomarker of lung cancer (14Tockman M.S. Gupta P.K. Myers J.D. Frost J.K. Baylin S.B. Gold E.B. Chase A.M. Wilkinson P.H. Mulshine J.L. J. Clin. Oncol. 1988; 6: 1685-1693Crossref PubMed Scopus (209) Google Scholar, 15Zhou J. Mulshine J.L. Unsworth E.J. Scott F.M. Avis I.M. Vos M.D. Treston A.M. J. Biol. Chem. 1996; 271: 10760-10766Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 16Tockman M.S. J. Cell. Biochem. 1996; 25 (suppl.): 177-184Crossref Scopus (26) Google Scholar, 17Tockman M.S. Mulshine J.L. Piantodosi S. Erozan Y.S. Gupta P.K. Ruckdeschel J.C. Taylor P.R. Zhukov T. Zhou W.H. Qiao Y.L. Yao S.X. Clin. Cancer Res. 1997; 3: 2237-2246PubMed Google Scholar), is identified as a potential target gene of NUDR regulation by the presence of a NUDR binding consensus sequence within the promoter region. We show that NUDR represses transcription of the hnRNP A2/B1 promoter through a DNA binding-dependent mechanism and that NUDR binding motifs within the 5′-UTR are involved in this regulation. We hypothesize that elevated levels of hnRNP A2/B1 found in some cancers may be a consequence of the inactivation or deregulation of NUDR. The bacterial expression and purification of recombinant proteins for full-length human NUDR (hNUDR) and monkey NUDR (sNUDR) have been described previously (1Huggenvik J.I. Michelson R.J. Collard M.W. Ziemba A.J. Gurley P. Mowen K.A. Mol. Endocrinol. 1998; 12: 1619-1639Crossref PubMed Scopus (68) Google Scholar). To facilitate the construction of various deletion proteins and peptides, sNUDR was used to derive the peptide constructs G, H, I, J, K, and L, whereas hNUDR was used to derive all other constructs. The full-length proteins of sNUDR and hNUDR differ by only five amino acids and have virtually indistinguishable binding characteristics. For the deletion constructs B, C, D, and E, cDNA fragments of hNUDR were excised from the parent vector pBSSK (Stratagene, La Jolla, CA) with BspEI and SphI (B), BspEI and HincII (C), BspEI andAatII (D), and XcmI and EcoRI (E) followed by T4 DNA polymerase fill-in, ligation of BamHI linkers, and BamHI digestion. The resulting DNA fragments were ligated into the BamHI-digested pET-16b vector (Novagen, Inc. Madison, WI) for production of N-terminal histidine-tagged proteins. For the internal deletion construct F, the cDNA in pBSSK was digested with NcoI andAflII, filled in with T4 DNA polymerase, and religated. For the internal deletion construct G, a portion of the cDNA was excised with EcoNI and AatII and replaced with an SV40 nuclear localization signal (18Dingwall C. Laskey R.A. Trends Biochem. Sci. 1991; 16: 478-481Abstract Full Text PDF PubMed Scopus (1711) Google Scholar) formed by hybridization of the following two oligonucleotides: 5′-cCCAAAAAAGAAGAGAAAGGTAgacgt-3′ and 5′-cTACCTTTCTCTTCTTTTTTGGgct-3′, with the lowercase letters denotingBsmI and AatII cohesive ends. The cDNAs for constructs F and G were excised with BspEI andEcoRI and treated as described above to add BamHI linkers and then subcloned into the pET-16b vector. Recombinant histidine-tagged fusion proteins were purified as described previously for the full-length proteins except that the pH of the renaturation buffer was changed from 8.0 to 9.1 to adjust for differences in the isoelectric points of the deletion proteins. For construction of the glutathione S-transferase (GST) fusion peptides H, I, J, K, and L, cDNA fragments of sNUDR were excised from the parent plasmid with EcoNI andAatII (H), NcoI and AatII (I),ApaI and AatII (J), NcoI andAflII (K), and ApaI and AflII (L), treated as described above to add BamHI linkers, and subcloned into the BamHI site of pGEX-2T (Amersham Pharmacia Biotech). Recombinant GST fusion proteins were purified as described previously for GST-sNUDR (1Huggenvik J.I. Michelson R.J. Collard M.W. Ziemba A.J. Gurley P. Mowen K.A. Mol. Endocrinol. 1998; 12: 1619-1639Crossref PubMed Scopus (68) Google Scholar). To determine the concentration of each protein preparation, the recombinant proteins were subjected to SDS-PAGE, stained with Coomassie Blue, and compared with a bovine serum albumin standard curve using a Densitometer SI (Molecular Dynamics, Sunnyvale, CA). Recombinant proteins were incubated on ice with either nonspecific or specific oligonucleotide competitors (as indicated) in a 20-μl reaction containing 500 ng of poly(dI-dC), 100 mm KCl, 20 mm HEPES (pH 8.1), 2 mm dithiothreitol, 7% glycerol, and 0.05% Tween 20. The glucocorticoid response element (GRE) oligonucleotide used for nonspecific competitor (Fig.2 B) was formed by hybridization of two synthetic oligonucleotides, 5′-TCGACTGTACAGGATGTTCTAGCTACT-3′ and 5′-TCGAAGTAGCTAGAACATCCTGTACAG-3′ (19Kuiper G.G. de Ruiter P.E. Trapman J. Jenster G. Brinkmann A.O. Biochem. J. 1993; 296: 161-167Crossref PubMed Scopus (16) Google Scholar), and the N42–78 oligonucleotide was formed by hybridization of 5′-cgggatccTTCGGACTGATTCGGCTTCCCACTTCG-3′ and 5′-cgggatccCGAAGTTCCCCGAAGTGGGAAGCCGAA-3′. The lowercase letters denote BamHI restriction sites used for subcloning. Radioactive oligonucleotide probes were produced by fill-in reactions with Klenow and [α-32P]dATP. After 15 min, the reactions were mixed with 120–240 fmol of 32P-labeled probe and incubated an additional 15 min at 25 °C. Protein-DNA complexes were separated on 4% nondenaturing polyacrylamide gels (acrylamide:bis, 40:0.8, in 1× Tris-borate EDTA) at 120 volts for 3 h, and results were imaged using a 445 SI PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The following DNA fragments were isolated and radiolabeled by fill-in reaction with [α-32P]dATP and Klenow DNA polymerase:EcoRI/BspMI fragment of hNUDR8 cDNA (Fig.2 A), EcoRI/HpaI fragment of N42–78 that was inserted in the BamHI site of pBLCAT5 (Fig.2 C), HindIII/SmaI fragment of the hnRNP A2/B1 gene from hnRNPCAT (Fig. 5 A), andEcoRI/HincII fragment of the hnRNP A2/B1 gene from hnRNP in pBSKSII (Stratagene) (Fig. 5 B). DNase I protection assays were performed as described (1Huggenvik J.I. Michelson R.J. Collard M.W. Ziemba A.J. Gurley P. Mowen K.A. Mol. Endocrinol. 1998; 12: 1619-1639Crossref PubMed Scopus (68) Google Scholar), except that poly(dI-dC) was not used in Fig. 5. The synthesis of the photoreactive nucleotide analog AB-dUTP (Fig. 4 A) has been described elsewhere (20Bartholomew B. Kassavetis G.A. Braun B.R. Geiduschek E.P. EMBO J. 1990; 9: 2197-2205Crossref PubMed Scopus (136) Google Scholar, 21Bartholomew B. Tinker R.L. Kassavetis G.A. Geiduschek E.P. Methods Enzymol. 1995; 262: 476-494Crossref PubMed Scopus (34) Google Scholar). The N42–78 oligonucleotide of hNUDR8 was subcloned into the BamHI site of pBLCAT5 and then excised with HindIII and EcoRI. After biotinylating the DNA fragment, one of the DNA strands was selectively immobilized on paramagnetic beads and used as the template in the synthesis of the photoaffinity probe (22Lannutti B.J. Persinger J. Bartholomew B. Biochemistry. 1996; 35: 9821-9831Crossref PubMed Scopus (32) Google Scholar). Approximately one pmol of template was used in a reaction containing 6 pmol of the specific oligonucleotide 5′-CGGCTTCCCACTTCGGGG-3′, ∼4.5 pmol [α-32P]dCTP, 0.6 μm AB-dUTP, 0.6 μm dATP, and 0.25 units of exonuclease-free Klenow fragment of DNA polymerase I (Amersham Pharmacia Biotech) in a final volume of 20 μl. After 5 min at 37 °C, 2.5 μl of 5 mm unlabeled dNTPs were added and incubated at 37 °C for an additional 10 min. A second oligonucleotide, complementary to the multiple cloning region of pBLCAT5 and 5′ of the first oligonucleotide, was annealed to the immobilized DNA, and double-stranded DNA was synthesized with T4 DNA polymerase and subsequent treatment with T4 DNA ligase to seal the nicks. The double-stranded DNA photoaffinity probe was removed from the solid support by digestion with HincII. The binding reaction conditions for cross-linking were identical to those described for electrophoretic mobility shift assays (EMSAs), except that 2 fmol of the photoreactive probe were used. The cross-linking of the DNA and protein was performed by irradiation with UV light at 380 μW/cm2 for 2 min at a distance of 20 cm. The cross-linked samples were treated with DNase I and S1 nuclease as described (23Bartholomew B. Kassavetis G.A. Geiduschek E.P. Mol. Cell. Biol. 1991; 11: 5181-5189Crossref PubMed Scopus (125) Google Scholar) to remove all but the four labeled pyrimidines attached to the protein (labeled Intact, Fig. 4 D). An aliquot of the cross-linked sample was treated with 70% formic acid and 2% diphenylamine at 70 °C for 20 min to cleave the acid-labile Asp-Pro linkage at position 195–196 (labeled Asp-Pro cleavage, Fig.4 D). Samples were separated by SDS-PAGE on 10% polyacrylamide gels, followed by autoradiography. A 743-bp DNA fragment containing the hnRNP A2/B1 promoter (positions 1844–2586) was amplified by 35 cycles of PCR (GeneAmp 9600, Perkin-Elmer) using 600 ng of genomic DNA isolated from the human JEG-3 cell line, and the primers 5′-ACTTTCAGCAGCGAACTCTCC-3′ and 5′-AGTCGCTTCAGCCCGATTTC-3′. The PCR product was subcloned into theEcoRV site of pBSKSII (Stratagene) before excision withBamHI and HindIII and ligation into theBamHI/HindIII site of pBLCAT6 (24Boshart M. Kluppel M. Schmidt A. Schutz G. Luckow B. Gene. 1992; 110: 129-130Crossref PubMed Scopus (230) Google Scholar) to produce the reporter plasmid, hnRNPCAT. The hnRNP PCR product in pBSKSII was digested with BspEI, followed by a fill-in reaction and ligation of BamHI linkers. The DNA fragment containing the hnRNP promoter was excised with BamHI and HindIII and ligated into the BamHI/HindIII site of pBLCAT6 to produce the reporter hnRNPΔICAT. The reporter plasmids hnRNPΔIICAT and hnRNPΔI,IICAT were produced by excision of aXhoI DNA fragment from the plasmids hnRNPCAT and hnRNPΔICAT and subsequent religation. The 5′-UTR DNA was excised from hNUDR8 withEcoRI/BspEI, followed by a fill-in reaction and the addition of BamHI linkers. The 356-bp DNA fragment was ligated into the BamHI site or BglII site of pBLCAT5 (24Boshart M. Kluppel M. Schmidt A. Schutz G. Luckow B. Gene. 1992; 110: 129-130Crossref PubMed Scopus (230) Google Scholar) to produce the reporter constructs (h8N1–356)TKCAT and TK(h8N1–356)CAT, respectively. The 121-bpEcoRI/BspMI fragment of hNUDR8 was treated similarly and ligated into the BglII site of pBLCAT5 to produce TK(h8N1–121)CAT. To achieve high levels of protein expression in mammalian cells, the cDNAs for hNUDR, sNUDR, and hNUDR-R302T/K304T were subcloned into an expression plasmid that utilized the human cytomegalovirus immediate early gene promoter (CMV), as described previously (1Huggenvik J.I. Michelson R.J. Collard M.W. Ziemba A.J. Gurley P. Mowen K.A. Mol. Endocrinol. 1998; 12: 1619-1639Crossref PubMed Scopus (68) Google Scholar). The zinc finger homology region of hNUDR was deleted by HpaI andBsu36I digestion of the cDNA in pBSSKII, followed by fill-in and religation of the plasmid. The cDNA was excised withEcoRI, and BamHI linkers were added, digested with BamHI, and subcloned into the BglII site of pCMVNeo for the construct, CMVhNUDRaa1–505. TheNcoI/EcoRI DNA fragment containing the C-terminal portion of hNUDR was subcloned into the BglII site as above to produce the construct CMVhNUDRaa243–565. The primers 5′-CGCGGATCCACCATGGCAGCTCCCCTCAC-3′ and 5′-CTACCGGATCCTAGACGTCGCCCTGGGC-3′ were used in a 20-cycle PCR reaction with hNUDR as the template. The PCR product was digested withBamHI and subcloned into the BglII site of pCMVNeo for the construct, CMVhNUDRaa167–368. An EcoRI fragment of hNUDR from construct G in pBSSK was treated as above for the construct CMVhNUDRΔ255–367/SV40NLS. For each construct, the orientation and DNA sequence of the sites flanking an insertion or deletion were determined. Protein expression was confirmed in transfected CV-1 cells by immunofluorescence detection of NUDR, and the percentage of cells showing nuclear or cytoplasmic localization was estimated: hNUDRaa243–565 was 100% nuclear; hNUDR, hNUDRaa1–505 and hNUDRaa167–368 were 80% nuclear; hNUDRΔ255–367/SV40NLS was 64% nuclear; and hNUDR-R302T/K304T was 100% cytoplasmic (immunofluorescence data not shown). CV-1 cells were transfected with various reporter constructs and expression plasmids, cell extracts were prepared in 250 μl of homogenization buffer, and chloramphenicol acetyl transferase (CAT) activities were determined and normalized as described previously (1Huggenvik J.I. Michelson R.J. Collard M.W. Ziemba A.J. Gurley P. Mowen K.A. Mol. Endocrinol. 1998; 12: 1619-1639Crossref PubMed Scopus (68) Google Scholar). The normalized CAT activity determined for the indicated reporter construct was set at 100% and the effects of cotransfecting different expression plasmids with the reporter are shown relative to this activity. In a search for genes that contain the NUDR binding consensus sequence or TTCG motifs, we noted that multiple TTCG motifs occurred in the cDNA corresponding to the 5′-untranslated region (5′-UTRDNA) of human NUDR8 and monkey NUDR. Computer analysis of the NUDR cDNA for TTCG motifs demonstrates the presence of 14 motifs in the 5′-UTRDNA of hNUDR8, four motifs in the coding region, and the absence of motifs in the 3′-UTRDNA(Fig. 1 A). To test whether NUDR protein could bind these motifs, we performed an EMSA using radiolabeled DNA sequences from the 5′-UTRDNA (99 bp) and 3′-UTRDNA (130 bp) of hNUDR8. Low mobility complexes were observed with the 5′-UTRDNA probe when combined with 10 and 30 pmol of recombinant hNUDR protein, whereas no complexes were formed with the 3′-UTRDNA probe (Fig. 1 B). These data indicate that NUDR protein could potentially bind multiple TTCG motifs within its own 5′-UTRDNA in vivo. To examine the specific sequences within the 5′-UTRDNA that NUDR was binding, we utilized DNase I protection assays. In the presence of NUDR protein, a large 74-base pair region in the upper half of the 5′-UTRDNA was protected from nuclease digestion (Fig. 2 A). This protected region contained, but was not limited to, the TTCG motifs. Within this large region was a smaller area that was protected by the lowest protein concentration and contained two sets of TTCG pairs separated by six nucleotides (shown in bold capitals letters in Fig.2 D). Comparison of the two sets showed that 9 of the 15 nucleotides of each set were identical to each other and can be represented by the sequence, TTCGGNNNNNTTCGN. In addition, 9 nucleotides within each set of TTCG pairs (15 nucleotides in length) were identical to the derived NUDR binding consensus sequence, TTCGGGNNTTTCCGG. The six-nucleotide spacing between a pair of TTCGs would align the TTCG sequences on the same face of the DNA double helix within one turn and may allow optimal binding or interaction of one or more NUDR molecules. Other areas in the lower half of the 5′-UTRDNA of hNUDR8 were also protected from nuclease digestion by NUDR binding; some of these contained TTCG motifs whereas others did not (data not shown). Because the sequences and boundaries of the DNA protected by NUDR binding were not limited to TTCG or TTCG-like motifs, protein-protein interactions may extend the protection from DNase I to flanking sequences or may alter the DNA binding specificity. To facilitate subsequent studies, an oligonucleotide spanning the two sets of TTCG pairs from the 5′-UTRDNA of hNUDR8 (nucleotides N42–78, shown in bold capital letters in Fig.2 D) and including BamHI restriction sites at both ends was synthesized. In EMSA, radiolabeled N42–78 oligonucleotide was shifted by the addition of recombinant NUDR protein (Fig.2 B). DNA binding specificity of NUDR was shown by DNA binding competition with an excess of unlabeled N42–78, whereas no competition was observed with an excess of unlabeled oligonucleotide containing a glucocorticoid response element (Fig. 2 B). The N42–78 oligonucleotide was subcloned into a plasmid, and a DNA fragment containing this sequence was used in DNase I protection assays. As shown in Fig. 2 C, NUDR protein protected the entire N42–78 sequence from nuclease digestion. In addition, NUDR protein binding also produced DNase I hypersensitive sites in the sequence flanking N42–78 (Fig. 2 C) and in small regions of the 5′-UTRDNA of hNUDR8 (data not shown). We examined NUDR for a potential DNA binding domain, and the cysteine-rich, C terminus of the protein appeared as the most likely candidate. There are at least 20 protein sequences in the GenBank™ data base that have homology to this region of NUDR (1Huggenvik J.I. Michelson R.J. Collard M.W. Ziemba A.J. Gurley P. Mowen K.A. Mol. Endocrinol. 1998; 12: 1619-1639Crossref PubMed Scopus (68) Google Scholar), and several investigators have suggested that this arrangement of cysteines and histidines may constitute a zinc finger motif capable of interacting with DNA (2Gross C.T. McGinnis W. EMBO J. 1996; 15: 1961-1970Crossref PubMed Scopus (129) Google Scholar, 25Gamou T. Kitamura E. Hosoda F. Shimizu K. Shinohara K. Hayashi Y. Nagase T. Yokoyama Y. Ohki M. Blood. 1998; 91: 4028-4037Crossref PubMed Google Scholar, 26Zeng H. Jackson D.A. Oshima H. Simons Jr., S.S. J. Biol. Chem. 1998; 273: 17756-17762Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 27Feinstein P.G. Hogness D.S. Kornfeld K. Mann R.S. Genetics. 1995; 140: 573-586Crossref PubMed Google Scholar, 28Miyoshi H. Kozu T. Shimizu K. Enomoto K. Maseki N. Kaneko Y. Kamada N. Ohki M. EMBO J. 1993; 12: 2715-2721Crossref PubMed Scopus (458) Google Scholar, 29Kuroda Y. Suzuki N. Kataoka T. Science. 1993; 259: 683-686Crossref PubMed Scopus (120) Google Scholar). To investigate the region(s) of the protein responsible for DNA binding, we constructed various N-terminal, C-terminal, and internal deletions of NUDR (Fig. 3) and inserted them into bacterial expression vectors to produce fusion proteins with GST or an N-terminal histidine tag (see “Experimental Procedures”). Recombinant proteins were purified and assayed for their ability to bind the radiolabeled N42–78 sequence in EMSAs. We found deletion of the last 84 amino acids, which includes the potential zinc finger motif, had little effect on the DNA binding of NUDR (Fig. 3,construct B). Similarly, deletion and site-directed mutations of the zinc finger motif in DEAF-1 had no effect on its DNA binding properties (2Gross C.T. McGinnis W. EMBO J. 1996; 15: 1961-1970Crossref PubMed Scopus (129) Google Scholar). Furthermore, removal of up to 195 amino acids from the C terminus of NUDR (Fig. 3, constructs B-D) and up to 138 amino acids from the N terminus (data not shown) had little effect on NUDR binding of the N42–78 probe. DNA binding was compromised but not abolished by the deletion of the f" @default.
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• W2067229881 date "1999-10-01" @default.
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• W2067229881 title "Nuclear DEAF-1-related (NUDR) Protein Contains a Novel DNA Binding Domain and Represses Transcription of the Heterogeneous Nuclear Ribonucleoprotein A2/B1 Promoter" @default.
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• W2067229881 doi "https://doi.org/10.1074/jbc.274.43.30510" @default.
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