SAFB2, a New Scaffold Attachment Factor Homolog and Estrogen Receptor Corepressor
2003; Elsevier BV; Volume: 278; Issue: 22 Linguagem: Inglês
10.1074/jbc.m212988200
ISSN1083-351X
AutoresSteven M. Townson, Klaudia M. Dobrzycka, Adrian V. Lee, Mamie Air, Wanleng Deng, Kaiyan Kang, Shiming Jiang, Noriyuki Kioka, Kai Michaelis, Steffi Oesterreich,
Tópico(s)Circadian rhythm and melatonin
ResumoWe have characterized previously the nuclear matrix protein/scaffold attachment factor (SAFB) as an estrogen receptor corepressor and as a potential tumor suppressor gene in breast cancer. A search of the human genome for other potential SAFB family members revealed that KIAA00138 (now designated as SAFB2) has high homology to SAFB (now designated as SAFB1). SAFB1 and SAFB2 are mapped adjacent to each other on chromosome 19p13.3 and are arranged in a bidirectional divergent configuration (head to head), being separated by a short (<500 bp) GC-rich intergenic region that can function as a bidirectional promoter. SAFB1 and SAFB2 share common functions but also have unique properties. As shown previously for SAFB1, SAFB2 functions as an estrogen receptor corepressor, and its overexpression results in inhibition of proliferation. SAFB1 and SAFB2 interact directly through a C-terminal domain, resulting in additive repression activity. They are coexpressed in a number of tissues, but unlike SAFB1, which is exclusively nuclear, SAFB2 is found in the cytoplasm as well as the nucleus. Consistent with its cytoplasmic localization, we detected an interaction between SAFB2 and vinexin, a protein involved in linking signaling to the cytoskeleton. Our findings suggest that evolutionary duplication of the SAFB gene has allowed it to retain crucial functions, but also to gain novel functions in the cytoplasm and/or nucleus. We have characterized previously the nuclear matrix protein/scaffold attachment factor (SAFB) as an estrogen receptor corepressor and as a potential tumor suppressor gene in breast cancer. A search of the human genome for other potential SAFB family members revealed that KIAA00138 (now designated as SAFB2) has high homology to SAFB (now designated as SAFB1). SAFB1 and SAFB2 are mapped adjacent to each other on chromosome 19p13.3 and are arranged in a bidirectional divergent configuration (head to head), being separated by a short (<500 bp) GC-rich intergenic region that can function as a bidirectional promoter. SAFB1 and SAFB2 share common functions but also have unique properties. As shown previously for SAFB1, SAFB2 functions as an estrogen receptor corepressor, and its overexpression results in inhibition of proliferation. SAFB1 and SAFB2 interact directly through a C-terminal domain, resulting in additive repression activity. They are coexpressed in a number of tissues, but unlike SAFB1, which is exclusively nuclear, SAFB2 is found in the cytoplasm as well as the nucleus. Consistent with its cytoplasmic localization, we detected an interaction between SAFB2 and vinexin, a protein involved in linking signaling to the cytoskeleton. Our findings suggest that evolutionary duplication of the SAFB gene has allowed it to retain crucial functions, but also to gain novel functions in the cytoplasm and/or nucleus. Scaffold attachment factor B (SAFB, 1The abbreviations used are: SAFB, scaffold attachment factor B; DSP, dithiobis-(succinimidylproprionate); ER, estrogen receptor; ERE, estrogen response element; FACS, fluorescence-activated cell sorter; GFP, green fluorescent protein; GST, glutathione S-transferase; HA, hemagglutinin; LUC, luciferase; NLS, nuclear localization signal; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid RRM, RNA recognition motif; RT-PCR, reverse transcription-PCR; TK, thymidine kinase; SD, synthetic dropout.1The abbreviations used are: SAFB, scaffold attachment factor B; DSP, dithiobis-(succinimidylproprionate); ER, estrogen receptor; ERE, estrogen response element; FACS, fluorescence-activated cell sorter; GFP, green fluorescent protein; GST, glutathione S-transferase; HA, hemagglutinin; LUC, luciferase; NLS, nuclear localization signal; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid RRM, RNA recognition motif; RT-PCR, reverse transcription-PCR; TK, thymidine kinase; SD, synthetic dropout. also named HET and HAP in the literature) was originally identified based on its ability to bind to scaffold/matrix attachment regions (1Renz A. Fackelmayer F.O. Nucleic Acids Res. 1996; 24: 843-849Crossref PubMed Scopus (110) Google Scholar) and as a protein binding to the small heat shock protein hsp27 gene promoter (2Oesterreich S. Lee A.V. Sullivan T.M. Samuel S.K. Davie J.R. Fuqua S.A. J. Cell. Biochem. 1997; 67: 275-286Crossref PubMed Scopus (86) Google Scholar). SAFB is a large protein (130 kDa) with a number of putative functional domains. The function of many of these domains is still unclear but can be inferred from the roles of similar domains in other proteins. The N terminus contains an SAF box (3Kipp M. Gohring F. Ostendorp T. van Drunen C.M. van Driel R. Przybylski M. Fackelmayer F.O. Mol. Cell. Biol. 2000; 20: 7480-7489Crossref PubMed Scopus (160) Google Scholar) (also called an SAP domain (4Aravind L. Koonin E.V. Trends Biochem. Sci. 2000; 25: 112-114Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar)), which is a homeodomain-like DNA binding motif. This motif is believed to play a role in chromatin organization and specifically in organizing the interaction between nuclear matrix proteins and scaffold/matrix attachment regions. SAF boxes are found in proteins involved in very diverse processes such as transcription, RNA processing, apoptotic chromatin degradation, and DNA repair (3Kipp M. Gohring F. Ostendorp T. van Drunen C.M. van Driel R. Przybylski M. Fackelmayer F.O. Mol. Cell. Biol. 2000; 20: 7480-7489Crossref PubMed Scopus (160) Google Scholar). Amino acids 409–484 harbor an RNA recognition motif (RRM), which is often found in mRNA-processing proteins. SAFB can interact with a number of proteins from the RNA-processing machinery, such as AUF1/hnRNP D, hnRNP A1, htra2-β1, ASF/SF2, SRp30c, and CLK2 (5Weighardt F. Cobianchi F. Cartegni L. Chiodi I. Villa A. Riva S. Biamonti G. J. Cell Sci. 1999; 112: 1465-1476Crossref PubMed Google Scholar, 6Arao Y. Kuriyama R. Kayama F. Kato S. Arch. Biochem. Biophys. 2000; 380: 228-236Crossref PubMed Scopus (60) Google Scholar, 7Nayler O. Stratling W. Bourquin J.P. Stagljar I. Lindemann L. Jasper H. Hartmann A.M. Fackelmayer F.O. Ullrich A. Stamm S. Nucleic Acids Res. 1998; 26: 3542-3549Crossref PubMed Scopus (149) Google Scholar). Consistent with this, SAFB is able to alter the splice site selection of an E1A minigene (7Nayler O. Stratling W. Bourquin J.P. Stagljar I. Lindemann L. Jasper H. Hartmann A.M. Fackelmayer F.O. Ullrich A. Stamm S. Nucleic Acids Res. 1998; 26: 3542-3549Crossref PubMed Scopus (149) Google Scholar). Because SAFB was also shown to interact with the C-terminal domain of RNA polymerase II, it has been suggested to be part of a "transcriptosome" complex, coupling transcription and RNA processing (7Nayler O. Stratling W. Bourquin J.P. Stagljar I. Lindemann L. Jasper H. Hartmann A.M. Fackelmayer F.O. Ullrich A. Stamm S. Nucleic Acids Res. 1998; 26: 3542-3549Crossref PubMed Scopus (149) Google Scholar). SAFB has a nuclear localization signal (NLS), and using biochemical fractionation experiments, we and others have shown that SAFB is a nuclear protein that copurifies with chromatin (1Renz A. Fackelmayer F.O. Nucleic Acids Res. 1996; 24: 843-849Crossref PubMed Scopus (110) Google Scholar) and nuclear matrix protein fractions (2Oesterreich S. Lee A.V. Sullivan T.M. Samuel S.K. Davie J.R. Fuqua S.A. J. Cell. Biochem. 1997; 67: 275-286Crossref PubMed Scopus (86) Google Scholar). Previously we have published a number of studies showing that SAFB plays an important role in human breast cancer. First, it functions as an estrogen receptor (ER) corepressor (8Oesterreich S. Zhang Q. Hopp T. Fuqua S.A. Michaelis M. Zhao H.H. Davie J.R. Osborne C.K. Lee A.V. Mol. Endocrinol. 2000; 14: 369-381Crossref PubMed Scopus (80) Google Scholar). ER and SAFB interact both in vitro and in vivo, and overexpression of SAFB results in inhibition of ER activity. Second, overexpression of SAFB results in growth inhibition and generation of multinucleated cells. Finally, we discovered an exceptionally high rate of loss of heterozygosity (9Oesterreich S. Allredl D.C. Mohsin S.K. Zhang Q. Wong H. Lee A.V. Osborne C.K. O'Connell P. Br. J. Cancer. 2001; 84: 493-498Crossref PubMed Scopus (51) Google Scholar) at the SAFB chromosomal locus on 19p13 in human breast cancer (8Oesterreich S. Zhang Q. Hopp T. Fuqua S.A. Michaelis M. Zhao H.H. Davie J.R. Osborne C.K. Lee A.V. Mol. Endocrinol. 2000; 14: 369-381Crossref PubMed Scopus (80) Google Scholar). Importantly, we have also identified SAFB mutations in those tumors but not in the adjacent normal tissue (9Oesterreich S. Allredl D.C. Mohsin S.K. Zhang Q. Wong H. Lee A.V. Osborne C.K. O'Connell P. Br. J. Cancer. 2001; 84: 493-498Crossref PubMed Scopus (51) Google Scholar). Given that SAFB is a multifunctional gene that seems to play an important role in breast tumorigenesis, we set out to determine whether SAFB would also be part of a larger family. Searching GenBank we identified one gene that showed high homology to SAFB, termed KIAA0138. Herein we propose the nomenclature SAFB2 for KIAA0138 and SAFB1 for SAFB/HET/HAP. In this study we define similarities and differences in sequences and domain structures, chromosomal localizations, expression and subcellular localizations, and functions of SAFB1 and SAFB2. We believe that SAFB1 and SAFB2, like many other genes in the human genome, probably arose through duplication of a single ancestral gene. Plasmid Constructs and Chemicals—The cloning of the HET/SAF-B (SAFB1) expression construct (2Oesterreich S. Lee A.V. Sullivan T.M. Samuel S.K. Davie J.R. Fuqua S.A. J. Cell. Biochem. 1997; 67: 275-286Crossref PubMed Scopus (86) Google Scholar), of pEGFP-SAFB1 (11Townson S.M. Sullivan T. Zhang Q. Clark G.M. Osborne C.K. Lee A.V. Oesterreich S. Clin. Cancer Res. 2000; 6: 3788-3796PubMed Google Scholar), and of the mammalian expression vector for full-length ER (12Castles C.G. Oesterreich S. Hansen R. Fuqua S.A. J. Steroid Biochem. Mol. Biol. 1997; 62: 155-163Crossref PubMed Scopus (65) Google Scholar) has been described previously. The human KIAA0138 cDNA (clone HA03743) (SAFB2) was kindly provided by the Kazusa DNA Research Institute (Kisarazu, Chiba, Japan). The 3,233-bp fragment with 57-bp poly(A) stretches was inserted into the EcoRV-NotI site of the Bluescript SK+ vector (Stratagene, La Jolla, CA). To generate an expression vector, the cDNA was cut with the restriction enzymes KpnI and NotI and cloned into the KpnI and NotI sites of the pcDNA3 vector (Invitrogen). pEGFP-SAFB2 was constructed by excising full-length SAFB2 from pcDNA3-SAFB2 with NotI, which was made blunt, and digested with KpnI. SAFB2 was then ligated into the KpnI and a blunt BamHI site of pEGFP-C1 (BD Biosciences). The putative SAFB1/2 promoter fragment was amplified by PCR using platinum Taq PFX (Invitrogen) from genomic DNA (MCF-7) with the following primers containing XhoI sites: forward 5′-GGA ACT GCA GGT CTT CGC CAC CGA CTC AGT CG-3′ and 5′-GGA ACT GCA GCC GCC CAC TTT CCA CAG AAG-3′. The fragment was cloned in both orientations into the XhoI site of the pGL3 Basic reporter plasmid (Promega) upstream of the luciferase gene. The clones were sequenced to verify both the orientation of the insert and sequence identity with the corresponding NCBI human genome sequences. All other constructs were verified using restriction digests and/or extension capillary sequencing (SeqWright, Houston, TX). All primers were purchased from MWG Biotech Inc. (High Point, NC), and PCRs were carried out using the MJ Research PT-200 Peltier Thermal Cycler (Waltham, MA). Chemicals were purchased from Sigma unless stated otherwise. Restriction and general molecular biology enzymes were purchased from Invitrogen. Cell Culture, Stable and Transient Transfection—Human breast cancer cell lines (MCF-7, MDA-MB-231, and T47D), a human osteosarcoma cell line (Saos-2 Endo), a human myeloblastoma cell line (KG-1), a transformed embryonic kidney cell line (293), and a monkey kidney cell line (CV-1) were maintained in improved modified Dulbecco's medium supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 200 units/ml penicillin, 200 μg/ml streptomycin, and 6 ng/ml insulin. For reporter assays, cells were transiently transfected using FuGENE 6 (Roche Applied Science) following the manufacturer's protocol. One day before transfection, cells were plated at 1.5 × 105 cells/well in six-well plates. For estradiol induction experiments, the cells were plated in serum-free medium, which consisted of phenol red-free improved modified Dulbecco's medium, 10 mm HEPES pH 7.4, 1 μg/ml fibronectin (Invitrogen), trace elements (Biofluids), and 1 μg/ml transferrin (Invitrogen). Cotransfections were performed using 1 μg of reporter plasmid (ERE-TK-LUC), 25 ng of β-galactosidase expression vector, and expression plasmids as indicated in the figure legends for each experiment. 24 h after transfection, the medium was replaced with serum-free medium containing the appropriate ligand. 48 h later cells were washed twice with PBS, and luciferase activity was measured using the luciferase kit from Promega (Madison, WI). β-Galactosidase activity was measured as described previously (2Oesterreich S. Lee A.V. Sullivan T.M. Samuel S.K. Davie J.R. Fuqua S.A. J. Cell. Biochem. 1997; 67: 275-286Crossref PubMed Scopus (86) Google Scholar), and the luciferase activities were normalized by dividing by the β-galactosidase activity to give relative luciferase units. For FACS analysis, cells were harvested 48 h after transfection, washed, and fixed in 70% ethanol. Immediately before analysis on a FACS STAR PLUS (BD Biosciences), propidium iodide and RNase were added to final concentrations of 0.1 and 0.5 mg/ml, respectively. Data were analyzed using CellQuest software. To assay the activity of the putative SAFB1/2 promoter elements, 24 h before transfection, 1.5 × 105 MCF-7 cells were plated/well in a six-well plate in improved modified Dulbecco's medium. Cells were transfected using FuGENE 6 reagent with 1 μg of promoter or control vectors (empty pGL3 vector, TK-LUC) along with 25 ng of β-galactosidase control. Cells were harvested 36 h later, and luciferase and β-galactosidase activity were measured as described above. To generate doxycycline-inducible SAFB1-overexpressing cells, MCF-7 cells were transfected with pUHD172-1neo plasmid (13Gossen M. Freundlieb S. Bender G. Mueller G. Hillen W. Bujard H. Science. 1995; 268: 1766-1769Crossref PubMed Scopus (2011) Google Scholar) encoding a fusion product of the VP16 activation domain of herpes simplex virus and a mutated Escherichia coli tetracycline repressor protein. Neomycin-resistant clones were tested for responsiveness to doxycycline, a tetracycline derivative, by transient transfection of a luciferase reporter gene. A parental clone (RTA-16) was subsequently transfected with HA-tagged SAFB1 subcloned into pTRE2-hygro plasmid (Clontech) downstream from the tetracycline resistance operator and a minimal cytomegalovirus promoter. Stable clones were selected in the presence of 200 μg of hygromycin and tested for inducible expression of HA-SAFB1 by immunoblotting using anti-HA and anti-SAFB antibodies. Reverse Transcription-PCR (RT-PCR) and RNase Protection Assay— Total cellular RNA from cell lines was extracted using the RNeasy kit (Qiagen, Valencia, CA). Human tissue RNA for RNase protection assay analysis was purchased from Clontech. Reverse transcription of the RNA was performed in a final volume of 20 μl using 2 μg of RNA and 25pmol of primer (5′-GTG GCC ATG GCG CTC ATC TCC-3′). The reaction mixture was incubated at 70 °C for 5min and then put immediately on ice. 4 μl of 5 × incubation buffer, 40 units of RNase inhibitor, 625 μm each dNTP, and 200 units of Moloney murine leukemia virus reverse transcriptase (Promega) were added, and the reaction was incubated at 42 °C for 1 h. Subsequent PCR was performed using SAFB2-specific primers (5′-CAA AGG GAG AGA GAG CGC CAG-3′, and 5′-GTG GCC ATG GCG GTC ATC TCC-3′) and 200 μm dNTPs, 1.5 mm MgSO4, 0.5 × enhancer solution, 10 × Pfx DNA polymerase buffer (Invitrogen), 1.25 units platinum Pfx DNA polymerase. The RNase protection assay was performed as described previously (11Townson S.M. Sullivan T. Zhang Q. Clark G.M. Osborne C.K. Lee A.V. Oesterreich S. Clin. Cancer Res. 2000; 6: 3788-3796PubMed Google Scholar, 14Oesterreich S. Weng C.-N. Qiu M. Hilsenbeck S.G. Osborne C.K. Fuqua S.A.W. Cancer Res. 1993; 53: 4443-4448PubMed Google Scholar). In addition to the SAFB1 (11Townson S.M. Sullivan T. Zhang Q. Clark G.M. Osborne C.K. Lee A.V. Oesterreich S. Clin. Cancer Res. 2000; 6: 3788-3796PubMed Google Scholar) and 36B4 (15Laborda J. Nucleic Acids Res. 1991; 19: 3998Crossref PubMed Scopus (433) Google Scholar) probes, we also used an SAFB2-specific probe that was generated by cutting the SAFB2-pBSII SK+ with HindIII restriction enzyme. The released 700-bp fragment was cloned into pGEM-11zf (Promega), and the probe was made using T7 polymerase. Immunoblotting and Immunofluorescence—Proteins were resolved on 6% or 8% SDS-PAGE and transferred to nitrocellulose. The membrane was blocked in PBS and 0.1% Tween 20 (PBST) + 5% milk for 1 h at room temperature. Mouse monoclonal anti-SAFB (16Oesterreich S. Zhang Q.P. Lee A.V. Eur. J. Cancer. 2000; 36: S43-S44Abstract Full Text Full Text PDF PubMed Google Scholar) (Upstate Biotechnology), rabbit polyclonal anti-Gal4 DNA binding region (Upstate Biotechnology), mouse anti-GFP (BD Biosciences), polyclonal anti-vinexin (17Kioka N. Sakata S. Kawauchi T. Amachi T. Akiyama S.K. Okazaki K. Yaen C. Yamada K.M. Aota S. J. Cell Biol. 1999; 144: 59-69Crossref PubMed Scopus (155) Google Scholar), and mouse anti-HA (Covance, Princeton, NJ) were diluted at 1:1,000 in PBST + 5% milk. After washing six times for 5 min with PBST, the membranes were incubated with horseradish peroxidase-linked anti-mouse or anti-rabbit IgG at 1:1,000 (Amersham Biosciences) in PBST + 5% milk, washed six times for 5 min, and the signal was developed using enhanced chemiluminescence according to the manufacturer's instructions (Pierce). To produce SAFB2-specific antibodies, the SAFB2 peptide GLLDSFCDSKEYVAAQLRQ, corresponding to amino acids 154–172, was used to produce rabbit polyclonal anti-SAFB2 antiserum (Research Genetics, Huntsville, AL), which was subsequently purified using immobilized peptide. This peptide is within a region of low homology between SAFB1 and SAFB2. For immunofluorescence, 5 × 104 cells (293, HeLa, MCF-7) were seeded onto poly-l-lysine-coated coverslips (Biocoat) (BD Biosciences) in 24-well plates 1 day before transfection. The cells were then transfected with GFP-vinexin β (17Kioka N. Sakata S. Kawauchi T. Amachi T. Akiyama S.K. Okazaki K. Yaen C. Yamada K.M. Aota S. J. Cell Biol. 1999; 144: 59-69Crossref PubMed Scopus (155) Google Scholar) using LipofectAMINE Plus reagent (Invitrogen) overnight. The next day the medium was changed to Iscove's modified Dulbecco's medium + 10% fetal bovine serum, and cells were allowed to recover for 24 h. Cells were washed twice with PBS, fixed for 30 min in PEM (80 mm PIPES, pH 6.8, 5 mm EGTA, 2 mm MgCl2) and 4% paraformaldehyde, and then washed three times in PEM. The cells were permeabilized with PEM and 0.5% Triton X-100 buffer for 30 min, washed three times with PEM, and then blocked with PBST and 5% normal goat serum (Jackson Immunoresearch Laboratories Inc.). The cells were stained using our polyclonal anti-SAFB2 or the monoclonal anti-SAFB (Upstate Biotechnology) antibodies at 1:250 dilution in PBST and 5% normal goat serum overnight at 4 °C, and then washed five times for 5 min in PBST buffer. Cells were then stained with goat anti-rabbit or anti-mouse Texas Red antibodies (Jackson) at 1:100 dilution in PBST and 5% normal goat serum followed by five washes for 5 min in PBST buffer. The coverslips were mounted on slides using mounting medium (Vectorshield, Vector Laboratories, Burlingame, CA). Confocal microscopy was performed with a digital scanning confocal microscope (Nikon Eclipse E1000) equipped with a 60 ×/numerical aperture = 1.40/oil immersion objective. Images were exported to Adobe Photoshop (Adobe Systems, San Jose, CA), and final figures were composed in PowerPoint (Microsoft). In Vitro (GST Pull-down) and in Vivo (Coimmunoprecipitation) Protein-Protein Interaction—GST pull-down experiments were performed as described previously (8Oesterreich S. Zhang Q. Hopp T. Fuqua S.A. Michaelis M. Zhao H.H. Davie J.R. Osborne C.K. Lee A.V. Mol. Endocrinol. 2000; 14: 369-381Crossref PubMed Scopus (80) Google Scholar) using bacterially expressed GST fusion proteins and in vitro translated proteins. Coimmunoprecipitation studies were performed using transfected and nontransfected cell lines (i.e. coimmunoprecipitation of endogenous proteins). For the transient transfections, 293 and CV-1 cells were plated at 5 × 106 cells in 10-cm dishes. The next day the cells were transiently transfected with the appropriate plasmids using LipofectAMINE Plus (Invitrogen) for 7 h. The cells were maintained for an additional 20 h and then lysed in low stringency (PBS, 0.1% Nonidet P-40, protease inhibitors) or high stringency buffer (20 mm Tris, pH 7.4, 50 mm NaCl, 1 mm EDTA, 0.5% Nonidet P-40, 0.5% SDS, 0.5% deoxycholate, protease inhibitors). 500 μg of lysate was precleared with 50 μl of protein G-agarose for 30 min at 4 °C and incubated overnight with 4 μg of the appropriate antibodies at 4 °C. Protein G-agarose was added for another 4 h, and the beads were pelleted and washed three times with low or high stringency buffer, respectively. Bound proteins were eluted in SDS sample buffer, subjected to SDS-PAGE, and analyzed by immunoblotting. To detect interaction of endogenous proteins, MCF-7 cells were allowed to grow for 48 h in 20-cm dishes, washed with PBS, and proteins were cross-linked for 30 min with 1 mm DSP (Pierce) which is membrane-permeable. After subsequent washing of the cells, they were lysed in high stringency buffer, and immunoprecipitation was performed as described above. Yeast Two-hybrid Assays—We used the Matchmaker Two-Hybrid System 3 (BD Biosciences) with the yeast strain AH109, which includes three reporter genes, Ade2, His3, and α-galactosidase to reduce the incidence of false positive clones, for our yeast two-hybrid assays. Yeast manipulations were undertaken according the Clontech Matchmaker System 3 and Yeast Protocols Handbooks (BD Biosciences). The C-terminal SAFB1 domain (amino acids 599–915) was subcloned in-frame into the EcoRI-BamHI sites of pGBKT7. This SAFB1 clone results in expression of an SAFB1-C-Gal4DBD fusion protein of the expected size of 60 kDa (in vitro transcription and translation, TNT Kit, Promega) using the T7 promoter site of pGBKT7, as seen by Western blotting using polyclonal rabbit anti-Gal4 DNA binding region antibodies (Upstate Biotechnology) of the protein expressed in AH109 (data not shown). Similarly, the C terminus of SAFB2 (amino acids 600–953) was subcloned into pGADT7. SAFB1 bait and prey constructs were generated using appropriate PCR primers containing EcoRI or BamHI restriction sites and cloned into pGBKT7 and pGADT7 using the EcoRI and BamHI restriction sites of these vectors. The vinexin β yeast two-hybrid clone 3-1 was obtained from a yeast two-hybrid screen using the SAFB1 C-terminal domain as bait against a normal mammary gland library (Clontech). 2S. M. Townson, K. Kang, A. V. Lee, and S. Oesterreich, manuscript in preparation. For yeast two-hybrid interactions, the bait and prey constructs were cotransformed into the yeast strain AH109 and plated onto both SD-Leu/Trp and high stringency SD-Leu/Trp/His/Ade dropout plates. Colonies from the SD-Leu/Trp/His/Ade plates were allowed to grow for 5–10 days and then streaked onto new SD-Leu/Trp/His/Ade dropout plates. Interacting proteins were assessed after the colonies had been streaked a second time onto SD-Leu/Trp/His/Ade plates with or without X-α-galactose as the substrate for α-galactosidase. Appropriate controls as suggested by the manufacturer were used to ensure the validity of the interactions. Colonies that contained interacting proteins were maintained on SD-Leu/Trp/His/Ade and SD-Leu/Trp plates, whereas colonies having noninteracting proteins were maintained only on SD-Leu/Trp plates. SAFB1 and SAFB2 Are Highly Homologous Proteins That Map to the Same Chromosomal Locus—We set out to analyze whether SAFB is part of a gene family. Therefore, we performed a homology search using GenBank and identified a second human gene with very high homology to SAFB, termed KIAA0138 (accession number NM_014649). For ease of understanding and to prevent further confusion in the literature where a number of SAFB synonyms exist, we suggest the use of the terminology SAFB1 and SAFB2 for the two highly homologous genes; SAFB1 is the gene previously termed SAFB, HET, HET/SAFB, and HAP; and SAFB2 is the gene originally designated KIAAA0138. SAFB genes are highly conserved in other mammalian organisms. Orthologous SAFB1 and SAFB2 genes are present in mice; human SAFB1 corresponds to mouse gene ENSMUSG000000428128, and human SAFB2 corresponds to mouse gene ENSMUSG00000042625. A data base search of expressed sequence tag sequences also identified SAFB genes in cattle (Bos taurus, e.g. GenBank BE666390), and in pig (Sus scrofa, e.g. GenBank BI181164). Proteins with an SAF box and RRM domain structure similar to SAFBs have been identified in the sequence data bases of other organisms such as Drosophila melanogaster (gene CG6995, Berkeley Drosophila genome data base, (18Adams M.D. Celniker S.E. Holt R.A. Evans C.A. Gocayne J.D. Amanatides P.G. Scherer S.E. Li P.W. Hoskins R.A. Galle R.F. George R.A. Lewis S.E. Richards S. Ashburner M. Henderson S.N. Sutton G.G. Wortman J.R. Yandell M.D. Zhang Q. Chen L.X. Brandon R.C. Rogers Y.-H.C. Blazej R.G. Champe M. Pfeiffer B.D. Wan K.H. Doyle C.D. Baxter E.G. Helt G. Nelson C.R. Miklos G.L.G. Abril J.F. Agbayani A. An H.-J. Andrews-Pfannkoch C. Baldwin D. Ballew R.M. Basu A. Baxendale J. Bayraktaroglu L. Beasley E.M. Beeson K.Y. Benos P.V. Berman B.P. Bhandari D. Bolshakov S. Borkova D. Botchan M.R. Bouck J.B. Broksein P. Brottier P. Burtis K.C. Busam D.A. Butler H. Cadieu E. Center A. Chandra I. Cherry J.M. Cawley S. Dahlke C. Davenport L.B. Davies P. de Pablos B. Delcher A. Deng Z. Mays A.D. Dew I. Dietz S.M. Dodson K. Doup L.E. Downes M. Dugan-Rocha S. Dunkov B.C. Dunn P. Durbin K.J. Evangelista C.C. Ferraz C. Ferriera S. Fleischmann W. Fosler C. Gabrielian A.E. Garg N.S. Gelbart W.M. Glasser K. Glodek A. Gong F. Gorrell J.H. Gu Z. Guan P. Harris M. Harris N.L. Harvey D. Heiman T.J. Hernandez J.R. Houck J. Hostin D. Houstin K.A. Howland T.J. Wei M.-H. Ibegwam C. Jalali M. Kalush F. Karpen G.H. Ke Z. Kennison J.A. Ketchum K.A. Kimmel B.E. Kodira C.D. Kraft C. Kravitz S. Kulp D. Lai Z. Lasko P. Lei Y. Levitsky A.A. Li J. Li Z. Liang Y. Lin X. Liu X. Mattei B. McIntosh T.C. McLeod M.P. McPherson D. Merkulov G. Milshina N.V. Mobarry C. Morris J. Moshrefi A. Mount S.M. Moy M. Murphy B. Murphy L. Muzny D.M. Nelson D.L. Nelson D.R. Nelson K.A. Nelson, Nixon K. Nusskern D.R. Pacleb J.M. Palazzolo M. Pittman G.S. Pan S. Pollard J. Puri V. Reese M.G. Reinert K. Remington K. Saunders R.D.C. Scheeler F. Shen H. Shue B.C. Sidén-Kiamos I. Simpson M. Skupski M.P. Smith T. Spier E. Spradling A.C. Stapleton M. Strong R. Sun E. Svirskas R. Tector C. Turner R. Venter E. Wang A.H. Wang X. Wang Z.-Y. Wassarman D.A. Weinstock G.M. Weissenbach J. Williams S.M. Woodage T. Worley K.C. Wu D. Yang S. Yao Q.A. Ye J. Yeh R.-F. Zaveri J.S. Zhan M. Zhang G. Zhao Q. Zheng L. Zheng X.H. Zhong F.N. Zhong W. Zhou X. Zhu S. Zhu X. Smith H.O. Gibbs R.A. Myers E.W. Rubin G.M. Venter J.C. Science. 2000; 287: 2185-2195Crossref PubMed Scopus (4741) Google Scholar)) and Caenorhabditis elegans. A search of the worm data base (19Stein L. Sternberg P. Durbin R. Thierry-Mieg J. Spieth J. Nucleic Acids Res. 2001; 29: 82-86Crossref PubMed Scopus (241) Google Scholar) at the Sanger Center revealed a putative gene CE43E11.1 (gene CE43E11.1 from clone C43E11, GenBank accession no. U80437) coding for the protein CE23592. Proteins with a domain structure similar to SAFB1 were not found in any of the public domain data bases for yeast, plants, bacteria, or protozoa. Further computer analysis of the translated protein sequences derived from the NCBI human genome sequence data base, expressed sequence tag data bases, and overlapping cDNAs indicated the presence of a third gene with high homology in its SAF box (also called SAP domain for SAF-A/B, Acinus, and PIAS) and RRM domains to both SAFB1 and SAFB2. The full-length open reading frame for this hypothetical protein can be deduced readily from the overlap of GenBank sequences AK000867 and NM_024755 (for hypothetical protein FLJ13213) to give a 1,034-amino acid protein. However, because the overall nucleotide homology of this hypothetical protein is only 36% to SAFB1 and 37% to SAFB2, suggesting that it is a more distantly related member of this gene family, we have limited our current studies to the characterization of SAFB1 and SAFB2. SAFB2 (KIAA0138 cDNA) was originally cloned from the myeloblast cell line KG-1 by Nagase et al. (20Nagase T. Seki N. Tanaka A. Ishikawa K. Nomura N. DNA Res. 1995; 2 (199–210): 167-174Crossref PubMed Scopus (137) Google Scholar), but up until now no further analysis of this gene or its gene product has been reported. At the amino acid level, human SAFB2 shows a 74% homology to human SAFB1 (Fig. 1A) and 75% homology at the nucleotide level. The SAF box and RRM are highly conserved between SAFB1 and SAFB2 (Fig. 1B). Furthermore, there are three other regions that share high homology but whose function is unknown. One such region maps to the N terminus (amino acids 210–385), and the other two map to
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