Barrier to Autointegration Factor Interacts with the Cone-Rod Homeobox and Represses Its Transactivation Function
2002; Elsevier BV; Volume: 277; Issue: 45 Linguagem: Inglês
10.1074/jbc.m207952200
ISSN1083-351X
AutoresXuejiao Wang, Siqun Xu, Carlo Rivolta, Lili Y. Li, Guang-Hua Peng, Prabodh K. Swain, Ching-Hwa Sung, Anand Swaroop, Eliot L. Berson, Thaddeus P. Dryja, Shiming Chen,
Tópico(s)Cellular transport and secretion
ResumoCrx (cone-rod homeobox) is a homeodomain transcription factor implicated in regulating the expression of photoreceptor and pineal genes. To identify proteins that interact with Crx in the retina, we carried out a yeast two-hybrid screen of a retinal cDNA library. One of the identified clones encodes Baf (barrier toautointegration factor), which was previously shown to have a role in mitosis and retroviral integration. Additional biochemical assays provided supporting evidence for a Baf-Crx interaction. The Baf protein is detectable in all nuclear layers of the mouse retina, including the photoreceptors and the bipolar cells where Crx is expressed. Transient transfection assays with a rhodopsin-luciferase reporter in HEK293 cells demonstrate that overexpression of Baf represses Crx-mediated transactivation, suggesting that Baf acts as a negative regulator of Crx. Consistent with this role for Baf, an E80A mutation of CRX associated with cone-rod dystrophy has a higher than normal transactivation potency but a reduced interaction with Baf. Although our studies did not identify a causative Baf mutation in retinopathies, we suggest that Baf may contribute to the phenotype of a photoreceptor degenerative disease by modifying the activity of Crx. In view of the ubiquitous expression of Baf, we hypothesize that it may play a role in regulating tissue- or cell type-specific gene expression by interacting with homeodomain transcription factors. Crx (cone-rod homeobox) is a homeodomain transcription factor implicated in regulating the expression of photoreceptor and pineal genes. To identify proteins that interact with Crx in the retina, we carried out a yeast two-hybrid screen of a retinal cDNA library. One of the identified clones encodes Baf (barrier toautointegration factor), which was previously shown to have a role in mitosis and retroviral integration. Additional biochemical assays provided supporting evidence for a Baf-Crx interaction. The Baf protein is detectable in all nuclear layers of the mouse retina, including the photoreceptors and the bipolar cells where Crx is expressed. Transient transfection assays with a rhodopsin-luciferase reporter in HEK293 cells demonstrate that overexpression of Baf represses Crx-mediated transactivation, suggesting that Baf acts as a negative regulator of Crx. Consistent with this role for Baf, an E80A mutation of CRX associated with cone-rod dystrophy has a higher than normal transactivation potency but a reduced interaction with Baf. Although our studies did not identify a causative Baf mutation in retinopathies, we suggest that Baf may contribute to the phenotype of a photoreceptor degenerative disease by modifying the activity of Crx. In view of the ubiquitous expression of Baf, we hypothesize that it may play a role in regulating tissue- or cell type-specific gene expression by interacting with homeodomain transcription factors. homeodomain barrier to autointegration factor (different from the BAF proteins in the SWI/SNF-like BAF chromatin remodeling complexes) DNA-binding domain cAMP-response element-binding protein glutathione S-transferase nitrilotriacetic acid electrophoretic mobility shift assay phosphate-buffered saline preintegration complex group of overlapping clones 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside Bardet-Biedl syndrome-1 Development and maintenance of photoreceptor function in mammalian retina requires the expression of photoreceptor-specific or photoreceptor-enriched genes. Under- or over-expression of these genes, such as the visual pigment rhodopsin (1Humphries M.M. Rancourt D. Farrar G.J. Kenna P. Hazel M. Bush R.A. Sieving P.A. Sheils D.M. McNally N. Creighton P. Erven A. Boros A. 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It acts synergistically with Nrl (5Chen S. Wang Q.-L. Nie Z. Sun H. Lennon G. Copeland N.G. Gilbert D.J. Jenkins N.A. Zack D.J. Neuron. 1997; 19: 1017-1030Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar), a bZIP transcription factor expressed specifically in rod photoreceptors (12Swain P.K. Hicks D. Mears A.J. Apel I.J. Smith J.E. John S.K. Hendrickson A. Milam A.H. Swaroop A. J. Biol. Chem. 2001; 276: 36824-36830Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The Crx protein includes a homeodomain (HD)1near its N terminus, followed by a glutamine-rich (Gln) region, a basic region, a WSP (SIWSPASESP) region, and an Otx tail region that all share homology with corresponding regions of Otx1 and Otx2 (5Chen S. Wang Q.-L. Nie Z. Sun H. Lennon G. Copeland N.G. Gilbert D.J. Jenkins N.A. Zack D.J. Neuron. 1997; 19: 1017-1030Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar). The Crx HD is of the K50 subtype (with lysine at its 50th residue) of the paired-like class (5Chen S. Wang Q.-L. Nie Z. Sun H. Lennon G. Copeland N.G. Gilbert D.J. Jenkins N.A. Zack D.J. Neuron. 1997; 19: 1017-1030Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar), and it is responsible for binding to target DNA (5Chen S. Wang Q.-L. Nie Z. Sun H. Lennon G. Copeland N.G. Gilbert D.J. Jenkins N.A. Zack D.J. Neuron. 1997; 19: 1017-1030Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar, 13Chen S. Wang Q.-L., Xu, S. Liu Y. Lili Y.L. Wang Y. Zack D.J. Hum. Mol. Genet. 2002; 11: 873-884Crossref PubMed Scopus (53) Google Scholar), the nuclear localization of the Crx protein (14Fei Y. Hughes T.E. Invest. Ophthalmol. Vis. Sci. 2000; 41: 2849-2856PubMed Google Scholar), and mediating a physical and functional interaction with Nrl (15Mitton K.P. Swain P.K. Chen S., Xu, S. Zack D.J. Swaroop A. J. Biol. Chem. 2000; 275: 29794-29799Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). In vitro protein-DNA binding assays demonstrated that the Crx HD binds to at least three target sites in the rhodopsin promoter, all with a (C/T)TAATCC consensus sequence, including a high affinity site called BAT-1 and two low affinity sites called Ret-1 and Ret-4 (5Chen S. Wang Q.-L. Nie Z. Sun H. Lennon G. Copeland N.G. Gilbert D.J. Jenkins N.A. Zack D.J. Neuron. 1997; 19: 1017-1030Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar). Transient transfection assays in HEK293 cells demonstrated that the C-terminal region of Crx (between amino acids 107 and 284) contains the transactivation domains AD-1 and AD-2, which are important for its ability to activate promoters (13Chen S. Wang Q.-L., Xu, S. Liu Y. Lili Y.L. Wang Y. Zack D.J. Hum. Mol. Genet. 2002; 11: 873-884Crossref PubMed Scopus (53) Google Scholar). Mutant mice that are homozygous for a null allele of Crx(Crx −/−) fail to develop outer segments of the photoreceptors, which subsequently undergo progressive degeneration (8Furukawa T. Morrow E.M., Li, T. Davis F.C. Cepko C.L. Nat. Genet. 1999; 23: 466-470Crossref PubMed Scopus (446) Google Scholar). The expression levels of many photoreceptor genes are altered in the Crx −/− mouse retina, indicating that these genes are either direct or indirect targets of Crx (8Furukawa T. Morrow E.M., Li, T. Davis F.C. Cepko C.L. Nat. Genet. 1999; 23: 466-470Crossref PubMed Scopus (446) Google Scholar, 16Blackshaw S. Fraioli R.E. Furukawa T. Cepko C.L. Cell. 2001; 107: 579-589Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 17Livesey F.J. Furukawa T. Steffen M.A. Church G.M. Cepko C.L. Curr. Biol. 2000; 10: 301-310Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). 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Genet. 1998; 18: 311-312Crossref PubMed Scopus (253) Google Scholar, 23Swaroop A. Wang Q.L., Wu, W. Cook J. Coats C., Xu, S. Chen S. Zack D.J. Sieving P.A. Hum. Mol. Genet. 1999; 8: 299-305Crossref PubMed Scopus (156) Google Scholar, 24Rivolta C. Peck N.E. Fulton A.B. Fishman G.A. Berson E.L. Dryja T.P. Hum. Mutat. 2001; 18: 550-551Crossref PubMed Scopus (28) Google Scholar). In vitro functional analyses of some of these mutations demonstrated reduced binding to and/ortrans-activation of the rhodopsin promoter (13Chen S. Wang Q.-L., Xu, S. Liu Y. Lili Y.L. Wang Y. Zack D.J. Hum. Mol. Genet. 2002; 11: 873-884Crossref PubMed Scopus (53) Google Scholar, 21Swain P.K. Chen S. Wang Q.-L. Affatigato L.M. Coats C.L. Brady K.D. Fishman G.A. Jacobson S.G. Swaroop A. Stone E.M. Sieving P.A. Zack D.J. Neuron. 1997; 19: 1329-1336Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 23Swaroop A. Wang Q.L., Wu, W. Cook J. Coats C., Xu, S. Chen S. Zack D.J. Sieving P.A. Hum. Mol. Genet. 1999; 8: 299-305Crossref PubMed Scopus (156) Google Scholar). These results, combined with the mouse studies, provide strong evidence that Crx is required for both the development and maintenance of photoreceptors by acting as an important regulator of photoreceptor gene expression. Several studies have identified Crx-interacting proteins, such as Nrl (15Mitton K.P. Swain P.K. Chen S., Xu, S. Zack D.J. Swaroop A. J. Biol. Chem. 2000; 275: 29794-29799Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar), CREB-binding protein/p300 (25Yanagi Y. Masuhiro Y. Mori M. Yanagisawa J. Kato S. Biochem. Biophys. Res. Commun. 2000; 269: 410-414Crossref PubMed Scopus (26) Google Scholar), phosducin and phosducin-like proteins (PhLP1 and PhLOP1) (26Zhu X. Craft C.M. Mol. Cell. Biol. 2000; 20: 5216-5226Crossref PubMed Scopus (64) Google Scholar), the nonhistone high mobility group protein HMGA1 (formerly HMG19Y) (27Chau K.Y. Munshi N. Keane-Myers A. Cheung-Chau K.W. Tai A.K. Manfioletti G. Dorey C.K. Thanos D. Zack D.J. Ono S.J. J. Neurosci. 2000; 20: 7317-7324Crossref PubMed Google Scholar), and ataxin-7 (28La Spada A.R., Fu, Y. Sopher B.L. Libby R.T. Wang X., Li, L.Y. Einum D.D. Huang J. Possin D.E. Smith A.C. Martinez R.A. Koszdin K.L. Treuting P.M. Ware C.B. Hurley J.B. Ptacek L.J. Chen S. Neuron. 2001; 31: 913-927Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Among these, Nrl, HMGA1, and CREB-binding protein/p300 enhance, whereas Phd (PhLPs) and ataxin-7 repress, the transactivation activity of Crx. To further enhance our understanding of Crx function, we carried out a protein-protein interaction screen of a bovine retinal cDNA library in yeast with Crx as bait. Here, we report the identification of barrier-to-autointegration factor (Baf) as a Crx-interacting protein and a detailed characterization of the physical and functional interaction of Crx and Baf. Our studies suggest a novel cellular function for Baf that is directly linked to transcriptional regulation of tissue-specific genes in addition to its reported role in chromatin decondensation and nuclear envelope assembly during mitosis (29Haraguchi T. Koujin T. Segura-Totten M. Lee K.K. Matsuoka Y. Yoneda Y. Wilson K.L. Hiraoka Y. J. Cell Sci. 2001; 114: 4575-4585Crossref PubMed Google Scholar, 30Shumaker D.K. Lee K.K. Tanhehco Y.C. Craigie R. Wilson K.L. EMBO J. 2001; 20: 1754-1764Crossref PubMed Scopus (168) Google Scholar, 31Segura-Totten M. Kowalski A.K. Craigie R. Wilson K.L. J. Cell Biol. 2002; 158: 475-485Crossref PubMed Scopus (154) Google Scholar). The two-hybrid assays in yeast (32Fields S. Song O. Nature. 1989; 340: 245-246Crossref PubMed Scopus (4880) Google Scholar) were carried out using the Matchmaker Two-Hybrid System 2 (BD-Biosciences CLONTECH, Palo Alto, CA) with a dual reporter strain, Y190, as described previously (15Mitton K.P. Swain P.K. Chen S., Xu, S. Zack D.J. Swaroop A. J. Biol. Chem. 2000; 275: 29794-29799Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 28La Spada A.R., Fu, Y. Sopher B.L. Libby R.T. Wang X., Li, L.Y. Einum D.D. Huang J. Possin D.E. Smith A.C. Martinez R.A. Koszdin K.L. Treuting P.M. Ware C.B. Hurley J.B. Ptacek L.J. Chen S. Neuron. 2001; 31: 913-927Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 33Bessant D.A. Payne A.M. Mitton K.P. Wang Q.L. Swain P.K. Plant C. Bird A.C. Zack D.J. Swaroop A. Bhattacharya S.S. Nat. Genet. 1999; 21: 355-356Crossref PubMed Scopus (152) Google Scholar). The "bait" construct Crx-HD-pAS2 contains the bovine Crx homeodomain and its flanking sequences (amino acid residues 34–107) fused in frame with the Gal4-DNA binding domain (dbd) in the pAS2-1 vector. A bovine retinal cDNA library (34Tai A.W. Chuang J.Z. Bode C. Wolfrum U. Sung C.H. Cell. 1999; 97: 877-887Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar) generated in pACTII (the prey vector with a Gal4 activation domain) was used for screening Crx-HD-interacting clones. The reporter strain Y190 was transformed with the bait vector Crx-HD and tested for a basal expression level of the dual reporter genes His3 andlacZ using 3-amino-1,2,4-triazole (3-AT; a competitive inhibitor of the His3 protein) and a colony lift X-gal filter assay, respectively, as described in the CLONTECH manual. The bait-containing Y190 cells were subsequently transformed with 20 μg of DNA from the retinal cDNA library. Colonies that grew on SD-Trp−, Leu−, His− medium supplemented with 15 mm 3-AT were considered as "positives," and they were verified using X-gal filter assays. Y190 transformants containing the known interacting protein partners, Snf1 (in pAS1) and Snf4 (in pACTII), were used as controls for a positive interaction (35Celenza J.L. Eng F.J. Carlson M. Mol. Cell. Biol. 1989; 9: 5045-5054Crossref PubMed Scopus (147) Google Scholar). Yeast DNA harboring a mixture of the bait and prey plasmids was prepared from each of the clones that tested positive by the dual reporter assay. The prey plasmids in the positive colonies were recovered by electroporation of the yeast DNA intoEscherichia coli strain DH5α, selection of E. coli colonies containing the plasmids, and subsequent amplification and purification of plasmid DNA. False positives were further eliminated by retransforming the prey DNA to the original bait strain and a strain harboring the unrelated bait Snf1. Library clones that were positive for interaction with Crx-HD but not with Snf1 were sequenced and characterized. To confirm the interaction of Crx and the product of the Bafgene identified by the yeast two-hybrid screening, an insert swap between the bait and prey was performed. The Baf insert was PCR-amplified and cloned into the pAS2 bait vector at theNdeI site (filled in) with the predicted open reading frame fused in-fame with Gal4-dbd. The full-length coding region of bovineCrx was cloned in-frame with Gal4 activation domain in pACTII at the BamHI (5′) and XhoI (3′) site. The resulting Baf bait and Crx prey constructs were co-transformed into the yeast Y190 for 3-AT and X-gal assays as described above. To express and purify the bovine Baf (bBaf) protein from E. coli, a PCR-amplified cDNA corresponding to the open reading frame ofbBaf was cloned in frame with the His6 tag of pTrcHisA (Invitrogen) at the BamHI (5′) and EcoRI (3′) site. For mammalian expression and in vitrotranscription/translation, a PCR-amplified cDNA containing thebBaf coding region fused in frame with an N-terminal Myc tag was generated and cloned into pcDNA3.1(+) (Invitrogen) at theHindIII and XbaI site (bBaf-pcDNA3.1(+)/myc). All of the PCR amplifications were performed using the high fidelity Pfu DNA polymerase (Stratagene, La Jolla, CA). The frame of each fusion protein was confirmed by sequencing using an ABI Prism DNA sequencing kit and ABI Prism 310 Genetic Analyzer (PerkinElmer Life Sciences). The bacterial expression vector Crx-HD-GST containing the Crx homeodomain fused with the GST tag in pGEX-4T-2 (Amersham Biosciences), the mammalian expression vectors carrying the coding cDNA of the human (hCRX) and bovine (bCrx) Crx and its deletion series, in frame with the Xpress tag in pcDNA3.1/HisC (Invitrogen), and the hCRX constructs carrying missense mutations in the HD were described previously (5Chen S. Wang Q.-L. Nie Z. Sun H. Lennon G. Copeland N.G. Gilbert D.J. Jenkins N.A. Zack D.J. Neuron. 1997; 19: 1017-1030Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar, 15Mitton K.P. Swain P.K. Chen S., Xu, S. Zack D.J. Swaroop A. J. Biol. Chem. 2000; 275: 29794-29799Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). The GST and Crx-HD-GST proteins were expressed in E. coli and purified using glutathione-Sepharose beads as described previously (5Chen S. Wang Q.-L. Nie Z. Sun H. Lennon G. Copeland N.G. Gilbert D.J. Jenkins N.A. Zack D.J. Neuron. 1997; 19: 1017-1030Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar). The His6-tagged Baf (Baf-His) and the His6 tag alone (His) were expressed in the E. coli BL21 strain (Stratagene) and purified using Ni2+-NTA-agarose resin (Qiagen, Valencia, CA) under denaturing conditions with 6 mguanidine-HCl according to the manufacturer's instructions with some modifications. In brief, bacterial cells were lysed in a lysis buffer (0.1 m NaH2PO4, 10 mmTris-Cl, 6 m guanidine HCl, pH 8.0), bound to the Ni2+-NTA resin using the batch method, washed with the lysis buffer supplemented with 5 mm imidazole, and eluted with an imidazole gradient (20–700 mm) in the lysis buffer. The fractions containing either Baf-His or His were pooled after SDS-PAGE analysis of each fraction. The affinity-purified proteins were renatured by dialysis against a storage buffer containing 25 mm Hepes, pH 7.6, 60 mm KCl, 0.1 mm EDTA, 2 mm dithiothreitol, 10% glycerol, and 0.1 mm phenylmethylsulfonyl fluoride. The resulting protein preparations were quantified using Bio-Rad Protein Assay Kit II and analyzed by SDS-PAGE and immunoblots with the anti-polyhistidine monoclonal antibody (Sigma-Aldrich) and an anti-Baf antibody (see below). Each protein preparation was also analyzed for possible DNA contamination by UV spectrum measurement (at 260 and 280 nm) and agarose gel electrophoresis (1% with ethidium bromide). P261, a rabbit polyclonal antibody to Crx, was described previously (28La Spada A.R., Fu, Y. Sopher B.L. Libby R.T. Wang X., Li, L.Y. Einum D.D. Huang J. Possin D.E. Smith A.C. Martinez R.A. Koszdin K.L. Treuting P.M. Ware C.B. Hurley J.B. Ptacek L.J. Chen S. Neuron. 2001; 31: 913-927Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). A polyclonal antibody against human BAF was generated in rabbits using purified recombinant human BAF expressed in E. coli. This antibody and its control serum (preimmune) were kindly provided by Robert Craigie. Crude protein lysates were prepared by homogenization of frozen tissue samples or cell pellets in a 3-fold volume of a sample buffer containing 62.5 mm Tris-HCl, pH 6.8, 4% SDS, 200 mmdithiothreitol, 10% glycerol, and 0.001% bromphenol blue using a Pro250 homogenizer (PRO Scientific Inc., Monroe, CT), followed by immediately boiling for 10 min. Eight μl of each protein sample were resolved by SDS-PAGE (15% gel) and transferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA), which was probed with the primary antibody against Baf at a 1:1000 dilution, and the signal was detected by a horseradish peroxidase-conjugated anti-rabbit-IgG secondary antibody at a 1:1000 dilution and the ECL kit (AmershamBiosciences). Co-immunoprecipitation assays with in vitrotranslated proteins were carried out essentially as described previously (28La Spada A.R., Fu, Y. Sopher B.L. Libby R.T. Wang X., Li, L.Y. Einum D.D. Huang J. Possin D.E. Smith A.C. Martinez R.A. Koszdin K.L. Treuting P.M. Ware C.B. Hurley J.B. Ptacek L.J. Chen S. Neuron. 2001; 31: 913-927Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar) with minor modifications. 1% Triton X-100 was included in the wash buffer (50 mm Tris-Cl (pH 7.5), 150 mm NaCl, 1% Triton X-100), and 2–4 μl of a specific antibody were used for co-immunoprecipitation. Antibodies used for co-immunoprecipitation include anti-Crx P261 (28La Spada A.R., Fu, Y. Sopher B.L. Libby R.T. Wang X., Li, L.Y. Einum D.D. Huang J. Possin D.E. Smith A.C. Martinez R.A. Koszdin K.L. Treuting P.M. Ware C.B. Hurley J.B. Ptacek L.J. Chen S. Neuron. 2001; 31: 913-927Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar), anti-Myc (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-Chx10 (a gift of Dr. Connie Cepko), and anti-Nrl (12Swain P.K. Hicks D. Mears A.J. Apel I.J. Smith J.E. John S.K. Hendrickson A. Milam A.H. Swaroop A. J. Biol. Chem. 2001; 276: 36824-36830Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Co-immunoprecipitated proteins were resolved by SDS-PAGE and quantified using a Storm 860 PhosphorImager system and ImageQuant 5.0 analytic program (Amersham Biosciences). For co-immunoprecipitation assays with tissue extracts, whole cell extracts were prepared by homogenizing tissue samples in a 3-fold volume of a whole cell lysis buffer (50 mm Tris-Cl (pH 7.5), 450 mm NaCl, 1% Triton X-100, and 10% glycerol with a mixture of protease inhibitors (Roche Molecular Biochemicals)). After a brief centrifugation for 5 min at 10,000 × g, 200 μl of the supernatants were incubated with 2 μl of the anti-Crx P261 antibody for 2 h at 4 °C, followed by the addition of 45 μl of 50% Protein A-Sepharose beads and gentle mixing on a rotator at 4 °C overnight. After being washed five times with the wash buffer (50 mm Tris-HCl (pH 7.5), 150 mm NaCl, 1% Triton X-100), the bound proteins were eluted and analyzed by SDS-PAGE and immunoblots with the anti-Baf antibody. For pull-down assays, 100 ng of the purified Crx-HD-GST protein was gently mixed with 45 μl of Ni2+-NTA beads coupled with the Baf-His or His protein in 100 μl of a binding buffer (1× PBS, 0.01% Nonidet P-40) at 4 °C overnight. In a reciprocal approach, 50 ng of the purified Baf-His protein in 100 μl of the binding buffer was incubated with glutathione-Sepharose beads coupled with Crx-HD-GST or GST. Proteins bound to the beads were washed five times with the wash buffer, eluted from the beads by boiling in 20 μl of a SDS-PAGE loading buffer, resolved by SDS-PAGE (11–15% gel), and detected by immunoblots with the anti-GST antibody (Sigma) (for assays with the His protein beads) or anti-Baf antibody (for assays with the GST protein beads). For EMSAs with Crx-HD peptides, the GST tag was removed from the Crx-HD-GST proteins by digestion with thrombin protease (Amersham Biosciences) at a concentration of 2 units/μg of fusion protein (room temperature for 4 h). EMSAs with recombinant proteins and bovine retinal nuclear extracts were performed as described (5Chen S. Wang Q.-L. Nie Z. Sun H. Lennon G. Copeland N.G. Gilbert D.J. Jenkins N.A. Zack D.J. Neuron. 1997; 19: 1017-1030Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar). For supershift EMSAs, the Crx-HD peptides or the bovine retinal nuclear extract were preincubated with increasing amounts (in μl) of the Baf-His protein or the His tag control in the reaction buffer for 10 min on ice prior to the addition of the probes. The reactions were incubated on ice for an additional 30 min and resolved by native PAGE (5% gel). HEK293 cells were cultured on 35-mm plates, transfected using the calcium phosphate method, and analyzed using dual luciferase assays as described by Chenet al. (13Chen S. Wang Q.-L., Xu, S. Liu Y. Lili Y.L. Wang Y. Zack D.J. Hum. Mol. Genet. 2002; 11: 873-884Crossref PubMed Scopus (53) Google Scholar). Typically, a total of 3.0 μg of DNA was used for each transfection, including 2 μg of the rhodopsin-luciferase reporter pBR130-luc (36Kumar R. Chen S. Scheurer D. Wang Q.L. Duh E. Sung C.H. Rehemtulla A. Swaroop A. Adler R. Zack D.J. J. Biol. Chem. 1996; 271: 29612-29618Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), 1 ng of the Renilla luciferase reporter pRL-CMV (Promega, Madison, WI) as an internal control for transfection efficiency, 100 ng of a mammalian vector expressing the transcription activator Crx (bCrx-pcDNA3.1/HisC), and/or Nrl (pMT-NRL), 50–800 ng of the Baf expression vector (bBaf-pcDNA3.1(+)/myc), and various amounts of the carrier DNA (pcDNA3.1/HisC) to keep the amount of total DNA constant. Each sample was done in duplicate, and at least four independent experiments were performed. The significance of the results was calculated using Student's t test, and it was assumed that each pair of samples under comparison had equal variances. For analyzing the effect of Baf on transactivation activity of the Gal4 fusion proteins and c-Jun/c-Fos, two different luciferase reporters were used: a Gal4-responsive luciferase construct pFR-luc (Stratagene) for assays with Gal4dbd-Crx-(111–299) or Gal4-VP16 (13Chen S. Wang Q.-L., Xu, S. Liu Y. Lili Y.L. Wang Y. Zack D.J. Hum. Mol. Genet. 2002; 11: 873-884Crossref PubMed Scopus (53) Google Scholar) and a collagenase promoter-luciferase construct (36Kumar R. Chen S. Scheurer D. Wang Q.L. Duh E. Sung C.H. Rehemtulla A. Swaroop A. Adler R. Zack D.J. J. Biol. Chem. 1996; 271: 29612-29618Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) for assays with c-Jun/c-Fos. HEK293 cells were cultured on poly-d-lysine (100 μg/ml; Sigma)-coated glass coverslips and co-transfected with 1 μg of each of the mammalian cell expression constructs bCrx-pcDNA3.1/HisC and bBaf-pcDNA3.1(+)/myc. At 24 h after transfection, the cells were fi
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