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The Embryonic Function of Germ Cell Nuclear Factor Is Dependent on the DNA Binding Domain

2002; Elsevier BV; Volume: 277; Issue: 52 Linguagem: Inglês

10.1074/jbc.m209586200

1083-351X

Zi‐Jian Lan, Arthur Chung, Xueping Xu, Francesco J. DeMayo, Austin J. Cooney,

Retinoids in leukemia and cellular processes

Germ cell nuclear factor (GCNF), an orphan nuclear receptor, is essential for mouse embryogenesis. GCNF specifically binds to a DR0 response element via its DNA binding domain (DBD) in vitro and functions as a repressor of gene transcription. To further study the role of GCNF during embryogenesis, we have employed a Cre/loxP strategy and generated a line ofGCNF mutant mice (GCNF lox/lox) in which the 243-base pair DBD-encoding exon has been deleted in the germline. However, the ligand binding domain (LBD) of GCNF is still expressed at the mRNA and protein levels in theGCNF lox/lox mice.GCNF lox/lox mice die at 9.5–10.5 days postcoitum. The tailbuds of these mutant embryos protrude outside the yolk sac. Expression of Oct-4 in the somatic cells ofGCNF lox/lox embryos at 8.25 days postcoitum was not silenced as in the GCNF +/+ embryos. Therefore, GCNF lox/lox mice phenocopy theGCNF −/− mice. Our results indicate that the DBD is essential for the function of GCNF during early mouse embryogenesis, and that the LBD does not mediate any function independent of the DBD at this stage of embryonic development. Our results also suggest that GCNF is indeed a transcriptional factor that represses gene transcription mediated via its DBD. Germ cell nuclear factor (GCNF), an orphan nuclear receptor, is essential for mouse embryogenesis. GCNF specifically binds to a DR0 response element via its DNA binding domain (DBD) in vitro and functions as a repressor of gene transcription. To further study the role of GCNF during embryogenesis, we have employed a Cre/loxP strategy and generated a line ofGCNF mutant mice (GCNF lox/lox) in which the 243-base pair DBD-encoding exon has been deleted in the germline. However, the ligand binding domain (LBD) of GCNF is still expressed at the mRNA and protein levels in theGCNF lox/lox mice.GCNF lox/lox mice die at 9.5–10.5 days postcoitum. The tailbuds of these mutant embryos protrude outside the yolk sac. Expression of Oct-4 in the somatic cells ofGCNF lox/lox embryos at 8.25 days postcoitum was not silenced as in the GCNF +/+ embryos. Therefore, GCNF lox/lox mice phenocopy theGCNF −/− mice. Our results indicate that the DBD is essential for the function of GCNF during early mouse embryogenesis, and that the LBD does not mediate any function independent of the DBD at this stage of embryonic development. Our results also suggest that GCNF is indeed a transcriptional factor that represses gene transcription mediated via its DBD. germ cell nuclear factor DNA binding domain ligand-binding domain days postcoitum embryonic stem reverse transcription phosphoglycerate kinase cytomegalovirus Germ cell nuclear factor (GCNF,1 NR6A1) is a novel orphan member of the nuclear receptor superfamily as it is more distantly related to other members and forms a sixth and separate subbranch of the family (1Nuclear Receptor Nomenclature Committee Cell. 1999; 97: 161-163Abstract Full Text Full Text PDF PubMed Scopus (934) Google Scholar, 2Cooney A.J. Katz D. Hummelke G. Jackson K. Amer. Zool. 1999; 39: 796-806Crossref Scopus (13) Google Scholar). GCNF was initially cloned by our laboratory using low stringency screening with a DNA binding domain (DBD) probe (3Chen F. Cooney A.J. Wang Y. Law S.W. O'Malley B.W. Mol. Endocrinol. 1994; 8: 1434-1444PubMed Google Scholar) and subsequently cloned by other laboratories and given other names, e.g. RTR (retinoid receptor-related testis-specific receptor (4Hirose T. O'Brien D.A. Jetten A.M. Gene. 1995; 152: 247-251Crossref PubMed Scopus (75) Google Scholar)) and NCNF (neuronal cellnuclear factor (5Bauer U.M. Schneider-Hirsch S. Reinhardt S. Pauly T. Maus A. Wang F. Heiermann R. Rentrop M. Maelicke A. Eur. J. Biochem. 1997; 249: 826-837Crossref PubMed Scopus (33) Google Scholar)). To date, homologs ofGCNF have been cloned from several other species including human, Xenopus, and zebrafish (2Cooney A.J. Katz D. Hummelke G. Jackson K. Amer. Zool. 1999; 39: 796-806Crossref Scopus (13) Google Scholar, 6Greschik H. Schule R. J. Mol. Med. 1998; 76: 800-810Crossref PubMed Scopus (27) Google Scholar, 7Lan Z.J. Cooney A.J. Recent Res. Devel. Endocrinol. 2001; 2: 295-307Google Scholar, 8Braat A.K. Zandbergen M.A., De Vries E. Van Der Burg B. Bogerd J. Goos H.J. Mol. Reprod. Dev. 1999; 53: 369-375Crossref PubMed Scopus (25) Google Scholar). The mouseGCNF gene contains 11 exons (9Susens U. Borgmeyer U. Genome Biol. 2000; 1 (, research 0006): 1-3Crossref PubMed Google Scholar) and is located on chromosome 2, 2A. J. Cooney, unpublished data.2A. J. Cooney, unpublished data. while the humanGCNF gene is located on chromosome 9 at the locus q33–34.1 (10Lei W. Hirose T. Zhang L.X. Adachi H. Spinella M.J. Dmitrovsky E. Jetten A.M. J. Mol. Endocrinol. 1997; 18: 167-176Crossref PubMed Scopus (33) Google Scholar, 11Agoulnik I.Y. Cho Y. Niederberger C. Kieback D.G. Cooney A.J. FEBS Lett. 1998; 424: 73-78Crossref PubMed Scopus (36) Google Scholar). Sequence analysis has shown that GCNF homologs have high homology in the DBD and the ligand binding domain (LBD) among different species (2Cooney A.J. Katz D. Hummelke G. Jackson K. Amer. Zool. 1999; 39: 796-806Crossref Scopus (13) Google Scholar, 8Braat A.K. Zandbergen M.A., De Vries E. Van Der Burg B. Bogerd J. Goos H.J. Mol. Reprod. Dev. 1999; 53: 369-375Crossref PubMed Scopus (25) Google Scholar). This high amino acid identity within the DBD and LBD across species indicates the functional conservation of GCNF protein in binding to its DNA-response element and its putative ligand during evolution. Indeed, the DNA binding specificity of GCNF is conserved among mouse, human and Xenopus (3Chen F. Cooney A.J. Wang Y. Law S.W. O'Malley B.W. Mol. Endocrinol. 1994; 8: 1434-1444PubMed Google Scholar, 12Yan Z.H. Medvedev A. Hirose T. Gotoh H. Jetten A.M. J. Biol. Chem. 1997; 272: 10565-10572Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 13Cooney A.J. Hummelke G.C. Herman T. Chen F. Jackson K.J. Biochem. Biophys. Res. Commun. 1998; 245: 94-100Crossref PubMed Scopus (46) Google Scholar, 14David R. Joos T.O. Dreyer C. Mech. Dev. 1998; 79: 137-152Crossref PubMed Scopus (33) Google Scholar, 15Schmitz T.P. Susens U. Borgmeyer U. Biochim. Biophys. Acta. 1999; 1446: 173-180Crossref PubMed Scopus (14) Google Scholar). GCNF can specifically bind to either a DR0 element, a direct repeat of the estrogen receptor half-site (AGGTCA) with zero base pair spacing between the half-sites, as a homodimer (3Chen F. Cooney A.J. Wang Y. Law S.W. O'Malley B.W. Mol. Endocrinol. 1994; 8: 1434-1444PubMed Google Scholar, 12Yan Z.H. Medvedev A. Hirose T. Gotoh H. Jetten A.M. J. Biol. Chem. 1997; 272: 10565-10572Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 13Cooney A.J. Hummelke G.C. Herman T. Chen F. Jackson K.J. Biochem. Biophys. Res. Commun. 1998; 245: 94-100Crossref PubMed Scopus (46) Google Scholar, 15Schmitz T.P. Susens U. Borgmeyer U. Biochim. Biophys. Acta. 1999; 1446: 173-180Crossref PubMed Scopus (14) Google Scholar, 16Borgmeyer U. Eur. J. Biochem. 1997; 244: 120-127Crossref PubMed Scopus (32) Google Scholar, 17Greschik H. Wurtz J.M. Hublitz P. Kohler F. Moras D. Schule R. Mol. Cell. Biol. 1999; 19: 690-703Crossref PubMed Scopus (38) Google Scholar), or an extended DR0 half-site (TCAAGGTCA) either as a monomer (12Yan Z.H. Medvedev A. Hirose T. Gotoh H. Jetten A.M. J. Biol. Chem. 1997; 272: 10565-10572Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), a homodimer (13Cooney A.J. Hummelke G.C. Herman T. Chen F. Jackson K.J. Biochem. Biophys. Res. Commun. 1998; 245: 94-100Crossref PubMed Scopus (46) Google Scholar, 17Greschik H. Wurtz J.M. Hublitz P. Kohler F. Moras D. Schule R. Mol. Cell. Biol. 1999; 19: 690-703Crossref PubMed Scopus (38) Google Scholar), or both a monomer and homodimer (17Greschik H. Wurtz J.M. Hublitz P. Kohler F. Moras D. Schule R. Mol. Cell. Biol. 1999; 19: 690-703Crossref PubMed Scopus (38) Google Scholar). Recently, two dimerization motifs in mouse GCNF, one located in the DBD including the adjacent TA box and the other in α-helix 3 of the LBD, have been characterized (17Greschik H. Wurtz J.M. Hublitz P. Kohler F. Moras D. Schule R. Mol. Cell. Biol. 1999; 19: 690-703Crossref PubMed Scopus (38) Google Scholar). The TA box is critically involved in homodimeric interactions on the DR0 element (16Borgmeyer U. Eur. J. Biochem. 1997; 244: 120-127Crossref PubMed Scopus (32) Google Scholar, 17Greschik H. Wurtz J.M. Hublitz P. Kohler F. Moras D. Schule R. Mol. Cell. Biol. 1999; 19: 690-703Crossref PubMed Scopus (38) Google Scholar), while both the TA box and the α-helix 3 are required for homodimeric binding of mouse GCNF to the extended DR0 half-site (17Greschik H. Wurtz J.M. Hublitz P. Kohler F. Moras D. Schule R. Mol. Cell. Biol. 1999; 19: 690-703Crossref PubMed Scopus (38) Google Scholar). Characterization of the DNA binding motif of GCNF led to the identification of several potential target genes for GCNF including the protamine 1 and 2 genes in the testis (12Yan Z.H. Medvedev A. Hirose T. Gotoh H. Jetten A.M. J. Biol. Chem. 1997; 272: 10565-10572Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 18Hummelke G.C. Meistrich M.L. Cooney A.J. Mol. Reprod. Dev. 1998; 50: 396-405Crossref PubMed Scopus (37) Google Scholar) and the Oct4 gene in the mouse embryo (19Fuhrmann G. Chung A.C. Jackson K.J. Hummelke G. Baniahmad A. Sutter J. Sylvester I. Scholer H.R. Cooney A.J. Dev. Cell. 2001; 1: 377-387Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). In the absence of a ligand, GCNF functions to repress the transcription of these target genes in vitro and in vivo (7Lan Z.J. Cooney A.J. Recent Res. Devel. Endocrinol. 2001; 2: 295-307Google Scholar,19Fuhrmann G. Chung A.C. Jackson K.J. Hummelke G. Baniahmad A. Sutter J. Sylvester I. Scholer H.R. Cooney A.J. Dev. Cell. 2001; 1: 377-387Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 20Hummelke G.C. Cooney A.J. Front. Biosci. 2001; 6: D1186-D1191Crossref PubMed Google Scholar). It has been shown that GCNF is not only expressed in mouse and Xenopus embryos after the onset of gastrulation (14David R. Joos T.O. Dreyer C. Mech. Dev. 1998; 79: 137-152Crossref PubMed Scopus (33) Google Scholar,21Joos T.O. David R. Dreyer C. Mech. Dev. 1996; 60: 45-57Crossref PubMed Scopus (42) Google Scholar, 22Susens U. Aguiluz J.B. Evans R.M. Borgmeyer U. Dev. Neurosci. 1997; 19: 410-420Crossref PubMed Scopus (75) Google Scholar, 23Song K. Takemaru K.I. Moon R.T. Dev. Biol. 1999; 213: 170-179Crossref PubMed Scopus (10) Google Scholar, 24Chung A.C. Katz D. Pereira F.A. Jackson K.J. DeMayo F.J. Cooney A.J. O'Malley B.W. Mol. Cell. Biol. 2001; 21: 663-677Crossref PubMed Scopus (91) Google Scholar) but also in the gametogenic cells of adult vertebrates (3Chen F. Cooney A.J. Wang Y. Law S.W. O'Malley B.W. Mol. Endocrinol. 1994; 8: 1434-1444PubMed Google Scholar, 4Hirose T. O'Brien D.A. Jetten A.M. Gene. 1995; 152: 247-251Crossref PubMed Scopus (75) Google Scholar,11Agoulnik I.Y. Cho Y. Niederberger C. Kieback D.G. Cooney A.J. FEBS Lett. 1998; 424: 73-78Crossref PubMed Scopus (36) Google Scholar, 18Hummelke G.C. Meistrich M.L. Cooney A.J. Mol. Reprod. Dev. 1998; 50: 396-405Crossref PubMed Scopus (37) Google Scholar, 25Bauer U.M. Schneider-Hirsch S. Reinhardt S. Benavente R. Maelicke A. FEBS Lett. 1998; 439: 208-214Crossref PubMed Scopus (32) Google Scholar, 26Zhang Y.L. Akmal K.M. Tsuruta J.K. Shang Q. Hirose T. Jetten A.M. Kim K.H. O'Brien D.A. Mol. Reprod. Dev. 1998; 50: 93-102Crossref PubMed Scopus (49) Google Scholar, 27Katz D. Niederberger C. Slaughter G.R. Cooney A.J. Endocrinology. 1997; 138: 4364-4372Crossref PubMed Scopus (69) Google Scholar). This expression pattern indicates that GCNF may play a role in gametogenesis and normal embryonic development during gastrulation. Recently, we have generated a GCNF knockout mouse model (GCNF −/−) using a conventional embryonic stem cell strategy to address the functions of GCNF during mammalian embryogenesis (24Chung A.C. Katz D. Pereira F.A. Jackson K.J. DeMayo F.J. Cooney A.J. O'Malley B.W. Mol. Cell. Biol. 2001; 21: 663-677Crossref PubMed Scopus (91) Google Scholar). Insertion of PGK-neomycin (PGK-neo) into the GCNF locus causes embryonic lethality at 9.5–10.5 dpc. The most remarkable phenotype of theseGCNF mutant embryos is that the posterior tailbud develops outside of the yolk sac (24Chung A.C. Katz D. Pereira F.A. Jackson K.J. DeMayo F.J. Cooney A.J. O'Malley B.W. Mol. Cell. Biol. 2001; 21: 663-677Crossref PubMed Scopus (91) Google Scholar). These mutant embryos have serious defects in posterior and trunk development and somitogenesis (24Chung A.C. Katz D. Pereira F.A. Jackson K.J. DeMayo F.J. Cooney A.J. O'Malley B.W. Mol. Cell. Biol. 2001; 21: 663-677Crossref PubMed Scopus (91) Google Scholar). In addition, expression of the POU domain transcription factor,Oct4, is not repressed in the somatic cells of theseGCNF mutant embryos at 8.25–8.75 dpc when it is silenced in normal embryos (19Fuhrmann G. Chung A.C. Jackson K.J. Hummelke G. Baniahmad A. Sutter J. Sylvester I. Scholer H.R. Cooney A.J. Dev. Cell. 2001; 1: 377-387Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). It appears that GCNF is required for normal anteroposterior development, somitogenesis, and Oct4expression in mouse embryos. However, recent studies in several other laboratories have shown that the introduction of the PGK-neointo the mouse genome by conventional ES cell targeting can cause unexpected phenotypes in the resulting animal models (28Holzenberger M. Leneuve P. Hamard G. Ducos B. Perin L. Binoux M. Le Bouc Y. Endocrinology. 2000; 141: 2557-2566Crossref PubMed Scopus (80) Google Scholar, 29Olson E.N. Arnold H.H. Rigby P.W. Wold B.J. 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Therefore, generation and phenotypic analysis of a line ofGCNF mutant mice, in which the PGK-neo cassette is removed, will definitely clarify whether the phenotypes observed in the conventional GCNF knockout mice are due to the ablation of the GCNF gene or to the insertion of the PGK-neo cassette in the GCNF allele, causing misexpression of neighboring genes such as the steroidogenic factor 1 (SF-1) gene, which is located 3′ to the GCNFgene. As a member of the nuclear receptor superfamily, the DBD of GCNF is required to bind to its response elements and regulate gene transcription. Deletion of the 55 amino acid residues containing the DBD zinc finger region in the N terminus of Xenopus GCNF abolishes its DNA binding activity, and overexpression of this truncated GCNF protein in Xenopus embryos causes abnormal head development (14David R. Joos T.O. Dreyer C. Mech. Dev. 1998; 79: 137-152Crossref PubMed Scopus (33) Google Scholar). How much of the function of GCNF is mediated by the DBD in mouse embryonic development remains to be determined. GCNF may have DNA binding-independent activities, similar to other nuclear receptors such as glucocorticoid receptor (33Reichardt H.M. Kaestner K.H. Tuckermann J. Kretz O. Wessely O. Bock R. Gass P. Schmid W. Herrlich P. Angel P. Schutz G. Cell. 1998; 93: 531-541Abstract Full Text Full Text PDF PubMed Scopus (915) Google Scholar) and TR3 (34Li H. Kolluri S.K., Gu, J. Dawson M.I. Cao X. Hobbs P.D. Lin B. Chen G., Lu, J. Lin F. Xie Z. Fontana J.A. Reed J.C. Zhang X. Science. 2000; 289: 1159-1164Crossref PubMed Scopus (587) Google Scholar). In this study, we have generated a line of GCNF mutant mice lacking the 243-bp DBD-encoding exon 4 of the GCNF gene (9Susens U. Borgmeyer U. Genome Biol. 2000; 1 (, research 0006): 1-3Crossref PubMed Google Scholar) using the Cre/loxP system (35Rossant J. McMahon A. Genes Dev. 1999; 13: 142-145Crossref PubMed Scopus (95) Google Scholar). We found that these mutant mice have the same phenotypes as the conventional GCNF knockout mice (19Fuhrmann G. Chung A.C. Jackson K.J. Hummelke G. Baniahmad A. Sutter J. Sylvester I. Scholer H.R. Cooney A.J. Dev. Cell. 2001; 1: 377-387Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 24Chung A.C. Katz D. Pereira F.A. Jackson K.J. DeMayo F.J. Cooney A.J. O'Malley B.W. Mol. Cell. Biol. 2001; 21: 663-677Crossref PubMed Scopus (91) Google Scholar). Unlike the conventional GCNF knockout mice, the LBD of GCNF was still expressed in these mutant embryos at both the mRNA and protein levels. Therefore, the DBD of GCNF is essential for mediating the function of GCNF during mouse embryonic development. Genomic clones of the murine GCNF gene have been isolated from a 129Sv strain mouse genomic library (24Chung A.C. Katz D. Pereira F.A. Jackson K.J. DeMayo F.J. Cooney A.J. O'Malley B.W. Mol. Cell. Biol. 2001; 21: 663-677Crossref PubMed Scopus (91) Google Scholar). The targeting vector was constructed from a genomic clone containing exon 4 encoding the GCNF DBD. First, oligonucleotides containing an XhoI restriction site and a loxP site were synthesized and then inserted into a neo/tkselection cassette (pNeoTKLOX, generously provided by Dr. Allan Bradley) flanked by two loxP sites to generate the plasmid ZJ-1. A second plasmid, ZJ-2, was constructed when the 3.6-kbBglI/ApaI fragment, downstream of the DBD exon from the genomic clone, was ligated into the BstXI site of the plasmid ZJ-1 in the presence of linkers. Then, the 1.3-kbBsiEI/BglI fragment flanking exon 4 from the genomic clone was inserted into the SalI site of the plasmid ZJ-2 to generate the plasmid ZJ-4. Finally the targeting vector, ZJ-5, was constructed when the 5.1-kb EcoRV/BsiEI fragment from the genomic clone 5′ of the DBD exon was inserted into the KpnI site of the ZJ-4 plasmid in the presence of linkers. This targeting vector (ZJ-5) contains 6.4 kb of homologous DNA on the 5′-long arm and 3.6 kb of homologous sequence on the 3′-short arm flanking the neo/tk cassette (Fig. 1 A). GCNF lox mice were generated by homologous recombination in the AB1.2 ES cell line (provided by Dr. Allan Bradley) using the Cre/loxP system. TheKpnI-linearized targeting vector ZL-5 (20 μg) was electroporated into 107 ES cells, and stably transfected clones were isolated after selection with 400 μg/ml G418 for 10 days. Homologously recombined clones were identified by mini-Southern blot analysis using 5′-GCNF and neo probes (24Chung A.C. Katz D. Pereira F.A. Jackson K.J. DeMayo F.J. Cooney A.J. O'Malley B.W. Mol. Cell. Biol. 2001; 21: 663-677Crossref PubMed Scopus (91) Google Scholar). Subsequently, correctly recombined clones were amplified and then transiently transfected with a Cre expression vector (pOG231, 1 μg) (36O'Gorman S. Dagenais N.A. Qian M. Marchuk Y. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14602-14607Crossref PubMed Scopus (383) Google Scholar). After negative selection in FIAU (1-(2′-deoxy-2′-fluoro-1-β-d-arabinofuranosyl-5-iodo)uracil), surviving clones were picked and analyzed for recombination of the two loxP sites using mini-Southern blot analysis with the 5′-GCNF and neo probes. Correctly recombined clones were expanded and microinjected into mouse C57BL/6 blastocysts to generate chimeric animals. Male chimeric mice (greater than 90% agouti coat color) were bred with C57BL/6 female mice to generate heterozygous GCNF lox mice. Two clones produced chimeric males that demonstrated germ line transmission of theGCNF lox allele. HeterozygousGCNF lox mice were intercrossed to generate homozygous GCNF lox/lox embryos. Genomic DNA was extracted from either mouse tails or embryos. Genotypes of weaned mice or embryos were determined by Southern blot analysis using a 5′-GCNF probe or by PCR using two separate sets of primers. The primer set (Primer 1, 5′-CAGTGCTGACTTATCCATG-3′; Primer 2, 5′-TTCCTGTTCATGCCCATCT-3′, a 264-bp DNA product) and its PCR reactions for determining the wild typeGCNF allele were described previously (24Chung A.C. Katz D. Pereira F.A. Jackson K.J. DeMayo F.J. Cooney A.J. O'Malley B.W. Mol. Cell. Biol. 2001; 21: 663-677Crossref PubMed Scopus (91) Google Scholar). The primer set (Primer 3, 5′-CCAATTCCCCCCAAAGTGTC-3′; Primer 4, 5′-CAGGTCGAGGGACCTATAAC-3′, a 400-bp DNA product) was used to determine the GCNF lox allele. PCR reactions for determining the GCNF lox allele were carried out for 35 cycles (94 °C, 1 min; 54 °C, 2 min; 72 °C, 2 min) in a buffer containing 2.5 mm MgCl2. Total RNA from embryos was isolated using Trizol reagent (Invitrogen). For RT-PCR analysis, first strand cDNA synthesis was performed at 42 °C for 60 min using 1 μg of total RNA as template and hexanucleotides as primers using the SuperScript™ First-Strand Synthesis System for RT-PCR kit (Invitrogen). To determine the presence of the DBD and LBD regions of the GCNF mRNA and β-actin mRNA in the embryos, PCR reactions with 1/10 of the cDNA were carried out for 40 cycles (94 °C, 1 min; 52 °C, 1 min; 72 °C, 1 min) in a buffer containing 1.5 mm MgCl2 with the following primer sets: GCNF DBD primers (Primer 5, 5′-CTGAACAACGAACCTGTCTC-3′; Primer 6, 5′-ACATGACACAGTTCTTGTCAC-3′; a 150-bp DNA product); LBD primers (Primer 7, 5′-CAGCAAGCAGATCTTTGGG-3′; Primer 8, 5′-TGACAAATGTACCAATACCGC-3′, a 260-bp DNA product); actin primers (ACTIN-F, 5′-TTGAGACCTTCAACACCCC-3′; ACTIN-R, 5′-AGCCAGAGCAGTAATCTCC-3′, a 593-bp DNA product). RNase protection analysis was performed using an Ambion RPA-III system according to the manufacturer's protocol (Ambion, Inc., Austin, TX). The [α-32P]UTP-labeled cRNA probe for the LBD of theGCNF mRNA was synthesized from a plasmid containing 260 bp GCNF cDNA sequence (nucleotides 1256–1516). Proteins from mouse embryos at 8 and 9 dpc were extracted in an extraction buffer (20 mm Tris, pH 7.5, 100 mm NaCl, 0.01% Triton 100, 0.01% Nonidet P-40, 1× proteinase inhibitor). CMVGCNF protein and Cos-1 proteins were prepared from transiently transfected Cos-1 cells as described previously (18Hummelke G.C. Meistrich M.L. Cooney A.J. Mol. Reprod. Dev. 1998; 50: 396-405Crossref PubMed Scopus (37) Google Scholar). Western blot analysis was performed using a specific polyclonal antibody (LBD pAb) raised against a C-terminal polypeptide (amino acid residues from 477 to 495) of the GCNF protein (37Lan, Z. J., Gu, P., Xu, X. P., and Cooney, A. J. (2003) Biol. Reprod. in pressGoogle Scholar). Wild type and GCNF lox/lox embryos at the same somite stage were obtained on 7.5–8.5 dpc of pregnancies. The embryo yolk sacs were removed for genotyping. Whole-mount in situ hybridization was performed as previously described (24Chung A.C. Katz D. Pereira F.A. Jackson K.J. DeMayo F.J. Cooney A.J. O'Malley B.W. Mol. Cell. Biol. 2001; 21: 663-677Crossref PubMed Scopus (91) Google Scholar). The cRNA probe used for Oct4 was as described by Scholeret al. (38Scholer H.R. Dressler G.R. Balling R. Rohdewohld H. Gruss P. EMBO J. 1990; 9: 2185-2195Crossref PubMed Scopus (493) Google Scholar). Histological analysis of embryonic sections was performed as previously described (24Chung A.C. Katz D. Pereira F.A. Jackson K.J. DeMayo F.J. Cooney A.J. O'Malley B.W. Mol. Cell. Biol. 2001; 21: 663-677Crossref PubMed Scopus (91) Google Scholar). A Cre/loxP targeting strategy was used to delete the 243 bp DBD-encoding exon of the GCNF gene. As shown in Fig. 1 A, three loxP sites were introduced into a GCNF allele of ES cells by homologous recombination with the targeting vector. Mini-Southern blot analysis was performed to identify the ES clones that had gone under correct homologous recombination using 5′-GCNF and neoprobes (Fig. 1 B). Nineteen ES cell clones carrying three loxP sites were obtained from the screening of 160 ES cell clones. Subsequently, two independent ES cell clones carrying three loxP sites were amplified and then transiently transfected with a CMV-Cre expression plasmid to delete the GCNF DBD-encoding exon and theneo/tk cassette. ES cell clones carrying the recombinedGCNF lox allele were identified by the Southern blot analysis using the 5′-GCNF and neo probes (Fig. 1 C). Sixty-seven (of 80) ES cell clones carrying theGCNF lox allele were obtained, two of which were used to generate chimeric mice. Chimeric mice from each clone transmitted the recombined GCNF lox allele to their offspring. Heterozygous (GCNF +/lox) and homozygous (GCNF lox/lox) embryos were identified by Southern blot analysis and PCR (Fig. 1 D). At weaning, no homozygous GCNF lox/lox mice were obtained from the intercross of heterozygous mice (TableI), indicating that deletion of the DBD-encoding exon caused embryonic lethality, similar to theGCNF null mutant mice reported previously (24Chung A.C. Katz D. Pereira F.A. Jackson K.J. DeMayo F.J. Cooney A.J. O'Malley B.W. Mol. Cell. Biol. 2001; 21: 663-677Crossref PubMed Scopus (91) Google Scholar). To determine when homozygous GCNF lox/lox mutants die, heterozygous females were sacrificed at various days postcoitum after mating with heterozygous males, and the embryos were collected and then genotyped by either PCR or Southern blot analysis (Fig. 1 D). At 8.5–9.5 dpc, embryos obtained from theGCNF +/lox X GCNF +/loxmating showed the expected 1:2:1 Medelian ratio ofGCNF +/+, GCNF +/lox, andGCNF lox/lox genotypes (Table I). By 10.5–11 dpc, the litter size was reduced compared with that at 8.5–9.5 dpc, and no viable GCNF lox/lox embryos, except resorbing embryos, were obtained. These results confirm thatGCNF lox/lox embryos die at 9.5–10.5 dpc, similar to the GCNF null mutant embryos reported previously (24Chung A.C. Katz D. Pereira F.A. Jackson K.J. DeMayo F.J. Cooney A.J. O'Malley B.W. Mol. Cell. Biol. 2001; 21: 663-677Crossref PubMed Scopus (91) Google Scholar).Table IGenotypes of weaned mice and dissected embryosAgeNumbers of mice/embryos (% of totals)GCNF+/+GCNF+/loxGCNFlox/loxDay 21 (weaned pups)34 (33.7%)67 (66.3%)0 (0%)10.5–11 dpc7 (29.2%)15 (62.5%)2 (8.3%)1-aResorbing embryos.8.5–9.5 dpc28 (26.7%)51 (48.6%)26 (24.8%)1-a Resorbing embryos. Open table in a new tab Since the size of the DBD-encoding exon of the GCNF gene is 243 bp and the loxP sites were inserted into the intron surrounding the DBD exon, we postulated that deletion of the 243-bp DBD-encoding exon should not cause a reading frameshift in the GCNF downstream LBD inGCNF lox/lox mutants (Fig. 2 A). To determine whether the DBD of GCNF is deleted and the LBD of GCNF is still expressed in the GCNF lox/lox embryos, we performed RT-PCR analysis and RNase protection analysis. As shown in Fig. 2 B, the DBD of the GCNF transcript was completely deleted in the GCNF lox/lox mutant embryos. However, the LBD of the GCNF transcript was still expressed in the mutant embryos (Fig. 2 B). To determine whether the LBD protein is present in theGCNF lox/lox embryos, we performed Western blot analysis using antibodies raised against the LBD of GCNF. As shown in Fig. 2 C, a 58-kDa GCNF protein band was detected inGCNF +/+ and GCNF +/loxembryos but not in GCNF −/− embryos. A truncated GCNF protein band (50 kDa) was also detected in the heterozygous and homozygous GCNF lox embryos, indicating that the LBD is present inGCNF lox/lox embryos. Similar to GCNF −/− embryos, there was no gross morphological difference betweenGCNF +/+ and GCNF lox/loxembryos before 8.0 dpc. From 8.75 dpc, a protrusion of tissue started to appear at the base of the allantois ofGCNF lox/lox embryos (data not down). At 9.25–9.5 dpc, the tail bud continued to develop outside the yolk sac (Fig. 3 A). This phenotype was also observed in the GCNF −/− embryos. In addition, other phenotypes of these mutant embryos resembled those in GCNF −/− embryos (Fig. 3 A). TheGCNF lox/lox embryos did not undergo turning and remained in a lordotic position. Although the anterior neural tissue continued to grow, the anterior neural tube inGCNF lox/lox embryos remained open. Similar to GCNF −/− embryos, theGCNF lox/lox embryos also had a significant reduction of the trunk and posterior structures, with the somite number not greater than thirteen. Hindgut was never observed in these mutant embryos, and the pericardium was often dilated. The allantois inGCNF lox/lox embryos was often enlarged and was not always attached to the chorion, resulting in a lack of chorioallantoic development that probably contributed to the embryonic lethality. Another dramatic phenotype ofGCNF −/− embryos was the presence of a large invagination of neural epithelium within the primitive streak 8.5 dpc (Fig. 3 B). This large invagination was also observed inGCNF lox/lox embryos but not inGCNF +/+ embryos (Fig. 3 B). Oct4, a member of the POU homeodomain family of transcription factors, plays an essential role in the maintenance of embryonic stem cell potency and the establishment of the germ cell lineage (39Pesce M. Scholer H.R. Mol. Reprod. Dev. 2000; 55: 452-457Crossref PubMed Scopus (218) Google Scholar). It has been shown that the Oct4gene is expressed in the epiblast of mouse embryo at 6.5–7.5 dpc and then down-regulated from the anterior to the posterior during gastrulation (19Fuhrmann G. Chung A.C. Jackson K.J. Hummelke G. Baniahmad A. Sutter J. Sylvester I. Scholer H.R. Cooney A.J. Dev. Cell. 2001; 1: 377-387Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). A previous report has shown that GCNF can bind to the DR0 elements in the promoter of the Oct4 gene and repress the transcription of this gene and that the expression ofOct4 gene in large parts of theGCNF −/− embryos at 8.25-8.5 dpc is not silenced (19Fuhrmann G. Chung A.C. Jackson K.J. Hummelke G. Baniahmad A. Sutter J. Sylvester I. Scholer H.R. Cooney A.J. Dev. Cell. 2001; 1: 377-387Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). To determine whether the DBD of GCNF is essential for mediating GCNF-dependent repression of Oct4 gene transcription in vivo, we analyzed the expression ofOct4 in GCNF lox/lox embryos usingin situ hybridization assay. As shown in Fig. 4, Oct4 was expressed in theGCNF lox/lox embryos at 7.5 dpc, similar to theGCNF +/+ and GCNF −/−. By 8.25 dpc, Oct4 expression was restricted to primordial germ cells in the GCNF +/+ embryos. However,Oct4 derepressed expression domains were detected in the anterior and posterior of GCNF lox/lox andGCNF −/− embryos at 8.25 dpc (Fig. 4). These results suggest that the DBD of GCNF is required for GCNF to silence the expression of the Oct4 gene in somatic cells of mouse embryos. Nuclear receptors such as ER, PR, AR, TR, and RAR are well known transcription factors that bind their cognate DNA-responsive elements through their DNA binding domains and then regulate the expression of their target genes via their transactivation domains, which ultimately elicits their distinct physiological functions in response to their cognate hormones (40Mangelsdorf D.J. Thummel C. Beato M. Herrilich P. Schütz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 836-839Google Scholar, 41Achermann J.C. Ito M. Hindmarsh P.C. Jameson J.L. Nat. Genet. 1999; 22: 125-126Crossref PubMed Scopus (544) Google Scholar). Recently, DNA binding-independent functions of nuclear receptors such as GR (33Reichardt H.M. Kaestner K.H. Tuckermann J. Kretz O. Wessely O. Bock R. Gass P. Schmid W. Herrlich P. Angel P. Schutz G. Cell. 1998; 93: 531-541Abstract Full Text Full Text PDF PubMed Scopus (915) Google Scholar) and TR3 (34Li H. Kolluri S.K., Gu, J. Dawson M.I. Cao X. Hobbs P.D. Lin B. Chen G., Lu, J. Lin F. Xie Z. Fontana J.A. Reed J.C. Zhang X. Science. 2000; 289: 1159-1164Crossref PubMed Scopus (587) Google Scholar) have been described. As a member of the nuclear receptor superfamily, GCNF has been shown to bind to DR0 elements and repress gene transcription. Similar to other orphan nuclear receptors, such as SF-1 (41Achermann J.C. Ito M. Hindmarsh P.C. Jameson J.L. Nat. Genet. 1999; 22: 125-126Crossref PubMed Scopus (544) Google Scholar) and TR3 (34Li H. Kolluri S.K., Gu, J. Dawson M.I. Cao X. Hobbs P.D. Lin B. Chen G., Lu, J. Lin F. Xie Z. Fontana J.A. Reed J.C. Zhang X. Science. 2000; 289: 1159-1164Crossref PubMed Scopus (587) Google Scholar), the DBD of GCNF is required for its DNA binding activity as deletion of 55 amino acid residues at the N terminus ofXenopus GCNF (14David R. Joos T.O. Dreyer C. Mech. Dev. 1998; 79: 137-152Crossref PubMed Scopus (33) Google Scholar) or of 163 amino acid residues at the N terminus of mouse GCNF 3P. Gu and A. J. Cooney, unpublished data.abolishes its DNA binding activity. Replacement of the GCNFDBD-encoding exon by a PGK-neo cassette in the mouse germline using a conventional knockout strategy causes embryonic lethality at 9.5–10.5 dpc (24Chung A.C. Katz D. Pereira F.A. Jackson K.J. DeMayo F.J. Cooney A.J. O'Malley B.W. Mol. Cell. Biol. 2001; 21: 663-677Crossref PubMed Scopus (91) Google Scholar). These mouse embryos have defects in the correct formation of somites and anteroposterior axis postgastrulation. In addition, unsilenced expression of theOct4 gene in somatic cells is observed in these mutant embryos at 8.25 dpc. Even though the DBD region of the GCNFmRNA is completely deleted in these mutant mice (24Chung A.C. Katz D. Pereira F.A. Jackson K.J. DeMayo F.J. Cooney A.J. O'Malley B.W. Mol. Cell. Biol. 2001; 21: 663-677Crossref PubMed Scopus (91) Google Scholar), it is still unclear whether the phenotypes observed previously are due to the loss of GCNF or to the insertion of the PGK-neo cassette in theGCNF allele. Several studies in other laboratories have suggested that the introduction of PGK-neo cassettes can interfere with RNA processing of targeted transcripts (even when inserted in an intron) (28Holzenberger M. Leneuve P. Hamard G. Ducos B. Perin L. Binoux M. Le Bouc Y. Endocrinology. 2000; 141: 2557-2566Crossref PubMed Scopus (80) Google Scholar) and cause position effects on neighboring genes, particularly when inserted within gene clusters and locus control regions (29Olson E.N. Arnold H.H. Rigby P.W. Wold B.J. Cell. 1996; 85: 1-4Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar, 30Seidl K.J. Manis J.P. Bottaro A. Zhang J. Davidson L. Kisselgof A. Oettgen H. Alt F.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3000-3005Crossref PubMed Scopus (77) Google Scholar, 31Pham C.T. MacIvor D.M. Hug B.A. Heusel J.W. Ley T.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13090-13095Crossref PubMed Scopus (300) Google Scholar) leading to unexpected phenotypes in the resulting animal models. Additionally, bi-directional transcriptional activities of PGK-neo, normal sense PGK promoter activity, and aberrant antisense promoter activity in neomycin and PGK promoter regions, which drive the production of aberrant transcripts, have been reported in cultured cells (42Johnson P. Friedmann T. Gene. 1990; 88: 207-213Crossref PubMed Scopus (49) Google Scholar) and in vivo in mice (32Scacheri P.C. Crabtree J.S. Novotny E.A. Garrett-Beal L. Chen A. Edgemon K.A. Marx S.J. Spiegel A.M. Chandrasekharappa S.C. Collins F.S. Genesis. 2001; 30: 259-263Crossref PubMed Scopus (77) Google Scholar). Considering that the SF-1 gene, encoding another nuclear receptor, is immediately adjacent to the GCNF gene in the mouse genome and is known to be involved in adrenal and gonadal development (43Luo X. Ikeda Y. Parker K.L. Cell. 1994; 77: 481-490Abstract Full Text PDF PubMed Scopus (1363) Google Scholar), it is possible that insertion of the PGK-neo in the GCNF locus affects SF-1expression, complicating the interpretation of phenotypes observed in our conventional knockout embryos. Therefore, generation of a mouse line without the PGK-neo cassette in the GCNFlocus is necessary to determine the role of GCNF during embryonic development by comparison of the phenotypes of the conventional knockout embryos to those mice without the PGK-neoinsertion. In this study, we have successfully generatedGCNF lox/lox mice that do not contain the DBD-encoding exon of the GCNF gene nor the selection marker gene, PGK-neo, using Cre/loxP technology. Similarly to the conventional GCNF knockout mice,GCNF lox/lox mice died in utero at 9.5–10.5 dpc with defects in posterior embryonic development and the formation of somites and the anteroposterior axis. Expression of theOct4 gene in GCNF lox/lox embryos was not silenced in somatic cells at 8.25 dpc. Therefore,GCNF lox/lox mice phenocopy the conventionalGCNF knockout mice (19Fuhrmann G. Chung A.C. Jackson K.J. Hummelke G. Baniahmad A. Sutter J. Sylvester I. Scholer H.R. Cooney A.J. Dev. Cell. 2001; 1: 377-387Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 24Chung A.C. Katz D. Pereira F.A. Jackson K.J. DeMayo F.J. Cooney A.J. O'Malley B.W. Mol. Cell. Biol. 2001; 21: 663-677Crossref PubMed Scopus (91) Google Scholar), indicating that the phenotypes observed in the conventional knockout embryos result from the ablation of GCNF, not from the insertion of the PGK-neo cassette into the GCNF locus, confirming that GCNF is essential for normal embryonic development. As no RNA splicing acceptor consensus sequences, which are located upstream of the DBD-encoding exon of the GCNF gene, were deleted in our knockout strategy and no consensus RNA splicing acceptor elements or transcription termination signals were included in the loxP site and its surrounding DNA sequences, it was possible that transcription of the GCNF gene inGCNF lox/lox mice could be initiated and that normal RNA splicing can occur between exon 3 and exon 5 forming a truncated GCNF message, which encodes a protein that does not contain the DBD but does have an intact LBD. Indeed, we found that the LBD region, but not the DBD region, of the GCNF mRNA was expressed in the GCNF lox/lox mice detected either by RT-PCR or by RNase protection assay (Fig. 2). Since the size of the DBD exon of the GCNF gene is 243 bp (9Susens U. Borgmeyer U. Genome Biol. 2000; 1 (, research 0006): 1-3Crossref PubMed Google Scholar), deletion of this exon should not cause a reading frameshift in the downstream LBD. Using a specific GCNF polyclonal antibody raised against a 19-amino acid peptide at the C terminus, we found that a 58-kDa GCNF protein band was detected in the GCNF +/+ andGCNF +/lox mice but not in the conventionalGCNF knockout mice (Fig. 2) andGCNF lox/lox mice (Fig. 2). Instead, a 50- kDa-truncated protein band was detected in theGCNF lox (GCNF +/lox andGCNF lox/lox) embryos (Fig. 2). These studies suggest that the truncated GCNF transcript in theGCNF lox mice is translated into a 50-kDa protein that contains an intact LBD but not a DBD. These results clearly indicate that we have successfully deleted the DBD of GCNF in vivo. Although the truncated GCNF protein lacking only the DBD was observed in the GCNF lox mice (Fig. 2), phenotypes of these GCNF lox mice (Figs. 3 and 4) were the same as those of conventional GCNF knockout mice (19Fuhrmann G. Chung A.C. Jackson K.J. Hummelke G. Baniahmad A. Sutter J. Sylvester I. Scholer H.R. Cooney A.J. Dev. Cell. 2001; 1: 377-387Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 24Chung A.C. Katz D. Pereira F.A. Jackson K.J. DeMayo F.J. Cooney A.J. O'Malley B.W. Mol. Cell. Biol. 2001; 21: 663-677Crossref PubMed Scopus (91) Google Scholar), which did not express GCNF protein (Fig. 2). These results suggest that the DBD of GCNF is essential for mediating the function of GCNF during early embryonic development. The results also suggest that at this stage of embryonic development the LBD of GCNF does not mediate any functions independent of the DBD. It should be emphasized that GCNF can bind to the DR0 element in the promoter of the Oct4 gene and repress the transcription of the Oct4 gene in vitro (19Fuhrmann G. Chung A.C. Jackson K.J. Hummelke G. Baniahmad A. Sutter J. Sylvester I. Scholer H.R. Cooney A.J. Dev. Cell. 2001; 1: 377-387Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Loss of GCNF in the conventional GCNFknockout mice causes loss of repression of Oct4 expression in somatic cells (19Fuhrmann G. Chung A.C. Jackson K.J. Hummelke G. Baniahmad A. Sutter J. Sylvester I. Scholer H.R. Cooney A.J. Dev. Cell. 2001; 1: 377-387Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Similar results were obtained in theGCNF lox/lox mice. Considering that the LBD of GCNF does not bind to DR0 element,3 we can conclude that GCNF is indeed a transcription factor that represses gene transcription in mouse embryos. In addition, the expressed GCNF LBD does not have a dominant negative effect as the GCNF +/lox mice are normal. The lack of a dominant negative effect is probably due to an inability of the wild type GCNF and the GCNF LBD to heterodimerize. In summary, we have generated a line of mice that expresses a truncated GCNF protein lacking only the DBD. These mice phenocopy the conventional GCNF knockout mice. This study clarifies that the phenotypes observed in the conventional GCNF knockout mice are due to the loss of GCNF, not to the insertion of the PGK-neo cassette into the GCNF locus, and that GCNF is essential for normal mouse embryonic development. More importantly, our results suggest that the DBD of GCNF is essential for the function of GCNF during embryonic development, that GCNF does not have DNA-binding independent activity as reported for other nuclear receptors such as GR and TR3 (33Reichardt H.M. Kaestner K.H. Tuckermann J. Kretz O. Wessely O. Bock R. Gass P. Schmid W. Herrlich P. Angel P. Schutz G. Cell. 1998; 93: 531-541Abstract Full Text Full Text PDF PubMed Scopus (915) Google Scholar, 34Li H. Kolluri S.K., Gu, J. Dawson M.I. Cao X. Hobbs P.D. Lin B. Chen G., Lu, J. Lin F. Xie Z. Fontana J.A. Reed J.C. Zhang X. Science. 2000; 289: 1159-1164Crossref PubMed Scopus (587) Google Scholar) during mouse embryogenesis, and that GCNF is indeed a transcription factor that represses gene transcription in vivo. We thank Drs. A. Bradley and S. O'Gorman for providing the floxed Neo-TK cassette and pOG231 plasmid, respectively. We also thank Kathy Jackson for the help in ES cell culture.