Ajuba Functions as a Histone Deacetylase-dependent Co-repressor for Autoregulation of the Growth Factor-independent-1 Transcription Factor
2008; Elsevier BV; Volume: 283; Issue: 46 Linguagem: Inglês
10.1074/jbc.m802320200
ISSN1083-351X
AutoresDiego E. Montoya–Durango, Chinavenmeni S. Velu, Avedis Kazanjian, Meghan E. B. Rojas, Chris Jay, Gregory D. Longmore, H. Leighton Grimes,
Tópico(s)Cell death mechanisms and regulation
ResumoGrowth factor independent-1 (Gfi1) is a zinc finger protein with a SNAG-transcriptional repressor domain. Ajuba is a LIM domain protein that shuttles between the cytoplasm and the nucleus. Ajuba functions as a co-repressor for synthetic Gfi1 SNAG-repressor domain-containing constructs, but a role for Ajuba co-repression of the cognate DNA bound Gfi1 protein has not been defined. Co-immunoprecipitation of synthetic and endogenous proteins and co-elution with gel filtration suggest that an endogenous Ajuba·Gfi1·HDAC multiprotein complex is possible. Active histone deacetylase activity co-immunoprecipitates with Ajuba or Gfi1, and both proteins depend upon histone deacetylases for full transcriptional repression activity. Ajuba LIM domains directly bind to Gfi1, but the association is not SNAG domain-dependent. ChIP analysis and reciprocal knockdown experiments suggest that Ajuba selectively functions as a co-repressor for Gfi1 autoregulation. The data suggest that Ajuba is utilized as a corepressor selectively on Gfi1 target genes. Growth factor independent-1 (Gfi1) is a zinc finger protein with a SNAG-transcriptional repressor domain. Ajuba is a LIM domain protein that shuttles between the cytoplasm and the nucleus. Ajuba functions as a co-repressor for synthetic Gfi1 SNAG-repressor domain-containing constructs, but a role for Ajuba co-repression of the cognate DNA bound Gfi1 protein has not been defined. Co-immunoprecipitation of synthetic and endogenous proteins and co-elution with gel filtration suggest that an endogenous Ajuba·Gfi1·HDAC multiprotein complex is possible. Active histone deacetylase activity co-immunoprecipitates with Ajuba or Gfi1, and both proteins depend upon histone deacetylases for full transcriptional repression activity. Ajuba LIM domains directly bind to Gfi1, but the association is not SNAG domain-dependent. ChIP analysis and reciprocal knockdown experiments suggest that Ajuba selectively functions as a co-repressor for Gfi1 autoregulation. The data suggest that Ajuba is utilized as a corepressor selectively on Gfi1 target genes. Group III LIM domain proteins, which have three to four tandem LIM domains at the C terminus, are involved in intracellular shuttling of interacting proteins. This suggests that these proteins may be involved in assembling multiple protein complexes with a wide range of functions such as cell development, differentiation, and signaling in different cellular compartments (for review see Refs. 1Bach I. Mech. Dev. 2000; 91: 5-17Crossref PubMed Scopus (483) Google Scholar, 2Dawid I.B. Breen J.J. Toyama R. Trends Genet. 1998; 14: 156-162Abstract Full Text Full Text PDF PubMed Scopus (518) Google Scholar, 3Retaux S. Bachy I. Mol. Neurobiol. 2002; 26: 269-281Crossref PubMed Scopus (55) Google Scholar). The group III LIM protein family includes two subfamilies of proteins, which shuttle between the cytoplasm and the nucleus: Ajuba (Jub), LIMD1, WTIP, and Zyxin, LPP, and Trip6 (4Goyal R.K. Lin P. Kanungo J. Payne A.S. Muslin A.J. Longmore G.D. Mol. Cell. Biol. 1999; 19: 4379-4389Crossref PubMed Google Scholar, 8Feng Y. Zhao H. Luderer H.F. Epple H. Faccio R. Ross F.P. Teitelbaum S.L. Longmore G.D. J. Biol. Chem. 2007; 282: 39-48Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). In the cytoplasm and at sites of cell adhesion, group-III LIM proteins function as adapter proteins in signal transduction (1Bach I. Mech. Dev. 2000; 91: 5-17Crossref PubMed Scopus (483) Google Scholar, 4Goyal R.K. Lin P. Kanungo J. Payne A.S. Muslin A.J. Longmore G.D. Mol. Cell. Biol. 1999; 19: 4379-4389Crossref PubMed Google Scholar, 9Ren Y. Meng S. Mei L. Zhao Z.J. Jove R. Wu J. J. Biol. Chem. 2004; 279: 8497-8505Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). For example, Ajuba directly interacts with Grb2 and stimulates Ras signaling (4Goyal R.K. Lin P. Kanungo J. Payne A.S. Muslin A.J. Longmore G.D. Mol. Cell. Biol. 1999; 19: 4379-4389Crossref PubMed Google Scholar). In the nucleus, group III LIM proteins are suggested to regulate transcription. Specifically, the Trip6 protein functions as a transcriptional co-activator for the REL oncoprotein (10Wang Y. Gilmore T.D. Biochim. Biophys. Acta. 2001; 1538: 260-272Crossref PubMed Scopus (75) Google Scholar). However, whereas nuclear Ajuba was shown to interact with the TTF1 transcription factor, the Ajuba and TTF1 interaction did not influence TTF1 transcriptional activity (11Missero C. Pirro M.T. Simeone S. Pischetola M. Di Lauro R. J. Biol. Chem. 2001; 276: 33569-33575Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Growth Factor Independence-1 (Gfi1) 4The abbreviations used are: GFI1, growth factor independent-1; HDAC, histone deacetylase; ELA2, elastase 2, neutrophil; CSF1, colony stimulating factor-1; SCN, severe congenital neutropenia; TSA, trichostatin A; NaB, sodium butyrate; TK, herpes simplex virus thymidine kinase minimal promoter; CAT, chloramphenicol acetyltransferase; SNAG, Snail + Gfi1 repressor domain; SV40SWAP, Gfi1 mutant with the SNAG domain replaced with the SV40 large T antigen nuclear localization motif; shRNA, short hairpin RNA; TBS, Tris-buffered saline; HRP, horseradish peroxidase; ChIP, chromatin immunoprecipitation. is a 55-kDa nuclear transcriptional repressor protein that was identified as a target for proviral insertion of the Moloney murine leukemia virus (12Gilks C. Bear S. Grimes H. Tsichlis P. Mol. Cell. Biol. 1993; 113: 1759-1768Crossref Scopus (201) Google Scholar). Gfi1 regulates genes involved in hemato-poietic stem cell maintenance, and myelopoiesis (reviewed in Ref. 13Kazanjian A. Gross E.A. Grimes H.L. Crit. Rev. Oncol./Hematol. 2006; 59: 85-97Crossref PubMed Scopus (44) Google Scholar). Moreover, Gfi1 acts as a molecular switch in regulating granulopoiesis (14Zarebski A. Velu C.S. Baktula A.M. Bourdeau T. Horman S.R. Basu S. Bertolone S.J. Horwitz M. Hildeman D.A. Trent J.O. Grimes H.L. Immunity. 2008; 28: 370-380Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Gfi1 target genes include GFI1, GFI1B (15Doan L.L. Porter S.D. Duan Z. Flubacher M.M. Montoya D. Tsichlis P.N. Horwitz M. Gilks C.B. Grimes H.L. Nucleic Acids Res. 2004; 32: 2508-2519Crossref PubMed Scopus (75) Google Scholar, 18Zweidler-Mckay P. Grimes H. Flubacher M. Tsichlis P. Mol. Cell. Biol. 1996; 16: 4024-4034Crossref PubMed Scopus (273) Google Scholar), and CSF1 (14Zarebski A. Velu C.S. Baktula A.M. Bourdeau T. Horman S.R. Basu S. Bertolone S.J. Horwitz M. Hildeman D.A. Trent J.O. Grimes H.L. Immunity. 2008; 28: 370-380Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Gfi1 contains six C2-H2 zinc fingers that bind to the core DNA sequence 5′-TAAATCAC(A/T)GCA-3′ (18Zweidler-Mckay P. Grimes H. Flubacher M. Tsichlis P. Mol. Cell. Biol. 1996; 16: 4024-4034Crossref PubMed Scopus (273) Google Scholar). The N-terminal 20 amino acids of GFI1 encode a transferable repressor domain termed "SNAG," because it is conserved between SNAIL and GFI1-related proteins (19Grimes H. Chan T. Zweidler-McKay P. Tong B. Tsichlis P. Mol. Cell. Biol. 1996; 16: 6263-6272Crossref PubMed Scopus (238) Google Scholar). A yeast two-hybrid assay with the GFI1 SNAG domain identified Ajuba as an interacting protein (20Ayyanathan K. Peng H. Hou Z. Fredericks W.J. Goyal R.K. Langer E.M. Longmore G.D. Rauscher 3rd, F.J. Cancer Res. 2007; 67: 9097-9106Crossref PubMed Scopus (48) Google Scholar). Synthetic constructs in which the Gfi1 SNAG domain was fused to a heterologous DNA binding domain suggested that Ajuba functions as a co-repressor for Gfi1 (20Ayyanathan K. Peng H. Hou Z. Fredericks W.J. Goyal R.K. Langer E.M. Longmore G.D. Rauscher 3rd, F.J. Cancer Res. 2007; 67: 9097-9106Crossref PubMed Scopus (48) Google Scholar); however, analysis of the cognate DNA-bound Gfi1 protein was not performed, leaving open the question of Ajuba as a co-repressor for Gfi1. Here we show that Ajuba functions as an HDAC-dependent co-repressor for a subset of Gfi1 target genes. Specifically, Ajuba functionally mediates GFI1 autoregulation. Cell Culture—EL4.IL-2 T cells (ATCC TIB-181) were grown in RPMI 1640 with 10% horse serum, 1% l-Gln, 1% Pen/Strep (Invitrogen). Jurkat T cells (clone E6-1, ATCC TIB-152) and human promyelocytic leukemia cells (HL60, ATCC CCL-240) were grown in RPMI 1640 with 10% fetal bovine serum, 1% l-Gln, 1% Pen/Strep (Invitrogen). Human embryonic kidney (HEK) 293T, and Phoenix cells were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 1% l-Gln, 1% Pen/Strep (Invitrogen). All cell lines were kept at 37 °C with 5% CO2 in a humidified atmosphere. Plasmids and Subcloning—Chloramphenicol acetyl transferase (CAT) reporter constructs and Gfi1 expression plasmids used in transient transcription assays were described previously (15Doan L.L. Porter S.D. Duan Z. Flubacher M.M. Montoya D. Tsichlis P.N. Horwitz M. Gilks C.B. Grimes H.L. Nucleic Acids Res. 2004; 32: 2508-2519Crossref PubMed Scopus (75) Google Scholar, 19Grimes H. Chan T. Zweidler-McKay P. Tong B. Tsichlis P. Mol. Cell. Biol. 1996; 16: 6263-6272Crossref PubMed Scopus (238) Google Scholar). Luciferase versions were generated by cloning the B30 × 2TK promoter and TK promoter fragments into the pGL3 vector (Promega, Madison, WI). MIEV-Gfi1-FLAG retroviral construct was generated by digesting CMV14-Gfi1-FLAG with XhoI to release the Gfi1-FLAG fragment. The Gfi1-FLAG insert was subcloned into SalI/XhoI-digested pMIEV. Plasmids were sequenced and screened by restriction endonucleases to corroborate the proper sequence and orientation of the insert. To generate the LexA-Ajuba expression vector and their mutants LexA-LIM and LexA-PreLIM we first PCR-amplified LexA with primers (5′-AGAATTCAACAGCCAGTCGCCGTTGCG-3′ and 5′-CCAAGCTTACCATGAAAGCGTTAACGGCC-3′) and subcloned it into the TOPO vector by blunt-end ligation generating the vector LexA-TOPO. Next, EcoRI/HindIII digestion released a LexA insert, which was agarose-purified and subcloned into EcoRI/HindIII-linearized pCS2 (4Goyal R.K. Lin P. Kanungo J. Payne A.S. Muslin A.J. Longmore G.D. Mol. Cell. Biol. 1999; 19: 4379-4389Crossref PubMed Google Scholar) to generate the plasmid pCS2-LexA. Finally, pCS2-Ajuba (4Goyal R.K. Lin P. Kanungo J. Payne A.S. Muslin A.J. Longmore G.D. Mol. Cell. Biol. 1999; 19: 4379-4389Crossref PubMed Google Scholar) was digested with EcoRI to release the cDNA for Ajuba, which was ligated into the EcoRI-linearized pCS2-LexA. The mutant pCS2-LexA-LIM was generated by the same strategy. To generate LexA-PreLIM, the vector pCS2-LexA-Ajuba was digested with StuI, and the agarose-purified vector band was self-ligated. Plasmids were sequenced and digested with restriction endonucleases to corroborate the correct orientation of the products. Transient Transcription Assays—For transient transfections, 1.5 × 105 HEK 293T cells were plated on 24-well plates and transfected for 36 h with Lipofectamine 2000 reagent according to the manufacturer's directions (Invitrogen). Electroporation of EL4 cells was carried out with 20 μg of DNA, in 0.45-μm cuvettes (Bio-Rad), and 106 cells in 250 μl of media. Settings for pulses were 960 microfarads and 270 mV. Cells were cultured for 36 h before harvest. The chloramphenicol acetyltransferase assay was performed as previously described, utilizing a Gfi1-insensitive mutant of a cytomegalovirus-immediate-early promoter-driven β-galactosidase vector as a transfection efficiency control (19Grimes H. Chan T. Zweidler-McKay P. Tong B. Tsichlis P. Mol. Cell. Biol. 1996; 16: 6263-6272Crossref PubMed Scopus (238) Google Scholar). Cellular extracts were adjusted for equivalent β-galactosidase activity, then analyzed by the scintillation method for CAT activity (19Grimes H. Chan T. Zweidler-McKay P. Tong B. Tsichlis P. Mol. Cell. Biol. 1996; 16: 6263-6272Crossref PubMed Scopus (238) Google Scholar). Luciferase assay transfections were controlled by co-transfection of a Gfi1-insensitive Renilla luciferase vector (Promega). Firefly and Renilla luciferase activities were measured consecutively with Dual-Luciferase Reporter assay reagents (Promega) using Lumat LB 9507 (Berthold Technologies). A t statistic was calculated on the difference between the values of each measurement of CAT or Firefly luciferase activity to determine statistical significance for -fold repression. All assays shown were repeated at least three times with similar results unless other-wise stated. Retroviral Transduction—Phoenix-Ampho cells were transiently transfected with retroviral constructs using the CaPO4 method, then co-cultured with Jurkat cells at 33 °C for 16 h. GFP+ cells were sorted 1 week after transduction, and single cell clones were obtained by limiting dilution. Clonal populations were analyzed for the presence of the FLAG-tagged proteins. Commercially available vectors were purchased for Gfi1 or Ajuba knockdown (Sigma). Lentiviral stocks were generated by three plasmid packaging in HEK 293 cells, concentrated by ultracentrifugation, and tittered on MEL cells. HL60 cells were transduced with non-targeting and Gfi1- or Ajuba-targeting shRNA viruses at an multiplicity of infection of 5. The transduced cells were cultured in RPMI media with 10% fetal bovine serum and puromycin (5 μg/ml). Immunoblots and Immunoprecipitation—Nuclear extracts were prepared using a modified procedure of Dignam (21Dignam J. Lebovitz R. Roeder R. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9164) Google Scholar). Briefly, cells were rinsed twice in ice-cold phosphate-buffered saline (Invitrogen), trypsinized, and resuspended in buffer A (20 mm Tris-HCl, pH 7.4, 1.5 mm MgCl2, 10 mm KCl, 1 mm Complete inhibitor (Roche Applied Science), and 1 mm phenylmethylsulfonyl fluoride). After incubating 10 min on ice, cells were transferred into a glass Dounce homogenizer and processed for 30-40 strokes with pestle A. Nuclei were pelleted by centrifugation at 4 °C at 13,000 rpm for 10 min. Cytoplasmic extract was transferred to a clean tube and snap-frozen. The nuclei were resuspended in buffer C (20 mm Tris-HCl, pH 7.0, 25% glycerol, 0.42 m NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 1 mm Complete inhibitor (Roche Applied Science), and 1 mm phenylmethylsulfonyl fluoride), transferred into a glass Dounce homogenizer. Nuclear extract was prepared by processing with pestle B until the pellet was totally dispersed, then centrifuged for 10-min centrifugation at 13,000 rpm at 4 °C to pellet nuclear debris. For knockdown experiments, HL60 cell lysate was prepared using CompleteM lysis buffer (Roche Applied Science). Protein concentrations were determined by BCA assay (Pierce), and 20 μg of protein extract was separated on 10% SDS-polyacrylamide gel and electroblotted onto an Immobilon-P membrane (Millipore). The membranes were blocked with 5% Carnation instant milk in TBS (50 mm Tris-HCl, 150 mm NaCl, pH 7.5) at 4 °C for 16 h. The blocked membranes were incubated with primary antibodies in 5% milk in TTBS (0.05% Tween 20 in TBS) between 1 and 4 h at room temperature. Gfi1 was detected using goat polyclonal N-20 (1:500, Santa Cruz Biotechnology) and mouse monoclonal (2.5D17) (22Kazanjian A. Wallis D. Au N. Nigam R. Venken K.J. Cagle P.T. Dickey B.F. Bellen H.J. Gilks C.B. Grimes H.L. Cancer Res. 2004; 64: 6874-6882Crossref PubMed Scopus (66) Google Scholar) antibody. Rabbit polyclonal antisera against the histone deacetylases HDAC1 (Upstate Biotechnologies), HDAC2 (H-54, Santa Cruz Biotechnology), HDAC3 (Upstate Biotechnologies), Ajuba (Cell Signaling), and a FLAG-horseradish peroxidase (HRP)-conjugated antibody (Sigma) were utilized according to the manufacturer's directions. The secondary antibodies were donkey anti-goat HRP-conjugated IgG (1:5000, Santa Cruz Biotechnology), Sheep anti-mouse Ig-HRP (1:2500) and donkey anti-rabbit Ig-HRP (1:5000, Amersham Biosciences). Secondary antibodies were prepared in 5% milk TTBS and incubated with blots for 1 h at room temperature. The detection was performed with ECL-plus detection reagent according to manufacturer's instructions (Amersham Biosciences). For each immunoprecipitation, 200 μg of nuclear extracts was diluted to 150 mm NaCl and 5% glycerol. Extracts were precleared with a mix of protein A/G-agarose beads (Invitrogen) for 1 h under gentle rocking, then 15 μl of antisera was added, and the immunocomplexes were allowed to form for 1 h at 4 °C. To capture the immunocomplexes, 20 μl of protein A/G-agarose beads were added for 16 h at 4 °C. Beads were pelleted at 2000 rpm for 5 min and washed five times with 20 mm Tris-HCl, pH 7.0, 5% glycerol, 150 mm NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 0.5% Nonidet P-40. Samples were resuspended in 20 μl of 1× loading buffer, boiled for 10 min, and subjected to immunoblot analysis. Isotype-matched-control immunoglobulin was used as a control for the immunoprecipitations. FLAG and Myc-specific immunoprecipitations were carried out with M2 affinity resin (Sigma) and Myc-agarose beads (Santa Cruz Biotechnology), respectively, following the manufacturer's directions. Size Exclusion Chromatography—Nuclear extracts from 3 to 5 billion Jurkat cells were applied to a Sephacryl S-300 column on an AKTA10 purifier (Amersham Biosciences). The column was run using Dignam buffer C with 150 mm NaCl at a flow rate of 0.5 ml/min. Fractions (1.0 ml) were collected, and 80 μl of every fifth fraction was used for immunoblot analysis. HDAC Assays—Nuclear extracts from Jurkat cells transduced with Gfi1-FLAG or FLAG-Ajuba expressing or empty MSCV retroviral vectors were used for immunoprecipitation with the M2 affinity resin (Sigma) as described above. The beads were mixed with 200 μl of 50 μm HDAC assay substrate (Fluor-de-Lys HDAC assay, BioMol) and incubated for 1.5 h at room temperature. For each data point triplicate immunoprecipitations without and with TSA were analyzed. Aliquots were taken at 0.5 and 1.5 h, and the change in HDAC activity was determined according to the manufacturer's directions. TSA-sensitive relative-fluorescent units were generated by subtracting values for triplicate immunoprecipitations treated with TSA from values for triplicate immunoprecipitations treated with vehicle control. Chromatin Immunoprecipitation—The ChIP assay was performed as previously described (14Zarebski A. Velu C.S. Baktula A.M. Bourdeau T. Horman S.R. Basu S. Bertolone S.J. Horwitz M. Hildeman D.A. Trent J.O. Grimes H.L. Immunity. 2008; 28: 370-380Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Briefly, logarithmically growing HL60 cells (1 × 108 cells) were cross-linked using formaldehyde (final concentration, 1% v/v) in RPMI 1640 medium for 10 min on ice. Glycine was added to a final concentration of 0.125 m to stop cross-linking. Fixed cells were pelleted by centrifugation and sequentially washed three times with ice-cold phosphate-buffered saline with 1× Complete inhibitor (Roche Applied Science). The cells were then resuspended in lysis buffer (1% SDS, 50 mm Tris-HCl, pH 8.1, 10 mm EDTA, 1× Complete inhibitor) and were sonicated to make soluble chromatin using Sonicator 3000 cup horn (Misonix). An aliquot of total chromatin was taken at this point to use as a positive control in the PCRs (input chromatin). The cell lysates were precleared by incubation with protein A/G-Sepharose beads (Santa Cruz Biotechnology) and then incubated with Gfi1 monoclonal antibody (2.5D17) (22Kazanjian A. Wallis D. Au N. Nigam R. Venken K.J. Cagle P.T. Dickey B.F. Bellen H.J. Gilks C.B. Grimes H.L. Cancer Res. 2004; 64: 6874-6882Crossref PubMed Scopus (66) Google Scholar), Ajuba polyclonal antibody (4897) (Cell Signaling), and control mouse (GE Healthcare) or rabbit (sc-2027, Santa Cruz Biotechnology) IgG overnight at 4 °C. DNA·protein complexes were collected with protein A/G-Sepharose beads followed by several rounds of washing. Bound DNA·protein complexes were eluted from the antibodies with two incubations in elution buffer (100 mm NaHCO3, 1% SDS) at room temperature for 15 min. Cross-links were reversed by addition of sodium chloride followed by incubation at 65 °C overnight. RNase A and proteinase K were sequentially added and incubated for 1 h at 37 °C. DNA fragments were purified using a QIAquick PCR purification kit (Qiagen) and used for PCR amplifications. The PCR products were fractionated on 2% agarose gels, stained with ethidium bromide. PCR primer pairs for the ChIP were: GFI1 (5′-CACACCTTCATCCACACAGG-3′ and 5′-GATGAGCTTTGCACACTGGA-3′); GFI1B (5′-GGGCGATGCATTCATTTCC-3′ and 5′-CACCTCGATTTTGGATTTCTAG-3′); CSF1 (5′-GGGCCTCTGGGGTGTAGTAT-3′ and 5′-CCGAGGCAAACTTTCACTTT-3′). β-ACTIN (5′-AGCGCGGCTACAGCTTCA-3′ and 5′-CGTAGCACAGCTTCTCCTTAATGTC-3′) was used as the negative control. Each experiment was performed at least twice with similar results, and representative data are shown. Quantitative PCR—TRIzol (Invitrogen) extracted RNA from shRNA vector-transduced cells was quantified, and equal amounts of RNA were utilized in first strand cDNA synthesis reaction and then applied to TaqMan probe sets for GFI1B (Hs00180261_m1), GFI1 (Hs00382207_m1), and CSF1 (Hs00174164_m1) according to the method of the manufacturer (ABI). Gfi1 Mediates Transcriptional Repression through Titratable-associated Factors—Gfi1 transcriptional repression may require limiting and titratable-associated factors (14Zarebski A. Velu C.S. Baktula A.M. Bourdeau T. Horman S.R. Basu S. Bertolone S.J. Horwitz M. Hildeman D.A. Trent J.O. Grimes H.L. Immunity. 2008; 28: 370-380Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 19Grimes H. Chan T. Zweidler-McKay P. Tong B. Tsichlis P. Mol. Cell. Biol. 1996; 16: 6263-6272Crossref PubMed Scopus (238) Google Scholar). 293T cells are human epithelial kidney cells that express a low level of Gfi1 (15Doan L.L. Porter S.D. Duan Z. Flubacher M.M. Montoya D. Tsichlis P.N. Horwitz M. Gilks C.B. Grimes H.L. Nucleic Acids Res. 2004; 32: 2508-2519Crossref PubMed Scopus (75) Google Scholar). 293T cells were co-transfected with reporter constructs with or without "B30" (18Zweidler-Mckay P. Grimes H. Flubacher M. Tsichlis P. Mol. Cell. Biol. 1996; 16: 4024-4034Crossref PubMed Scopus (273) Google Scholar) high affinity Gfi1 binding sites (Fig. 1A), and increasing amounts of expression vectors encoding Gfi1 or a Gfi1·SNAG repressor domain mutant in which all 20 amino acids of the SNAG domain have been replaced with the SV40 nuclear localization motif (SV40SWAP) (19Grimes H. Chan T. Zweidler-McKay P. Tong B. Tsichlis P. Mol. Cell. Biol. 1996; 16: 6263-6272Crossref PubMed Scopus (238) Google Scholar). Whereas low levels of the Gfi1 expression construct induced a significant increase in repression, expression construct levels in excess of 5 ng decreased repression in a dose-dependent manner (Fig. 1B, upper panel). Immunoblot analysis confirmed increased protein levels (Fig. 1C). Notably, a titration of the Gfi1 SV40SWAP expression vector resulted in increased protein levels (Fig. 1C) but did not lead to significant repression at any level (Fig. 1B, lower panel). Thus, excess Gfi1 may disrupt functional Gfi1 transcription complexes, perhaps by sequestering limiting proteins required for an active Gfi1·SNAG repressor complex. The LIM Domain Protein Ajuba Increases Gfi1 Transcriptional Repression—Ajuba is a LIM domain protein that shuttles between the cytoplasm and the nucleus (6Kanungo J. Pratt S.J. Marie H. Longmore G.D. Mol. Biol. Cell. 2000; 11: 3299-3313Crossref PubMed Scopus (114) Google Scholar). In the nucleus, Ajuba interacted with a synthetic Gfi1·SNAG domain-containing construct to function as a corepressor (20Ayyanathan K. Peng H. Hou Z. Fredericks W.J. Goyal R.K. Langer E.M. Longmore G.D. Rauscher 3rd, F.J. Cancer Res. 2007; 67: 9097-9106Crossref PubMed Scopus (48) Google Scholar). To determine if Ajuba affects cognate DNA-bound Gfi1 transcriptional repression functions, we performed transient transcription assays with Gfi1-responsive reporter constructs (Fig. 1A) and expression constructs encoding Gfi1 and Ajuba. Co-expression of Ajuba and Gfi1 significantly increased Gfi1-mediated repression when compared with the repression induced by GFI1 alone (Fig. 1D). Notably, the activity of the TK-CAT reporter, which lacks Gfi1 binding sites, was not altered by the presence of either Gfi1, or co-expression of Gfi1 and Ajuba (Fig. 1D). Thus, Ajuba effects on transcription repression in this assay are dependent upon the presence of Gfi1 DNA binding sites. Moreover, the level of Gfi1 protein was not affected by the overexpression of Ajuba (Fig. 1F). In agreement with the low level of Gfi1 in 293T cells (15Doan L.L. Porter S.D. Duan Z. Flubacher M.M. Montoya D. Tsichlis P.N. Horwitz M. Gilks C.B. Grimes H.L. Nucleic Acids Res. 2004; 32: 2508-2519Crossref PubMed Scopus (75) Google Scholar), transfected B30 × 2 TK reporters demonstrate modest endogenous Gfi1 repression activity (Fig. 1, B, C, and G). When Ajuba levels were decreased by shRNA knockdown, the activity of the Gfi1-responsive vector was equivalent to that of the non-responsive control (Fig. 1G). Thus, repression mediated by endogenous Gfi1 upon the B30 × 2 TK reporter is completely abrogated by shRNA against Ajuba, and endogenous Gfi1 requires endogenous Ajuba to mediate repression of the B30 × 2 TK reporter in 293T cells. These results strongly suggest that Ajuba acts as a co-repressor for cognate Gfi1 and validate the 293T system to determine the mechanism. Gfi1 Associates with Ajuba—To further study the in vivo association of endogenous proteins, we performed immunoprecipitation from Jurkat cell line nuclear extracts. We employed two different antisera against Ajuba, an antisera against LPP (a related LIM domain protein), and isotype-matched IgG as controls. Immunoprecipitants were analyzed by immunoblot for the presence of Gfi1 (Fig. 2A). Notably, endogenous Gfi1 protein was co-immunoprecipitated with antiserum against Ajuba, but not LPP or the isotype IgG (Fig. 2A). The amount of Gfi1 that co-immunoprecipitated with Ajuba was <10% of the input. Thus, endogenous Ajuba and Gfi1 form a complex in Jurkat nuclear extracts. Next, we broadly defined the protein domains (Fig. 2B) of interaction between Ajuba and Gfi1 by immunoprecipitating FLAG-epitope-tagged Gfi1 (or the SV40SWAP mutant), then analyzing by immunoblot for the presence of Myc-epitope-tagged Ajuba (or Ajuba mutants). Both Gfi1 and the SV40SWAP mutant co-immunoprecipitated with Ajuba (Fig. 2C). Unlike full-length Ajuba, the N-terminal Ajuba-PreLIM region did not co-immunoprecipitate with Gfi1 (Fig. 2D). However, the C-terminal Ajuba-LIM region co-immunoprecipitated with Gfi1 (Fig. 2E). Therefore, Ajuba associates with Gfi1 through the Ajuba LIM region, but this interaction is not dependent upon the Gfi1 SNAG domain. The LIM Domain of Ajuba Functions as a Corepressor—To confirm a co-repressor function for Ajuba, we performed transient transcription assays with chimeric proteins in which the N terminus of the bacterial DNA-binding protein LexA is fused to Ajuba, Ajuba mutants (Fig. 3A), or the N terminus of Gfi1 (Fig. 3B). Immunoblot analysis revealed that all the LexA fusion proteins were synthesized in 293T cells at the expected molecular weight (Fig. 3C). We previously demonstrated that the N terminus of Gfi1 transfers active transcriptional repression to LexA (19Grimes H. Chan T. Zweidler-McKay P. Tong B. Tsichlis P. Mol. Cell. Biol. 1996; 16: 6263-6272Crossref PubMed Scopus (238) Google Scholar). Indeed, co-transfection of a TK-CAT reporter containing two Lex operons (Fig. 3B) and an expression vector encoding Gfi1-LexA resulted in transcriptional repression (Fig. 3D). In contrast, co-transfection of LexA-Ajuba with the reporter did not yield significant repression (Fig. 3D). However, co-transfection of LexA-Ajuba with Gfi1-LexA induced significant, additive, and dose-dependent repression of the reporter (Fig. 3D). These data could either indicate that Ajuba is not a co-repressor, or that the co-repressor function of Ajuba may only be revealed upon binding to a nuclear target. To explore these possibilities we tested the function of LexA fusion to the Ajuba preLIM or LIM domains. Like LexA-Ajuba, LexA-PreLIM did not repress the activity of the reporter (Fig. 3E). In contrast, LexA-LIM induced significant and dose-dependent repression of the reporter (Fig. 3E). We reasoned that if the transcriptional activity of full-length Ajuba is hindered by intramolecular interactions, then LexA-LIM (which is only a portion of the protein) might be unhindered and potently synergize with Gfi1-LexA. In fact, LexA-LIM and Gfi1-LexA demonstrated significant dose-dependent and synergistic transcriptional repression (Fig. 3F). In contrast, LexA-PreLIM and Gfi1-LexA together resulted in reporter activity similar to Gfi1-LexA alone (Fig. 3F). Utilizing B30-TK-CAT reporter vectors and Gfi1 and Ajuba protein expression vectors (without LexA fusion), we find that the Pre-LIM domain does not increase Gfi1 repression. However, expression constructs encoding full-length Ajuba or isolated LIM domains increased Gfi1 repression (supplemental Fig. S1). Thus, the Ajuba LIM domains function as a Gfi1 co-repressor. Gfi1 and Ajuba Interact Directly with HDACs—We next determined whether Gfi1 and Ajuba are in a multiprotein complex using gel-filtration chromatography. Gel filtration separates larger and smaller proteins in a complex mixture. Immunoblot analysis of nuclear extracts fractionated on a Sephacryl S-300 column revealed elution profiles. HDAC1, HDAC2, and HDAC3 are part of protein complexes that elute at large molecular size fractions (24Hassig C.A. Fleischer T.C. Billin A.N. Schreiber S.L. Ayer D.E. Cell. 1997; 89: 341-347Abstract Full Text Full Text PDF PubMed Scopus (661) Google Scholar, 25Li J. Wang J. Wang J. Nawaz Z. Liu J.M. Qin J. Wong J. EMBO J. 2000; 19: 4342-4350Crossref PubMed Scopus (512) Goo
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