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Direct Interaction of the KRAB/Cys2-His2Zinc Finger Protein ZNF74 with a Hyperphosphorylated Form of the RNA Polymerase II Largest Subunit

1997; Elsevier BV; Volume: 272; Issue: 44 Linguagem: Inglês

10.1074/jbc.272.44.27877

ISSN

1083-351X

Autores

Benoı̂t Grondin, Francine Côté, Martine Bazinet, Michel Vincent, Muriel Aubry,

Tópico(s)

Nuclear Structure and Function

Resumo

We previously identified ZNF74 as a developmentally expressed gene commonly deleted in DiGeorge syndrome.ZNF74 encodes an RNA-binding protein tightly associated with the nuclear matrix and belongs to a large subfamily of Cys2-His2 zinc finger proteins containing a KRAB (Kruppel-associated box) repressor motif. We now report on the multifunctionality of the zinc finger domain of ZNF74. This nucleic acid binding domain is shown here to function as a nuclear matrix targeting sequence and to be involved in protein-protein interaction. By far-Western analysis and coimmunoprecipitation studies, we demonstrate that ZNF74 interacts, via its zinc finger domain, with the hyperphosphorylated largest subunit of RNA polymerase II (pol IIo) but not with the hypophosphorylated form. The importance of the phosphorylation in this interaction is supported by the observation that phosphatase treatment inhibits ZNF74 binding. Double immunofluorescence experiments indicate that ZNF74 colocalizes with the pol IIo and the SC35 splicing factor in irregularly shaped subnuclear domains. Thus, ZNF74 sublocalization in nuclear domains enriched in pre-mRNA maturating factors, its RNA binding activity, and its direct phosphodependent interaction with the pol IIo, a form of the RNA polymerase functionally associated with pre- mRNA processing, suggest a role for this member of the KRAB multifinger protein family in RNA processing. We previously identified ZNF74 as a developmentally expressed gene commonly deleted in DiGeorge syndrome.ZNF74 encodes an RNA-binding protein tightly associated with the nuclear matrix and belongs to a large subfamily of Cys2-His2 zinc finger proteins containing a KRAB (Kruppel-associated box) repressor motif. We now report on the multifunctionality of the zinc finger domain of ZNF74. This nucleic acid binding domain is shown here to function as a nuclear matrix targeting sequence and to be involved in protein-protein interaction. By far-Western analysis and coimmunoprecipitation studies, we demonstrate that ZNF74 interacts, via its zinc finger domain, with the hyperphosphorylated largest subunit of RNA polymerase II (pol IIo) but not with the hypophosphorylated form. The importance of the phosphorylation in this interaction is supported by the observation that phosphatase treatment inhibits ZNF74 binding. Double immunofluorescence experiments indicate that ZNF74 colocalizes with the pol IIo and the SC35 splicing factor in irregularly shaped subnuclear domains. Thus, ZNF74 sublocalization in nuclear domains enriched in pre-mRNA maturating factors, its RNA binding activity, and its direct phosphodependent interaction with the pol IIo, a form of the RNA polymerase functionally associated with pre- mRNA processing, suggest a role for this member of the KRAB multifinger protein family in RNA processing. Zinc finger proteins of the TFIIIA/Kruppel type belong to the largest known family of transcription factors (1Pieler T. Bellefroid E. Mol. Biol. Rep. 1994; 20: 1-8Crossref PubMed Scopus (91) Google Scholar, 2Klug A. Schwabe J.W. FASEB J. 1995; 9: 597-604Crossref PubMed Scopus (546) Google Scholar). These proteins are characterized by Cys2-His2 zinc finger motifs often repeated in tandem that fold around zinc ions and function as nucleic acid binding domains (3Pavletich N.P. Pabo C.O. Science. 1991; 252: 809-817Crossref PubMed Scopus (1760) Google Scholar, 4Theunissen O. Rudt F. Guddat U. Mentzel H. Pieler T. 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A. 1994; 91: 4509-4513Crossref PubMed Scopus (521) Google Scholar, 9Witzgall R. O'Leary E. Leaf A. Onaldi D. Bonventre J.V. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4514-4518Crossref PubMed Scopus (314) Google Scholar, 10Vissing H. Meyer W.K. Aagaard L. Tommerup N. Thiesen H.J. FEBS Lett. 1995; 369: 153-157Crossref PubMed Scopus (128) Google Scholar, 11Pengue G. Lania L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1015-1020Crossref PubMed Scopus (52) Google Scholar, 12Kim S.-S. Chen Y.-M. O'Leary E. Witzgall R. Vidal M. Bonventre J.V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15299-15304Crossref PubMed Scopus (249) Google Scholar, 13Friedman J.R. Fredericks W.J. Jensen D.E. Speicher D.W. Huang X.P. Neilson E.G. Rauscher III, F.J. Genes Dev. 1996; 10: 2067-2978Crossref PubMed Scopus (545) Google Scholar, 14Moosmann P. Georgiev O. Le Douarin B. Bourquin J.P. Schaffner W. Nucleic Acids Res. 1996; 24: 4859-4867Crossref PubMed Scopus (249) Google Scholar). Since they encode a repression motif and a potential DNA binding domain, members from the large KRAB/Cys2-His2 protein family are presently thought to function as transcriptional regulators of gene expression. We previously cloned ZNF74, a gene that encodes a KRAB/Cys2-His2 protein (15Grondin B. Bazinet M. Aubry M. J. Biol. Chem. 1996; 271: 15458-15467Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). ZNF74lies a few kilobases proximal to a polymorphic CA repeat (D22S264) (16Marineau C. Aubry M. Julien J.P. Rouleau G.A. Nucleic Acids Res. 1992; 20: 1430Google Scholar) that was shown to be a distal marker for 22q11.2 deletions associated with increased susceptibility to schizophrenia (17Karayiorgou M. Morris M.A. Morrow B. Shprintzen R.J. Goldberg R. Borrow J. Gos A. Nestadt G. Wolyniec P.S. Lasseter V.K. Eisen H. Childs B. Kazazian H.H. Kucherlapati R. Antonarakis S.E. Pulver A.E. Housman D.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7612-7616Crossref PubMed Scopus (545) Google Scholar). ZNF74is also one of the few genes found hemizygously deleted in the majority of patients with the DiGeorge syndrome, a microdeletion disorder associated with a wide variety of congenital malformations including cardiac defects, thymic hypoplasia, and hypocalcemia (18Aubry M. Marineau C. Zhang F.R. Zahed L. Figlewicz D. Delattre O. Thomas G. de Jong P.J. Julien J.P. Rouleau G.A. Genomics. 1992; 13: 641-648Crossref PubMed Scopus (29) Google Scholar, 19Aubry M. Demczuk S. Desmaze C. Aikem M. Aurias A. Julien J.P. Rouleau G.A. Hum. Mol. Genet. 1993; 2: 1583-1587Crossref PubMed Scopus (53) Google Scholar, 20Glover T.W. Nat. Genet. 1995; 10: 257-258Crossref PubMed Scopus (29) Google Scholar). To date, however, both the role of embryologically expressed ZNF74 in the DiGeorge syndrome (19Aubry M. Demczuk S. Desmaze C. Aikem M. Aurias A. Julien J.P. Rouleau G.A. Hum. Mol. Genet. 1993; 2: 1583-1587Crossref PubMed Scopus (53) Google Scholar) and its biochemical and cellular functions remain unclear. Although the presence of a KRAB motif and of 12 Cys2-His2 zinc finger motifs suggest that ZNF74 may function as a transcription factor, we recently demonstrated that its zinc finger domain harbors an RNA binding activity in vitro (15Grondin B. Bazinet M. Aubry M. J. Biol. Chem. 1996; 271: 15458-15467Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). This result suggests that, in addition to its potential function in transcriptional regulation, ZNF74 may also be involved in RNA metabolism. We also previously reported that ZNF74 is tightly associated with the nuclear matrix, since detergent, DNase, RNase, and high salt treatments could not release it from this nuclear scaffold structure (15Grondin B. Bazinet M. Aubry M. J. Biol. Chem. 1996; 271: 15458-15467Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). In this study, we report that the ZNF74 zinc finger domain acts as a nuclear matrix targeting sequence. Furthermore, to further define ZNF74 function, we searched for proteins from the nuclear matrix that directly interact with ZNF74. We now show that ZNF74 interacts, via its multifinger domain, with a hyperphosphorylated form of the largest subunit of RNA polymerase II (pol IIo) and colocalizes with this protein in irregularly shaped subnuclear domains. Because the hyperphosphorylated form of this RNA pol II 1The abbreviations used are: pol, polymerase; HA, hemagglutinin; MBP, maltose-binding protein; aa, amino acids; mAb, monoclonal antibody; CTD, carboxyl-terminal domain; snRNP, small nuclear ribonucleoprotein; PMSF, phenylmethylsulfonyl fluoride; Pipes, 1,4-piperazinediethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; LS, largest subunit; SR protein family, family of proteins rich in serines and arginines. subunit has been shown to colocalize (21Bregman D.B. Du L. Ribisi S. Warren S.L. J. Cell Sci. 1994; 107: 387-396PubMed Google Scholar, 22Bregman D.B. Du L. Van der Zee S. Warren S.L. J. Cell Biol. 1995; 129: 287-298Crossref PubMed Scopus (311) Google Scholar) and associate (23Vincent M. Lauriault P. Dubois M.-F. Lavoie S. Bensaude O. Chabot B. Nucleic Acids Res. 1996; 24: 4649-4652Crossref PubMed Scopus (77) Google Scholar, 24Mortillaro M.J. Blencowe B.J. Wei X. Nakayasu H. Du L. Warren S.L. Sharp P.A. Berezney R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8253-8257Crossref PubMed Scopus (282) Google Scholar, 25Kim E. Du L. Bregman D.B. Warren S.L. J. Cell Biol. 1997; 136: 19-28Crossref PubMed Scopus (213) Google Scholar) with splicing factors and to be involved in pre-mRNA processing (26Du L. Warren S.L. J. Cell Biol. 1997; 136: 5-18Crossref PubMed Scopus (123) Google Scholar, 27McCracken S. Fong N. Yankulov K. Ballantyne S. Pan G. Greenblatt J. Patterson S.D. Wickens M. Bentley D.L. Nature. 1997; 385: 357-361Crossref PubMed Scopus (740) Google Scholar), our results indicate that ZNF74 might play a role in the regulation of gene expression not only by transcriptional but also by post-transcriptional mechanisms. pCGN, a cytomegalovirus enhancer-driven eukaryotic expression vector (28Tanaka M. Herr W. Cell. 1990; 60: 375-386Abstract Full Text PDF PubMed Scopus (517) Google Scholar) that encodes an N-terminal hemagglutinin (HA) epitope was used to generate HA fusion constructs. pMAL-c vector (New England Biolabs) was used to generate maltose-binding protein (MBP) fusion constructs for expression inEscherichia coli. The following constructs were previously described in Grondin et al. (15Grondin B. Bazinet M. Aubry M. J. Biol. Chem. 1996; 271: 15458-15467Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar): HA-ZNF74-(1–572), HA-ZNF74ΔKrab-(68–572), HA-NΔKrab-Zn-(68–509), HA-Zn-C-(175–572), HA-Zn-(175–509), MBP-ZNF74-(1–572), MBP-ZNF74-(106–572), MBP-Zn-(175–509), and MBP-β-gal-α (also called MBP herein). GAL4 DNA binding domain (GAL4-(1–147)) fusion proteins were obtained by introducing either the zinc finger domain (aa 175–509) or the N-terminal domain (aa 1–174) of ZNF74 in a Rous sarcoma virus enhancer-driven eukaryotic expression vector pRSVGAL4 (kindly provided by Dr. Robert Rehfuss). The zinc finger domain (aa 175–509) or the N-terminal domain (aa 1–174) of ZNF74 were PCR-amplified with oligonucleotide primers containing XbaI cloning sites. In each case, the 3′-primer included an in frame stop codon. The PCR-amplified products were subcloned in frame at the 3′-end of the GAL4 DNA binding domain in a unique XbaI site of the pRSVGAL4 vector. The mAb CC3 (IgG2a) was previously obtained by immunizing Balb/C mice with chick embryo proteins (29Thibodeau A. Vincent M. Exp. Cell Res. 1991; 195: 145-153Crossref PubMed Scopus (27) Google Scholar) and shown to recognize a phosphoepitope located on the carboxyl-terminal domain (CTD) of the hyperphosphorylated RNA polymerase II largest subunit (pol IIo) (23Vincent M. Lauriault P. Dubois M.-F. Lavoie S. Bensaude O. Chabot B. Nucleic Acids Res. 1996; 24: 4649-4652Crossref PubMed Scopus (77) Google Scholar, 30Dubois M.F. Vincent M. Vigneron M. Adamczewski J. Egly J.-M. Bensaude O. Nucleic Acids Res. 1997; 25: 694-700Crossref PubMed Scopus (55) Google Scholar). The mAb Pol3/3 recognizes a conserved region located outside of the CTD of the largest subunit of the RNA polymerase II (RNA pol II LS) and was kindly provided by E. K. F. Bautz (31Kramer A. Haars R. Kabish R. Will H. Bautz E.K. Mol. Gen. Genet. 1980; 180: 193-199Crossref PubMed Scopus (80) Google Scholar). The mouse hybridoma cell line CRL 2031, secreting the mAb SC35 (IgG1), was purchased from the American Type Culture Collection. The mAb SC35 recognizes a phosphorylated form of SC35 (32Fu X.D. Maniatis T. Nature. 1990; 343: 437-441Crossref PubMed Scopus (571) Google Scholar), a non-snRNP splicing protein, and possibly another structural protein that colocalizes to the same nuclear domains (33Xing Y. Johnson C.V. Moen Jr., P.T. McNeil J.A. Lawrence J.B. J. Cell Biol. 1995; 131: 1635-1647Crossref PubMed Scopus (199) Google Scholar). The mAb 12CA5 (34Niman H.L. Houghten R.A. Walker L.E. Reisfeld R.A. Wilson I.A. Hogle J.M. Lerner R.A. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 4949-4953Crossref PubMed Scopus (319) Google Scholar) or the rabbit polyclonal antibody Y-11 (Santa Cruz Biotechnology Inc.) was used to detect the HA epitope tag. The GAL4 (DBD) mouse monoclonal IgG antibody (RK5C1) (Santa Cruz Biotechnology) was used to detect the DNA binding domain (aa 1–147) of GAL4 protein. A rabbit anti-MBP polyclonal antiserum (New England Biolabs) was used to detect the maltose-binding protein. The mAb 131C1 (kindly provided by Dr. Yves Raymond) was used to detect lamin A and C. Monkey fibroblast-like COS-7 cells, mouse skin fibroblast L cells, mouse embryonal carcinoma P19 cells, human embryonal kidney HEK 293 cells, or 293T cells (35Pear W.S. Nolan G.P. Scott M.L. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8392-8396Crossref PubMed Scopus (2305) Google Scholar) were used to prepare the various cellular fractions. For experiments requiring transfection, cells were plated at a cell density of 4–8 × 105/100-mm plate, transfected 24 h later with various plasmid constructs (25 μg of DNA) (36Colbere-Garapin F. Garapin A.C. Dev. Biol. Stand. 1983; 55: 267-271PubMed Google Scholar), and harvested 30–36 h after transfection (3–6 × 106cells obtained from a 100-mm plate). For cellular fractionation and nuclear matrix isolation, transfected or untransfected cells (3–6 × 106 cells) were first submitted to a hypotonic lysis in 40 μl of RSB buffer (10 mm Tris, pH 7.4, 10 mm NaCl, 3 mm MgCl2, 0.5 mm PMSF), and nuclei were recovered following essentially the method of Cockerill and Garrard (37Cockerill P.N. Garrard W.T. Cell. 1986; 44: 273-282Abstract Full Text PDF PubMed Scopus (744) Google Scholar) as described previously (15Grondin B. Bazinet M. Aubry M. J. Biol. Chem. 1996; 271: 15458-15467Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Washed nuclei were subjected to subnuclear fractionation and nuclear matrix isolation using the method of He et al. (38He D.C. Nickerson J.A. Penman S. J. Cell Biol. 1990; 110: 569-580Crossref PubMed Scopus (369) Google Scholar) as previously detailed (15Grondin B. Bazinet M. Aubry M. J. Biol. Chem. 1996; 271: 15458-15467Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). In brief, the nuclei derived from a 100-mm plate were freed of the chromatin by a 50-min digestion at 30 °C with RNase-free DNase I (about 20 units/106 cell nuclei) (Life Technologies, Inc.) in 40 μl of digestion buffer (10 mm Pipes, pH 6.8, 50 mm NaCl, 300 mm sucrose, 3 mm MgCl2, 1 mm EGTA, 0.5% (v/v) Triton X-100, 1.2 mm PMSF, 2 μg/ml aprotinin, 2 μg/ml leupeptin, and 1 μg/ml pepstatin A), and then extracted by the addition of 1 m ammonium sulfate to a final concentration of 0.25 m. The 750 ×g supernatant containing digested chromatin corresponds to the soluble DNase I-treated nuclear fraction also called soluble nuclear fraction herein. The pellet was then submitted to an additional extraction with 50 μl of digestion buffer containing 2 mNaCl. The subsequent 750 × g pellet corresponding to insoluble nuclear matrix and associated ribonucleoproteins was subjected to RNase A (Quiagen) and RNase T (Boehringer Mannheim) (5 μg and 2 units, respectively) in 50 μl of digestion buffer. The resulting 750 × g pellet corresponds to the RNase-treated insoluble nuclear matrix fraction. To prepare the various nuclear fractions used in far-Western analysis, 2 mmorthovanadate was also included in the RSB buffer, and both 2 mm orthovanadate and 50 mm NaF were added to the digestion buffer. Furthermore, in some cases as indicated in the figure legends, a faster cell fractionation scheme was used to limit protein dephosphorylation by endogenous phosphatases; the centrifugation steps were reduced to 15 s at 15,000 ×g, the chromatin was digested for only 25 min with twice the amount of DNase I, and the RNase A and T digestion step was omitted. Before immunoprecipitations, the soluble DNase I-treated fractions (equivalent to 20–40 × 106cells/350 μl of digestion buffer) obtained as described under "Cellular Fractionation and Nuclear Matrix Isolation" were diluted with 2 volumes of lysis buffer (50 mm Tris, pH 7.5, 250 mm NaCl, 0.5% Triton X-100, 0.5% sodium deoxycholate, 5 mm EDTA, 2 mm orthovanadate, 50 mmNaF, 1 mm PMSF, 2 μg/ml aprotinin, 2 μg/ml leupeptin). To prepare nuclear matrix extracts for immunoprecipitations, the DNase/RNase-digested nuclear matrix pellets (from about 10–20 × 106 cells) obtained as described under "Cellular Fractionation and Nuclear Matrix Isolation" were extracted with 500 μl of SDS solubilizing buffer (50 mm Tris, pH 7.5, 100 mm NaCl, 0.8% SDS, 2 mm orthovanadate, 50 mm NaF) for 4 min at 95 °C (39Brancolini C. Schneider C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6936-6940Crossref PubMed Scopus (23) Google Scholar). The extracts were cooled at 4 °C for 4 min before adding 500 μl of Triton buffer (50 mm Tris, pH 7.5, 100 mm NaCl, 4% Triton X-100, 2 mm orthovanadate, 50 mm NaF) and then centrifuged at 15,000 × g for 2 min at 4 °C. To prepare total cell extracts (5–10 × 106 cells/ml) for immunoprecipitations, cells were either extracted in SDS solubilizing buffer and diluted in Triton buffer as described above or extracted in lysis buffer (50 mm Tris, pH 7.5, 250 mm NaCl, 0.5% Triton X-100, 0.5% sodium deoxycholate, 5 mm EDTA, 2 mm orthovanadate, 50 mmNaF, 1 mm PMSF, 2 μg/ml aprotinin, 2 μg/ml leupeptin). The extracts were passed through a 26G1/2 needle to reduce viscosity before centrifugation at 15,000 × g for 15 min at 4 °C. Total cell, nuclear matrix, and diluted soluble DNase I-treated extracts were precleared with protein A-Sepharose (Sigma) for 1 h at 4 °C. Precleared extracts were then recovered after removal of the beads by centrifugation (1 ml/20 μl stacked protein A beads). Immunoprecipitations were then carried out for 2 h at 4 °C (or 16 h for coimmunoprecipitation experiments) using 1 ml of precleared extracts, 10–50 μl of the appropriate antibody, and 30 μl of protein A-Sepharose. For the coimmunoprecipitation experiments, 10 mm EDTA and 10 mm EGTA were added during the preclearing and the immunoprecipitation steps to stabilize the pol IIo (40Kim W.-Y. Dahmus M.E. J. Biol. Chem. 1986; 261: 14219-14225Abstract Full Text PDF PubMed Google Scholar). The protein A-Sepharose beads were then washed 3 times either with 1 ml of lysis buffer for immunoprecipitations or with a buffer containing 25 mm Tris, pH 7.5, 100 mm NaCl, 0.2% SDS, and 1% Triton for coimmunoprecipitations. Washed immunoprecipitates were resuspended in Laemmli buffer, and the recovered proteins were analyzed by SDS-PAGE. In some cases, the supernatants of immunoprecipitation were first concentrated using Microcon 100 filter devices (Amicon) before dilution in Laemmli buffer. E. coli DH5α strain transformed with pMal-c-ZNF74 fusion constructs were grown to anA 600 of 0.3–0.5 (500 ml) and induced with 0.3 mm isopropyl-β-d-thiogalactopyranoside for 3 h. Crude soluble extracts containing the soluble MBP fusion proteins were prepared from bacterial cells resuspended in column buffer (200 mm Tris, pH 7.5, 200 mm NaCl, 10 mm β-mercaptoethanol) (50 ml). MBP fusion proteins from these extracts were immobilized on amylose resin (0.5 ml) (New England Biolabs). The resin was washed with 20 volumes of column buffer and with 40 volumes of elution buffer (20 mm Hepes, pH 7.5, 100 mm NaCl). Then, the immobilized MBP fusion proteins were eluted with 1.5 ml of elution buffer containing 10 mmmaltose. Depending on the preparation and on the fusion proteins, 100–1000 μg of eluted affinity-purified proteins were recovered as estimated by a Bradford protein assay (Bio-Rad) and as confirmed by Coomassie gel staining. The purified proteins were aliquoted and stored at −80 °C. Proteins were biotinylated according to Cicchetti and Baltimore (41Cicchetti P. Baltimore D. Methods Enzymol. 1995; 256: 140-148Crossref PubMed Scopus (6) Google Scholar). In brief, the MBP-ZNF74-(106–572) fusion protein eluted from the amylose resin was dialyzed against a borate buffer (100 mm, pH 8.0). The protein was incubated for 4 h at room temperature with biotinamidocaproateN-hydroxysuccinimide ester (Sigma) at a concentration of 50 μg of the biotin derivative for 1 mg of MBP-ZNF74-(106–572) fusion protein. The reaction was stopped by adding 1 mNH4Cl at a ratio of 4 μl for 50 μg of biotin derivative for 10 min at room temperature. The biotinylated protein was then dialyzed against a 50 mm Tris, pH 7.5, buffer containing 100 mm NaCl. Glycerol (final concentration 15%) was added to the dialyzed biotinylated protein before its storage at −20 °C. Thawed biotinylated proteins (1.5 μg/μl) were never kept longer than 2 weeks at 4 °C before use. Nuclear protein fractions or proteins immunoprecipitated from cell or nuclear extracts were resolved by SDS-PAGE on a 6% separating gel and electrophoretically transferred to 0.2-μm nitrocellulose membrane at 22 V for 12–16 h using a Mini Trans-Blot® electrophoretic transfer cell (Bio-Rad). After transfer, the blots were rinsed in TBST buffer (10 mm Tris, pH 7.5, 150 mm NaCl, 0.05% Tween 20) and used immediately or kept at 4 °C in the same buffer for a few days. The blots were incubated for 4–16 h in blocking buffer (50 mm Tris, pH 7.5, 150 mm NaCl, 2% bovine serum albumin). For far-Western analysis, the blots (9-cm2 strips) were probed for 12–16 h with affinity-purified MBP fusion proteins (0.5 μg/ml) or with biotinylated MBP-ZNF74-(106–572) fusion proteins (30 ng/ml) in overlay buffer (20 mm Hepes, pH 7.5, 100 mmNaCl, 1 mm EGTA, 1% Nonidet P-40, 0.5% bovine serum albumin, 0.25% gelatin) (2 ml). For competition experiments, the blots were incubated in the presence of the biotinylated MBP fusion protein and a 100-fold excess of unbiotinylated MBP fusion protein. All the above steps were performed at 4 °C. The blots were then washed in washing buffer (50 mm Tris, pH 7.5, 150 mmNaCl, 0.05% Tween 20, 0.2% gelatin) three times for 5 min at room temperature and then incubated for 1 h in TBST buffer containing 1% bovine serum albumin. Bound MBP fusion proteins were revealed by sequential incubation with a rabbit primary anti-MBP polyclonal antiserum (New England Biolabs), a secondary goat anti-rabbit horseradish peroxidase (Sigma), and a chemiluminescence reagent as described by the manufacturer (Renaissance® kit, NEN Life Science Products). Bound biotinylated MBP-ZNF74 fusion proteins were also revealed by chemiluminescence after an incubation step with avidin-horseradish peroxidase (Sigma). This protocol was modified from Bregman et al. (22Bregman D.B. Du L. Van der Zee S. Warren S.L. J. Cell Biol. 1995; 129: 287-298Crossref PubMed Scopus (311) Google Scholar). Proteins were immunoprecipitated as above with mAb CC3 coupled to protein A-Sepharose. For alkaline phosphatase treatment, the beads were washed with phosphatase buffer (10 mm Tris acetate, pH 7.5, 10 mm magnesium acetate, 50 mmpotassium acetate) and treated with or without 5 units of calf intestinal phosphatase (Pharmacia Biotech Inc.) in 25 μl (excluding bed volume) of phosphatase buffer for 10–60 min at 37 °C. The beads were resuspended in Laemmli buffer for analysis of the eluted proteins by SDS-PAGE. Subconfluent monkey COS-7 cells plated in 1 × 2-cm four-well Lab-Tek™ (Nunc) were transfected with appropriate plasmid constructs (2 μg) (36Colbere-Garapin F. Garapin A.C. Dev. Biol. Stand. 1983; 55: 267-271PubMed Google Scholar). About 40 h after transfection, the cells were either fixed and processed directly for immunofluorescence when indicated or first extracted in situas described by Bisotto et al. (42Bisotto S. Lauriault P. Duval M. Vincent M. J. Cell Sci. 1995; 108: 1873-1882PubMed Google Scholar) using the method of Heet al. (38He D.C. Nickerson J.A. Penman S. J. Cell Biol. 1990; 110: 569-580Crossref PubMed Scopus (369) Google Scholar). Briefly, for in situ sequential extraction, the cells were first washed in isotonic phosphate saline buffer (PBS buffer) and then extracted in cytoskeleton buffer (10 mm Pipes, pH 6.8, 100 mm NaCl, 300 mm sucrose, 3 mm MgCl2, 1 mm EGTA, 0.5% Triton X-100, 1 mm PMSF), digested with DNase I (Life Technologies), and washed with 0.25m ammonium sulfate for chromatin removal. When indicated, the transfected cells were also subjected to a high salt extraction with 2 m NaCl and an RNase A and T digestion. For immunofluorescence microscopy, untreated or in situextracted cells were treated sequentially at room temperature with 4% formaldehyde/PBS for 1 h, with 0.2% Tween-20, 4% formaldehyde/PBS for 1 h, with 50 mmNH4Cl/PBS for 30 min and with 0.2% cold fish gelatin (Sigma)/PBS for 30 min (15Grondin B. Bazinet M. Aubry M. J. Biol. Chem. 1996; 271: 15458-15467Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Primary and secondary antibody incubations were then performed essentially as described (15Grondin B. Bazinet M. Aubry M. J. Biol. Chem. 1996; 271: 15458-15467Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). For single immunolabeling, the mouse mAb 12CA5 (hybridoma supernatant diluted 1:500) was used as a primary antibody to detect HA epitope-tagged ZNF74 proteins, and a mouse anti-Gal 4 antibody (1:50) (Santa Cruz Biotechnology) was used to detect GAL4 proteins. A rabbit anti-mouse IgG conjugated to fluorescein isothiocyanate (Sigma) was used as a secondary antibody. For double immunolabeling of HA-ZNF74-(1–572) and pol IIo/CC3 antigen, a rabbit polyclonal anti-HA antibody (diluted 1:200) (Santa Cruz Biotechnology) and the mouse mAb CC3 antibody (ascitic fluid diluted 1:500) were sequentially added. For immunolabeling of both HA-ZNF74-(1–572) and SC35 antigen, the rabbit polyclonal anti-HA antibody and the mouse mAb SC35 (ascitic fluid diluted 1:200) were used as primary antibodies. A goat anti-rabbit IgG conjugated to fluorescein isothiocyanate (Sigma) and a goat anti-mouse IgG conjugated to Rhodamine (Pierce) were then used as secondary antibodies. The specificity of each of the secondary antibodies was tested either by omitting one of the primary antibodies or by incubating the primary antibody individually with each of the secondary antibodies. No cross-reaction was observed between the two sets of antibodies. DNA was stained by a 5-min treatment with 2.5 μg/ml Hoechst 33258 fluorochrome (Sigma). Preparations were examined under a Leika photomicroscope equipped for epifluorescence and photographed using NEOPAN 1600 (Fuji) or EPL 400 (Kodak) films. We previously reported that the protein encoded by ZNF74–2 cDNA (EMBL accession number X92715), called ZNF74 here, is an RNA-binding protein tightly associated with the nuclear matrix (15Grondin B. Bazinet M. Aubry M. J. Biol. Chem. 1996; 271: 15458-15467Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 19Aubry M. Demczuk S. Desmaze C. Aikem M. Aurias A. Julien J.P. Rouleau G.A. Hum. Mol. Genet. 1993; 2: 1583-1587Crossref PubMed Scopus (53) Google Scholar). To delimit the region required for ZNF74 association with the nuclear matrix, we assessed the subnuclear localization of various HA-tagged deletion mutants in transfected cells. Following subcellular fractionation of transfected L cells, all truncated ZNF74 proteins that included at least the zinc finger domain were found associated with the insoluble nuclear matrix fraction as assessed by immunoblot (Fig.1 A). As previously observed for the full-length protein (15Grondin B. Bazinet M. Aubry M. J. Biol. Chem. 1996; 271: 15458-15467Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), the attachment of all of these truncated ZNF74 proteins to the nuclear matrix was very tight, since none were detectable in the soluble nuclear fractions recovered after sequential DNase, high salt, and RNase treatments (not shown). Immunofluorescence studies using monolayers of transfected COS-7 cells confirmed that the multifinger region (aa 175–509) alone was targeted to the nuclear matrix, where it remained tightly bound followingin situ sequential DNase/RNase extractions (Fig.2 A).Figure 2Targeting of ZNF74 zinc finger region and a fused heterologous nucleoplasmic protein to the nuclear matrix as detected by immunolocalization. COS-7 cells were transfected inA with HA-tagged Zn-(175–509) and in B with the GAL4 DNA binding domain or the GAL4 DNA binding domain-Zn-(175–509) fusion construct. Cells were untreated or submitted to in

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