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• W2065571560 abstract "The NTF-like family of transcription factors have been implicated in developmental regulation in organisms as diverse asDrosophila and man. The two mammalian members of this family, CP2 (LBP-1c/LSF) and LBP-1a (NF2d9), are highly related proteins sharing an overall amino acid identity of 72%. CP2, the best characterized of these factors, is a ubiquitously expressed 66-kDa protein that binds the regulatory regions of many diverse genes. Consequently, a role for CP2 has been proposed in globin gene expression, T-cell responses to mitogenic stimulation, and several other cellular processes. To elucidate the in vivo role of CP2, we have generated mice nullizygous for the CP2 allele. These animals were born in a normal Mendelian distribution and displayed no defects in growth, behavior, fertility, or development. Specifically, no perturbation of hematopoietic differentiation, globin gene expression, or immunological responses to T- and B-cell mitogenic stimulation was observed. RNA and protein analysis confirmed that the nullizygous mice expressed no full-length or truncated version of CP2. Electrophoretic mobility shift assays with nuclear extracts from multiple tissues demonstrated loss of CP2 DNA binding activity in the −/− lines. However, a slower migrating complex that was ablated with antiserum to NF2d9, the murine homologue of LBP-1a, was observed with these extracts. Furthermore, we demonstrate that recombinant LBP-1a can bind to known CP2 consensus sites and form protein complexes with previously defined heteromeric partners of CP2. These results suggest that LBP-1a/NF2d9 may compensate for loss of CP2 expression in vivo and that further analysis of the role of the NTF family of proteins requires the targeting of the NF2d9 gene. The NTF-like family of transcription factors have been implicated in developmental regulation in organisms as diverse asDrosophila and man. The two mammalian members of this family, CP2 (LBP-1c/LSF) and LBP-1a (NF2d9), are highly related proteins sharing an overall amino acid identity of 72%. CP2, the best characterized of these factors, is a ubiquitously expressed 66-kDa protein that binds the regulatory regions of many diverse genes. Consequently, a role for CP2 has been proposed in globin gene expression, T-cell responses to mitogenic stimulation, and several other cellular processes. To elucidate the in vivo role of CP2, we have generated mice nullizygous for the CP2 allele. These animals were born in a normal Mendelian distribution and displayed no defects in growth, behavior, fertility, or development. Specifically, no perturbation of hematopoietic differentiation, globin gene expression, or immunological responses to T- and B-cell mitogenic stimulation was observed. RNA and protein analysis confirmed that the nullizygous mice expressed no full-length or truncated version of CP2. Electrophoretic mobility shift assays with nuclear extracts from multiple tissues demonstrated loss of CP2 DNA binding activity in the −/− lines. However, a slower migrating complex that was ablated with antiserum to NF2d9, the murine homologue of LBP-1a, was observed with these extracts. Furthermore, we demonstrate that recombinant LBP-1a can bind to known CP2 consensus sites and form protein complexes with previously defined heteromeric partners of CP2. These results suggest that LBP-1a/NF2d9 may compensate for loss of CP2 expression in vivo and that further analysis of the role of the NTF family of proteins requires the targeting of the NF2d9 gene. human immunodeficiency virus stage selector protein kilobase pair ribonuclease protection assay(s) electrophoretic mobility shift assay(s) yeast artificial chromosome days post-coitum Cellular diversity is generated by unique combinations of transcription factors interacting to specify patterns of gene expression in different cell types. This is particularly evident during development where biochemical and genetic studies have identified numerous proteins essential for the processes that govern embryogenesis. Many of these factors are highly conserved in evolution, playing critical roles in organisms as diverse as Drosophilaand man. One family of transcription factors that typifies these principles is the NTF-like group of proteins. The founding member of this family is the developmentally programmed Drosophilafactor, NTF-1 (neurogenic element bindingtranscription factor) (1Bray S.J. Kafatos F.C. Genes Dev. 1991; 5: 1672-1683Crossref PubMed Scopus (153) Google Scholar). NTF-1 (also known as Grainyhead or Elf-1) was first identified through its ability to bind acis element critical for expression of the Dopa decarboxylase gene (2Dynlacht B.D. Attardi L.D. Admon A. Freeman M. Tjian R. Genes Dev. 1989; 3: 1677-1688Crossref PubMed Scopus (95) Google Scholar, 3Attardi L.D. Tjian R. Genes Dev. 1993; 7: 1341-1353Crossref PubMed Scopus (84) Google Scholar). Subsequently, NTF-1 was shown to bind to promoters of other developmentally regulated genes includingUltrabithorax, fushi tarazu, andengrailed (2Dynlacht B.D. Attardi L.D. Admon A. Freeman M. Tjian R. Genes Dev. 1989; 3: 1677-1688Crossref PubMed Scopus (95) Google Scholar). NTF-1 has also been linked to dorsal/ventral and terminal patterning through the formation of multiprotein complexes that influence transcription from the decapentaplegic andtailless genes (4Liaw G.-J. Rudolph K.M. Huang J.-D. Dubnicoff T. Courey A.J. Lengyel J.A. Genes Dev. 1995; 9: 3163-3176Crossref PubMed Scopus (99) Google Scholar, 5Huang J.-D. Dubnicoff T. Liaw G.-J. Bai Y. Valentine S.A. Shirokawa J.M. Lengyel J.A. Courey A.J. Genes Dev. 1995; 9: 3177-3189Crossref PubMed Scopus (77) Google Scholar). More recently, tissue-specific isoforms of the protein have been described in Drosophila, and mutation of these isoforms or the ubiquitously expressed gene results in pupal lethality with gross developmental defects (1Bray S.J. Kafatos F.C. Genes Dev. 1991; 5: 1672-1683Crossref PubMed Scopus (153) Google Scholar, 6Uv A.E. Harrison E.J. Bray S.J. Mol. Cell. Biol. 1997; 17: 6727-6735Crossref PubMed Scopus (67) Google Scholar). In mammals, two highly related NTF-like genes have been identified. In humans they are known as LBP-1a and CP2 (LBP-1c/LSF), whereas the mouse homologues are referred to as NF2d9 and CP2, respectively (7Lim L.C. Swendeman S.L. Sheffery M. Mol. Cell. Biol. 1992; 12: 828-835Crossref PubMed Google Scholar, 8Shirra M.K. Zhu Q. Huang H.-C. Pallas D. Hansen U. Mol. Cell. Biol. 1994; 14: 5076-5087Crossref PubMed Scopus (51) Google Scholar, 9Yoon J.-B. Li G. Roeder R.G. Mol. Cell. Biol. 1994; 14: 1776-1785Crossref PubMed Scopus (99) Google Scholar, 10Sueyoshi T. Kobayasi R. Nishio K. Aida K. Moore R. Wada T. Handa H. Negishi M. Mol. Cell. Biol. 1995; 15: 4158-4166Crossref PubMed Scopus (52) Google Scholar). The human genes are 72% identical in overall amino acid sequence but share higher sequence identity (88%) in the N-terminal halves of the proteins than the C-terminal halves (52%). The homology with the NTF gene is also in the N-terminal region with three amino acid stretches, 148 to 159, 205 to 216, and 233 to 246 showing 66, 75, and 79% identity, respectively. The NTF-like gene family has been shown to have a variety of cellular and developmental functions in human and murine cells. The best characterized member of the family, CP2, was initially identified as a factor that binds to, and stimulates transcription from, the murine γ-fibrinogen promoter and the viral SV40 major late promoter (11Kim C.H. Heath C. Bertuch A. Hansen U. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6025-6029Crossref PubMed Scopus (58) Google Scholar, 12Chodosh L.A. Baldwin A.S. Carthew R.W. Sharp P.A. Cell. 1988; 53: 11-24Abstract Full Text PDF PubMed Scopus (435) Google Scholar). Binding sites for CP2 have also been defined in regulatory regions of the human immunodeficiency virus (HIV)1 where it acts in concert with YY1 to repress transcription (9Yoon J.-B. Li G. Roeder R.G. Mol. Cell. Biol. 1994; 14: 1776-1785Crossref PubMed Scopus (99) Google Scholar, 13Wu F.K. Garcia J.A. Harrich D. Gaynor R.B. EMBO J. 1988; 7: 2117-2129Crossref PubMed Scopus (109) Google Scholar, 14Jones K.A. Luciw P.A. Duchange N. Genes Dev. 1988; 2: 1101-1114Crossref PubMed Scopus (174) Google Scholar, 15Parada C.A. Yoon J. Roeder R.G. J. Biol. Chem. 1995; 270: 2274-2283Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 16Romerio F. Gabriel M.N. Margolis D.M. J. Virol. 1997; 71: 9375-9382Crossref PubMed Google Scholar). In the context of non-viral gene regulation, CP2 has been shown to bind homomerically to the human c-fos, ornithine decarboxylase, c-myc, and DNA polymerase promoters and the murine α-globin and fibrinogen promoters and activate transcription in vitro (17Volker J.L. Rameh L.E. Zhu Q. DeCaprio J. Hansen U. Genes Dev. 1997; 11: 1435-1446Crossref PubMed Scopus (51) Google Scholar, 18Shirra M.K. Hansen U. J. Biol. Chem. 1998; 273: 19260-19268Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Binding to the regulatory elements in thefos and ornithine decarboxylase promoters is modulated by cell growth signals. Mitogenic stimulation of resting T-cells is associated with rapid phosphorylation of CP2 by the mitogen-activated protein kinase pp44 (extracellular signal-regulated kinase 1) and a consequent increase in its DNA binding activity (17Volker J.L. Rameh L.E. Zhu Q. DeCaprio J. Hansen U. Genes Dev. 1997; 11: 1435-1446Crossref PubMed Scopus (51) Google Scholar). This modulation suggests that CP2 contributes to the regulation of early response genes and therefore plays a role as a cell growth regulator. A developmental role for CP2 has been identified in studies of globin gene regulation. In this context, CP2 binds to the stage selector element in the proximal γ-gene promoter as a heteromeric complex with a recently cloned fetal/erythroid-specific partner protein, NF-E4 (19Jane S.M. Nienhuis A.W. Cunningham J.M. EMBO J. 1995; 14: 97-105Crossref PubMed Scopus (104) Google Scholar). 2J. M. C. and S. M. J., submitted for publication. This complex, known as the stage selector protein (SSP), contributes to the preferential recruitment of the β-globin locus control region to the γ-promoter during fetal erythropoiesis (20Amrolia P.J. Cunningham J.M. Jane S.M. J. Biol. Chem. 1998; 273: 13593-13598Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 21Jane S.M. Ney P.A. Vanin E.F. Gumucio D.L. Nienhuis A.W. EMBO J. 1992; 11: 2961-2969Crossref PubMed Scopus (118) Google Scholar). SSP binding sites have also been defined in the ε-promoter and in the regions of DNase1 hypersensitivity that constitute the locus control region (19Jane S.M. Nienhuis A.W. Cunningham J.M. EMBO J. 1995; 14: 97-105Crossref PubMed Scopus (104) Google Scholar, 22Gumucio D.L. Shelton D.A. Bailey W.J. Slightom J.L. Goodman M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6018-6022Crossref PubMed Scopus (83) Google Scholar,23Gumucio D.L. Shelton D.A. Blanchard-McQuate K. Gray T. Tarle S. Heilstedt-Williamson H. Slightom J.L. Collins F. Goodman M. J. Biol. Chem. 1994; 269: 15371-15380Abstract Full Text PDF PubMed Google Scholar). Despite the extensive literature examining CP2 function in vitro and in cell lines, the in vivo role of this factor remains unknown. To address this, we have generated a CP2 null mutation in mice by homologous recombination. Mice lacking CP2 expression were examined for defects in growth and development, with a particular emphasis on hematopoiesis, immune, and neural function. We observed no significant abnormality in CP2−/− mice compared with wild type littermates. We have shown through DNA binding and protein-protein interaction studies that the lack of a discernible phenotype may be due to a complete rescue by NF2d9, the murine homologue of LBP1a. We isolated eight CP2 genomic clones by screening a 129-derived ES cell phage library with a full-length mouse CP2 cDNA probe. Duplicate lifts screened with a probe specific for exon two, containing the initiation ATG, identified one clone with a 12-kb insert that encodes the first two exons and the 5′ untranslated region. Detailed restriction endonuclease mapping of this fragment confirmed the previously reported genomic structure of murine CP2 with the exception of an EcoRI polymorphism detected in the 5′ untranslated region (see Fig. 1 A) (24Swendeman S.L. Spielholz C. Jenkins N.A. Gilbert D.J. Copeland N.G. Sheffery M. J. Biol. Chem. 1994; 269: 11663-11671Abstract Full Text PDF PubMed Google Scholar). Subsequently, a 6.6-kbNcoI fragment containing the 5′ untranslated region and a portion of the first exon was subcloned into pSL301. AXhoI-SalI fragment containing a phosphoglycerate kinase promoter-regulated hygromycin resistance expression cassette was cloned into a downstream SalI site. Finally, a 3.4-kbSalI-NotI fragment containing 2.4 kb of the second intron of CP2 and a 1-kb HSV-TK expression cassette fragment (a kind gift of Dr. J. vanDeursen) was cloned downstream of this region to provide 3′ homology and a negative selectable marker. This construct, pK01HygTK, was linearized with NotI and transfected by electroporation into RW8 embryonic stem cells (Genomic Systems Inc). The cells were cultured on primary irradiated embryonic STO feeder cells in the presence of 140 fg/ml hygromycin and 0.2 fm FIAU. Resistant clones were screened by Southern blot analysis using a unique 0.5-kbSalI-NcoI fragment located 5′ to the targeted sequence. Four karyotypically normal CP2+/− clones were microinjected into C57BL/6 blastocysts, of which three clones were transmitted through the germ line. Embryo sections were prepared as described previously (25Zindy F. Soares H. Herzog K.H. Morgan J. Sherr C.J. Roussel M.F. Cell Growth Differ. 1997; 8: 1139-1150PubMed Google Scholar). Briefly, C57/BL6 mice, overdosed with ketamine and rhompin, were perfused intracardially with paraformaldehyde, and the embryos from timed matings were postfixed in a similar solution. Sections of 10–14 μm were cut with a cryostat, mounted on glass slides, and stored at −20 °C. These slides were subsequently probed with sense and antisense riboprobes generated by [33P]UTP labeling from Bluescript plasmids encoding the complete cDNAs of CP2 and NF2d9 (the latter were a kind gift of Dr. M. Negishi) (10Sueyoshi T. Kobayasi R. Nishio K. Aida K. Moore R. Wada T. Handa H. Negishi M. Mol. Cell. Biol. 1995; 15: 4158-4166Crossref PubMed Scopus (52) Google Scholar). Specific signals were developed by dipping the slides in NTB2 emulsion (Kodak Scientific Imaging Systems) and exposed at 4 °C for two weeks. The sections were counterstained using 0.1% toluidine blue in distilled water and analyzed by phase-contrast microscopy. Tissues from normal and age-matched heterozygote and homozygote knockout mice were removed and fixed in formalin, and embedded paraffin sections were prepared. These sections were stained with hematoxylin and eosin and examined by light microscopy. Peripheral blood (150 μl) was obtained by retro-orbital puncture and blood cell counts, and erythrocyte parameters were determined utilizing an automated analyzer (Coulter). In addition an aliquot was stained with Wright's Giemsa or methylene blue to study hematopoietic cell morphology and reticulocytes, respectively. Bone marrow hematopoietic progenitors were cultured in methylcellulose in the presence of IL3, erythropoeitin, and stem cell factor (Terry Fox Laboratories, Vancouver, Canada). For immunological studies, single cell suspensions were prepared from spleen, lymph node, thymus, and bone marrow and stained with cell type-specific markers for granulocytes (Gr1), T-cells (CD8 and CD4), B-cells (B220 and IAb), and NK cells (NK1.1). Fluorescence analysis was performed utilizing a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). Similar cellular suspensions were utilized to assess the proliferative potential of T- and B-cells, culturing 105 cells in the presence of anti-CD3ε +/− phorbol 12-myristate 13-acetate, concanavalin A, lipopolysaccharide, phytohemagluttinin, ionomycin, or a combination of phorbol 12-myristate 13-acetate, phytohemagluttinin, and ionomycin for 48 h as described previously (17Volker J.L. Rameh L.E. Zhu Q. DeCaprio J. Hansen U. Genes Dev. 1997; 11: 1435-1446Crossref PubMed Scopus (51) Google Scholar). RNA was prepared from various tissues and from peripheral blood and bone marrow cells using RNAzol B or RNASTAT 60. Murine CP2 and NF2d9 cDNA fragments spanning exons 2 and 3 and a portion of exon 4 were subcloned into pSP73. RPA studies were performed using the Ambion RPAII kit as per the manufacturer's instructions. For studies of murine and transgenic human globin gene expression RPA was performed utilizing probes specific for the murine ζ, α, εy, βh1, and βmajor transcripts and the human ε-, Gγ-, and β-globin transcripts (a kind gift of Dr. K. Gaensler). Crude nuclear extracts were prepared from various primary tissues (26Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9159) Google Scholar, 27Lim L.C. Fang L. Swendeman S.L. Sheffery M. J. Biol. Chem. 1993; 268: 18008-18017Abstract Full Text PDF PubMed Google Scholar) and quantitated using the Bio-Rad protein assay system as per the manufacturer's instructions. EMSAs were performed by incubating varying amounts of nuclear extract with 105 cpm of [32P]dCTP end-labeled double stranded oligonucleotides encoding the CCAAT box region of the murine α-globin promoter, the γ-fibrinogen promoter, or the SV40 major late promoter in a 20-μl reaction containing 500 ng of poly[d(I·C)], 6 mm MgCl2, 16.5 mm KCl, and 100 μg of bovine serum albumin (21Jane S.M. Ney P.A. Vanin E.F. Gumucio D.L. Nienhuis A.W. EMBO J. 1992; 11: 2961-2969Crossref PubMed Scopus (118) Google Scholar,27Lim L.C. Fang L. Swendeman S.L. Sheffery M. J. Biol. Chem. 1993; 268: 18008-18017Abstract Full Text PDF PubMed Google Scholar). For antibody studies, 3 μl of preimmune serum or rabbit anti-mouse CP2 antibody were preincubated for 10 min with the binding reaction prior to addition of the probe. Polyclonal antiserum against NF2d9 was kindly provided by Dr. M. Negishi. After incubation at 4 °C for 10 min and 25 °C for 20 min, samples were electrophoresed on a 4% non-denaturing polyacrylamide gel in 0.5 × Tris borate-EDTA buffer for 90 min at 10 V/cm. Recombinant CP2 and NF2d9 were prepared as glutathione S-transferase fusion proteins as described previously (19Jane S.M. Nienhuis A.W. Cunningham J.M. EMBO J. 1995; 14: 97-105Crossref PubMed Scopus (104) Google Scholar). cDNA sequences encoding the C-terminal 250 amino acids (amino acids 250–500) of CP2 and the corresponding region of LBP-1a were inserted into the yeast expression vector pGBT9. The resultant plasmids encode hybrid proteins containing the DNA binding domain of GAL4 fused to CP2 or LBP-1a residues. The yeast reporter strain, HF7C, was transformed with this vector and a second plasmid encoding a hybrid protein of the GAL4 transactivation domain and either the NF-E4 cDNA or RING1B cDNA. The yeast were plated on leucine/tryptophan/histidine plates and incubated at 30 °C for 3 days (28Fields S. Song O. Nature. 1989; 340: 245-246Crossref PubMed Scopus (4860) Google Scholar). Protein interactions are indicated by growth on these plates. Transfection efficiency was monitored by plating of an aliquot of the transformation on leucine/tryptophan plates (data not shown). CP2−/− mice were bred with mice transgenic for a single copy of a yeast artificial chromosome (YAC) containing 250 kb of the human β-globin locus (β-YAC) (a kind gift of Karin Gaensler, University of California, San Francisco, CA) (29Gaensler K.M. Kitamura M. Kan Y.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11381-11385Crossref PubMed Scopus (126) Google Scholar). Timed pregnancies were set up utilizing CP2+/− females and CP2+/−YAC+males, where the day of plug formation was taken as 0.5 days post-coitum (E0.5 dpc). Embryos were collected on days E9.5 dpc, E10.5 dpc, E11.5 dpc, and E14.5 dpc and genotyped by standard methodologies utilizing the CP2 probes described above and a probe specific for IVSII of the human Aγ-globin gene (a kind gift of Dr. Karin Gaensler). RNA from yolk sac and fetal liver was prepared, and RPA analysis was performed as described above. To disrupt the murine CP2 gene, a targeting vector was designed that replaced the first untranslated exon and the entire second exon containing the initiation codon and the trans-activation domain with a hygromycin expression cassette (Fig. 1 A). In addition, this cassette introduced termination codons in all open reading frames. RW8 embryonic stem cells were electroporated with this construct and selected in hygromycin and FIAU. Southern analysis of resistant clones demonstrated a 9.0-kb EcoRI fragment in addition to the 10.5-kb wild type allele, at a mean frequency of one in 25 clones (data not shown). Four independently targeted clones, with normal karyotypes, were injected into C57BL/6J blastocysts, three of the clones being transmitted through the germ line. Interbreeding of mice heterozygous for the CP2 allele (CP2+/−) resulted in litters of normal size with the expected Mendelian frequency of genotypes. Of 256 total offspring tested, 72 were CP2+/+(28%), 125 were CP2+/− (49%), and 60 animals (23%) were nullizygous (CP2−/−) for the CP2 allele. To confirm the loss of CP2 gene expression in nullizygous animals, RNA was prepared from various tissues of both wild type and CP2−/− mice and analyzed by RNase protection analysis. A specific band of 380 nucleotides was observed in all tissues derived from wild type animals utilizing a riboprobe that hybridizes to exons 2–4 (Fig. 1 B, lanes 1–5). In contrast, no signal was observed utilizing RNA derived from the brain, heart, kidney, lung, and spleen of CP2−/− mice (Fig.1 B, lanes 7–11). An actin probe controlled for the integrity of the RNA (Fig. 1 C). To confirm the loss of CP2 expression, and to rule out a cryptic splicing event that might produce a functional CP2 transcript, RNA from wild type and CP2−/− tissues was assayed by RT polymerase chain reaction utilizing primers specific to the 3′ end of the mRNA transcript. Although a CP2-specific signal was observed in all wild type tissues tested, no signal was detected from RNA derived from CP2−/− animals (data not shown). Male and female knockout mice grew normally and were healthy up to 18 months of age. No abnormal behavioral patterns were observed. The fertility of CP2−/− animals was normal, and no increase in morbidity was observed when compared with littermate controls. Careful histopathological examination of brain, spleen, kidney, liver, thymus, lymph nodes, heart, skin, muscle, and bone from CP2−/−animals, performed at 3, 9, and 15 months, was identical to wild type littermate controls (data not shown). CP2 has been implicated in the regulation of several hematopoietic genes, particularly those of the globin loci (7Lim L.C. Swendeman S.L. Sheffery M. Mol. Cell. Biol. 1992; 12: 828-835Crossref PubMed Google Scholar, 19Jane S.M. Nienhuis A.W. Cunningham J.M. EMBO J. 1995; 14: 97-105Crossref PubMed Scopus (104) Google Scholar). To determine whether loss of CP2 expression resulted in changes in hematopoiesis, the hematological parameters of CP2−/−mice were assayed and compared with those of wild type littermates. No significant difference in total cell counts, hematocrits, reticulocytes, differential white cell counts, or the α-/β-globin ratio was observed (Table I). In addition, the numbers of bone marrow progenitors, as measured by colony-forming unit activity, were similar in CP2+/+ and CP2−/− animals (data not shown). Similar studies of lymphopoiesis were stimulated by recent studies implicating CP2 in the modulation of T-cell proliferative responses (17Volker J.L. Rameh L.E. Zhu Q. DeCaprio J. Hansen U. Genes Dev. 1997; 11: 1435-1446Crossref PubMed Scopus (51) Google Scholar). However, extensive analysis of T, B, and NK phenotypes, as well as functional assays of B- and T-cell function, failed to identify a difference between CP2+/+ and CP2−/− cells (data not shown).Table IHematological analysis of 18 CP2+/+ and 18 CP2−/− animalsParameter measuredCP2+/+ miceCP2−/− miceRCC × 109 cells/ml9.52 ± 0.209.44 ± 0.20Hematocrit48.13 ± 4.6750.83 ± 4.00Reticulocytes (%)2.63 ± 0.472.77 ± 0.63MCV52.3 ± 4.755.6 ± 5.46MCH16.49 ± 0.7316.67 ± 0.99RCC, red cell count; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin. Open table in a new tab RCC, red cell count; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin. It is possible that despite normal adult erythropoiesis, the loss of CP2 expression may affect either α- or β-globin gene expression during hematopoietic ontogeny. CP2 was initially identified as an α-globin CCAAT box binding activity, suggesting a possible role in α-globin gene expression. However, neither ζ- nor α-globin gene expression was perturbed in yolk sac or fetal liver cells (Fig.2 A). We have demonstrated that CP2 is a component of the γ-globin promoter-binding SSP complex and suggested that the γ to β switch in the β-globin subtype may be perturbed in a CP2 null environment. To test this hypothesis, we bred CP2−/− animals with mice transgenic for a 240-kb YAC containing the human β-globin locus (βYAC). Subsequently, we bred male progeny transgenic for the βYAC with CP2+/− females and examined the expression of both human and mouse β-globin-like genes at several developmental stages. As shown in Fig. 2 B, both human and murine β-globin-like gene expression in yolk sac, fetal liver, and bone marrow were identical in CP2+/− and CP2−/− embryos and adult mice, respectively. To examine CP2 DNA binding site occupancy in the null mice, we prepared crude nuclear extracts from lung, kidney, heart, and liver of wild type and CP2−/− animals and performed EMSA using a double stranded oligonucleotide probe containing the α-globin CCAAT box (7Lim L.C. Swendeman S.L. Sheffery M. Mol. Cell. Biol. 1992; 12: 828-835Crossref PubMed Google Scholar). Utilizing equal amounts of protein in each lane, a band of similar electrophoretic mobility was observed in all wild type tissues (Fig.3 A, compare lanes 1, 3, 5, and 7). In contrast, extracts from CP2−/− tissues failed to show the band seen with wild type extract and instead showed a DNA-protein complex with a slower migration pattern (Fig. 3 A, compare lanes 2, 4, 6, and 8 with 1,3, 5, and 7, respectively). This result was not dependent on the amount of protein added, as 2- to 8-fold more protein from CP2−/− liver extract incubated with the probe generated an identical band shift (Fig. 3 A, compare lane 7 with lanes 8–11). The lack of an obvious phenotype in the CP2−/− animals coupled with the persistent DNA site occupancy observed with extracts from nullizygous tissues suggested the presence of a ubiquitous CP2-like factor that could compensate for the lack of CP2. One candidate factor was Nf2d9, the murine homologue of the human NTF-like gene, LBP-1a (10Sueyoshi T. Kobayasi R. Nishio K. Aida K. Moore R. Wada T. Handa H. Negishi M. Mol. Cell. Biol. 1995; 15: 4158-4166Crossref PubMed Scopus (52) Google Scholar). Support for this hypothesis was obtained by studying the relative electrophoretic mobilities of CP2 and NF2d9. Both molecules bound the α-globin CCAAT box and γ-fibrogen probes, the NF2d9-DNA complex having a perceptibly slower mobility (Fig.3 B and data not shown). To determine whether the protein-DNA complex generated with CP2−/− extracts contained NF2d9, we performed competition experiments utilizing excess concentrations of unlabeled oligonucleotides that have been previously shown to bind CP2 and/or NF2d9(7, 10). These oligonucleotides were capable of ablating both wild type and mutant binding activity (data not shown). We also investigated the ability of monoclonal antisera generated against recombinant NF2d9 to disrupt binding activity. This antisera does not cross-react with CP2 as assessed by immunoblotting. 3A. T., J. M. C., and S. M. J., unpublished information. Addition of the antibody induced a partial supershift of wild type binding activity (Fig. 3 C, compare lanes 1 and 3). In contrast, mutant activity was completely supershifted (Fig.3 C, compare lanes 2 and 4). These data suggest that NF2d9 can maintain DNA site occupancy at CP2 binding sites. To determine whether the distribution of expression of CP2 and NF2d9 was similar, we performed in situ hybridization on embryo sections using antisense and sense probes specific for each mRNA transcript. Normal embryos were examined at E9.5, E11.5, and E13.5 dpc. Probes were of similar specific activity, and sense probes produced little background signal from embryos probed at all developmental stages (Fig. 4, A andC). However, utilizing an antisense probe, we observed CP2 expression in most tissues at similar levels at E13.5 dpc (Fig.4 B). In contrast, although expression of NF2d9 was observed in all tissues, it was markedly higher in the fetal liver (Fig.4 D, arrow). It is possible that loss of CP2 expression results in up-regulation of NF2d9 expression. To test this hypothesis, we determined the expression of NF2d9 in the brain, kidney, and heart of CP2+/+ and CP2 null animals. As shown in Fig. 4 E, no significant change in the relative expression of NF2d9 was observed in any of these tissues when compared with the actin control. The DNA binding, transactivation, and expression patterns of LBP-1a/NF2d9 suggested that this protein could potentially compensate for the loss of CP2. To evaluate this further, we examined whether LBP-1a could fulfill the protein-protein interaction role of CP2. We have recently defined two transcription factors that specifically interact with CP2. 4S. M. J. and J. M. C., submitted for publication." @default.
• W2065571560 created "2016-06-24" @default.
• W2065571560 creator A5005429562 @default.
• W2065571560 creator A5007043395 @default.
• W2065571560 creator A5040121512 @default.
• W2065571560 creator A5055253605 @default.
• W2065571560 creator A5059629859 @default.
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• W2065571560 date "2001-03-01" @default.
• W2065571560 modified "2023-09-28" @default.
• W2065571560 title "Targeted Disruption of the CP2 Gene, a Member of the NTF Family of Transcription Factors" @default.
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