Cooperation and Competition between the Binding of COUP-TFII and NF-Y on Human ε- and γ-Globin Gene Promoters
2001; Elsevier BV; Volume: 276; Issue: 45 Linguagem: Inglês
10.1074/jbc.m102987200
ISSN1083-351X
AutoresChiara Liberati, Maria Rosaria, Paola Secco, Claudio Santoro, Roberto Mantovani, Sergio Ottolenghi, Antonella Ronchi,
Tópico(s)RNA modifications and cancer
ResumoThe nuclear receptor COUP-TFII was recently shown to bind to the promoter of the ε- and γ-globin genes and was identified as the nuclear factor NF-E3. Transgenic experiments and genetic evidence from humans affected with hereditary persistence of fetal hemoglobin suggest that NF-E3 may be a repressor of adult ε and γ expression. We show that, on the ε-promoter, recombinant COUP-TFII binds to two sites, the more downstream of which overlaps with an NF-Y binding CCAAT box. Binding occurs efficiently to either the 5′ or the 3′ COUP-TFII site but not to both sites simultaneously. However, adding recombinant NF-Y induces the formation of a stable COUP-TFII·NF-Y-promoter complex at concentrations of COUP-TFII that would not give significant binding in the absence of NF-Y. Mutations of the promoter indicate that COUP-TFII cooperates with NF-Y when bound to the 5′ site, whereas binding at the 3′ site is mutually exclusive. Likewise, in the γ-promoter, COUP-TFII binds to a site overlapping the distal member of a duplicated CCAAT box, competing with NF-Y binding. Transfections in K562 cells show that both the mutation of the 5′ COUP-TFII or of the NF-Y site on the ε-promoter decrease the activity of a luciferase reporter; the mutation of the 3′ COUP-TFII site has little effect. These results, together with transgenic experiments suggesting a repressive activity of COUP-TFII on the ε-promoter and the observation that, on the 3′ site, COUP-TFII and NF-Y binding is mutually exclusive, suggest that COUP-TFII may exert different effects on ε transcription depending on whether it binds to the 5′ or to the 3′ site. At the 5′ site, COUP-TFII might cooperate with NF-Y, forming a stable complex, and stimulate transcription; at the 3′ site, COUP-TFII might compete for binding with NF-Y and, directly or indirectly, decrease gene activity. The nuclear receptor COUP-TFII was recently shown to bind to the promoter of the ε- and γ-globin genes and was identified as the nuclear factor NF-E3. Transgenic experiments and genetic evidence from humans affected with hereditary persistence of fetal hemoglobin suggest that NF-E3 may be a repressor of adult ε and γ expression. We show that, on the ε-promoter, recombinant COUP-TFII binds to two sites, the more downstream of which overlaps with an NF-Y binding CCAAT box. Binding occurs efficiently to either the 5′ or the 3′ COUP-TFII site but not to both sites simultaneously. However, adding recombinant NF-Y induces the formation of a stable COUP-TFII·NF-Y-promoter complex at concentrations of COUP-TFII that would not give significant binding in the absence of NF-Y. Mutations of the promoter indicate that COUP-TFII cooperates with NF-Y when bound to the 5′ site, whereas binding at the 3′ site is mutually exclusive. Likewise, in the γ-promoter, COUP-TFII binds to a site overlapping the distal member of a duplicated CCAAT box, competing with NF-Y binding. Transfections in K562 cells show that both the mutation of the 5′ COUP-TFII or of the NF-Y site on the ε-promoter decrease the activity of a luciferase reporter; the mutation of the 3′ COUP-TFII site has little effect. These results, together with transgenic experiments suggesting a repressive activity of COUP-TFII on the ε-promoter and the observation that, on the 3′ site, COUP-TFII and NF-Y binding is mutually exclusive, suggest that COUP-TFII may exert different effects on ε transcription depending on whether it binds to the 5′ or to the 3′ site. At the 5′ site, COUP-TFII might cooperate with NF-Y, forming a stable complex, and stimulate transcription; at the 3′ site, COUP-TFII might compete for binding with NF-Y and, directly or indirectly, decrease gene activity. hereditary persistence of fetal hemoglobin electrophoresis mobility shift assay glutathione S-transferase The non-α-globin genes are clustered in several species within a relatively small chromosomal region. The expression of these genes is precisely regulated both spatially and quantitatively during embryonic, fetal, and postnatal development to match the expression of α-globin genes, resulting in a perfectly balanced α/non-α synthetic ratio (1Stamatoyannopoulos G. Nienhuis z A.W. The Molecular Basis of Blood Disease. W. B. Saunders Co., New York1994: 107-155Google Scholar, 2Weatherall D.J. Clegg J.B. The Thalassemia Syndromes. Blackwell Scientific Ltd., Oxford1981Google Scholar, 3Forget B.G. Ann. N. Y. Acad. Sci. 1998; 850: 38-44Crossref PubMed Scopus (174) Google Scholar). In man, the predominant non-α-globin chain during the embryonic period is ε-globin, which around the third month of gestation (embryonic-fetal switch) is replaced by γ-globin (encoded by two non-allelic genes, Gγ- and Aγ-globin) and finally, around birth, by β-globin (fetal-adult switch). DNA sequences regulating globin gene expression have been extensively investigated; in addition to the upstream locus control region, essential for the correct activity of all the genes in the cluster (4Grosveld F. van Assendelft B. Greaves D.R. Kollias G. Cell. 1987; 51: 975-985Abstract Full Text PDF PubMed Scopus (1429) Google Scholar, 5Enver T. Raich N. Ebens A.J. Papayannopoulos T. Constantini F. Stamatoyannopoulos G. Nature. 1990; 344: 309-313Crossref PubMed Scopus (234) Google Scholar, 6Behringer R.R. Ryan T.M. Palmiter R.D. Brinster R.L. Palmiter R.D. Townes T. 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In particular, no transcription factor has been detected whose activity during development varies in a way consistent with the changes observed in globin gene expression at the various stages; even erythroid Krüppel-like protein, although necessary for β-globin (11Nuez B. Michalovich D. Bygrave A. Ploemacher R. Grosveld F. Nature. 1995; 375: 316-318Crossref PubMed Scopus (475) Google Scholar, 12Perkins A.C. Sharpe A.H. Orkin S.H. Nature. 1995; 375: 318-322Crossref PubMed Scopus (523) Google Scholar, 13Tewari R. Gillemans N. Wijgerde M. Nuez B. von Lindern M. Grosveld F. Philipsen S. EMBO J. 1998; 17: 2334-2341Crossref PubMed Scopus (69) Google Scholar, 14Wijgerde M. Gribnau J. Trimborn T. Nuez B. Philipsen S. Grosveld F. Fraser P. Genes Dev. 1996; 10: 2894-2902Crossref PubMed Scopus (180) Google Scholar) but not ε- and γ-globin expression, is present and active during the embryonic and early fetal stages, when β-globin is not yet expressed (13Tewari R. Gillemans N. Wijgerde M. Nuez B. von Lindern M. Grosveld F. Philipsen S. EMBO J. 1998; 17: 2334-2341Crossref PubMed Scopus (69) Google Scholar). Some clues to the nature of the DNA sequences controlling the temporal expression of γ-globin genes have come from inherited conditions usually observed in heterozygous individuals and known as hereditary persistence of fetal hemoglobin (HPFH)1 (1Stamatoyannopoulos G. Nienhuis z A.W. The Molecular Basis of Blood Disease. W. B. Saunders Co., New York1994: 107-155Google Scholar, 2Weatherall D.J. Clegg J.B. The Thalassemia Syndromes. Blackwell Scientific Ltd., Oxford1981Google Scholar, 3Forget B.G. Ann. N. Y. Acad. Sci. 1998; 850: 38-44Crossref PubMed Scopus (174) Google Scholar). Such individuals present, postnatally, moderate or high levels of fetal hemoglobin (α2γ2). Some HPFHs are caused by point mutations in either the Gγ orAγ-globin gene; the mutated gene is selectively overexpressed in adults. Six different mutations causing HPFH cluster around the double CCAAT box region and affect the binding of several proteins (3Forget B.G. Ann. N. Y. Acad. Sci. 1998; 850: 38-44Crossref PubMed Scopus (174) Google Scholar, 15Ottolenghi S. Mantovani R. Nicolis S. Ronchi A. Giglioni B. Hemoglobin. 1989; 13: 523-541Crossref PubMed Scopus (37) Google Scholar, 16Collins F.S. Metherall J.E. Yamakawa M. Pan J. Weissman S.M. Forget B.G. Nature. 1985; 313: 325-326Crossref PubMed Scopus (95) Google Scholar, 17Gelinas R. Endlich B. Pfeiffer C. Yagi M. Stamatoyannopoulos G. Nature. 1985; 313: 323-325Crossref PubMed Scopus (108) Google Scholar, 18Fucharoen S. Shimizu K. Fukumaki Y. Nucleic Acids Res. 1990; 18: 5245-5253Crossref PubMed Scopus (81) Google Scholar, 19Gilman J. Mishima N. Wen X.J. Stoming T.A. Lobel J. Huisman T.H.J. Nucleic Acids Res. 1988; 16: 10635-10642Crossref PubMed Scopus (41) Google Scholar, 20Indrak K. Indrakova J. Popsilova D. Suslovska I. Baysal E. Huisman T.H.J. Ann. Hematol. 1991; 63: 1-5Crossref PubMed Scopus (67) Google Scholar, 21Zertal-Zidani S. Merghoub T. Ducrocq R. Gerard N. Satta D. Krishnamoorthy R. Hemoglobin. 1999; 23: 159-169Crossref PubMed Scopus (11) Google Scholar, 22Motum P.I. Deng Z.M. Huong L. Trent R.J. Br. J. Haematol. 1994; 86: 219-221Crossref PubMed Scopus (14) Google Scholar, 23Ronchi A. Bottardi S. Mazzucchelli C. Ottolenghi S. Santoro C. J. Biol. Chem. 1995; 270: 21934-21941Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 24Berry M. Grosveld F. Dillon N. Nature. 1992; 358: 499-502Crossref PubMed Scopus (108) Google Scholar). Among them, all of the four different HPFH mutations studied so far greatly diminish or abolish the binding of a protein called NF-E3 (23Ronchi A. Bottardi S. Mazzucchelli C. Ottolenghi S. Santoro C. J. Biol. Chem. 1995; 270: 21934-21941Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). These results suggested that NF-E3 might be a γ-globin repressor and that the inability of the γ-globin promoter to bind it might be the underlying cause of the HPFH phenotype. Given the large number of proteins binding to this region, it has not been possible to design an artificial mutation that fully reproduces the binding abnormalities observed with spontaneous HPFH mutations and also causes HPFH in transgenic models; however, two mutations causing HPFH in humans have the same effect in transgenic mice (24Berry M. Grosveld F. Dillon N. Nature. 1992; 358: 499-502Crossref PubMed Scopus (108) Google Scholar, 25Ronchi A. Berry M. Raguz S. Imam A. Yannoutsos N. Ottolenghi S. Grosveld F. Dillon N. EMBO J. 1996; 15: 143-149Crossref PubMed Scopus (45) Google Scholar). Additional evidence that NF-E3 may be a repressor was also provided by more recent experiments showing that an extensive mutation of both the distal and the proximal NF-E3 binding sites on the ε-globin promoter causes persistent expression of an ε-globin transgene in adult erythroid cells (26Filipe A. Li Q. Deveaux S. Godin I. Romeo P.H. Stamatoyannopoulos G. Mignotte V. EMBO J. 1999; 18: 687-697Crossref PubMed Scopus (59) Google Scholar). In these studies it was shown that COUP-TFII, an orphan nuclear receptor, is either NF-E3 or, more likely, a part of an NF-E3 complex, whose composition varies during mouse development. In both the ε- and γ-globin promoters, the NF-E3 binding sites partially overlap an NF-Y binding site. NF-Y is a trimeric transcription factor composed of three subunits (A, B, C) that binds to the CCAAT box motif (27Mantovani R. Nucleic Acids Res. 1998; 26: 1135-1143Crossref PubMed Scopus (442) Google Scholar). Some observations suggest the relevance of NF-Y for γ-globin promoter activity in HPFH. (i) A mutation of the proximal γ-globin CCAAT box within a transgenic γ-globin HPFH construct carrying the −117 HPFH G → A mutation, adjacent but not overlapping the distal CCAAT box, almost completely suppresses the overexpression of γ-globin in adult cells, which is normally caused by the HPFH mutation (25Ronchi A. Berry M. Raguz S. Imam A. Yannoutsos N. Ottolenghi S. Grosveld F. Dillon N. EMBO J. 1996; 15: 143-149Crossref PubMed Scopus (45) Google Scholar). Note that the mutation of both CCAAT boxes has no effect on the embryonic expression of a normal γ-globin gene, indicating that at this stage factors other than NF-Y are responsible for the high level of γ-globin activity. (ii) HPFH point mutations, which mutate the CCAAT box, thus decreasing NF-Y binding, induce much lower levels of fetal hemoglobin expression in adults than the −117 HPFH G → A mutation, which does not affect the CCAAT box. In this paper, we have investigated by in vitroelectrophoresis mobility shift assay (EMSA) the simultaneous binding of recombinant COUP-TFII·NF-E3 and NF-Y to normal and mutated ε- and γ-globin promoters. We show that COUP-TFII and NF-Y binding can be either mutually exclusive or cooperative, depending on the particular binding site used. On the basis of these observations and transfection experiments, we propose that COUP-TFII may act as a modulator of ε-globin transcription by cooperating and/or interfering with NF-Y binding. NF-YA and NF-YB cDNA fragments were cloned into the Escherichia coli expression vector pET3b (29Mantovani R. Pessara U. Tronche F. Li X-Y. Knapp A.M. Pasquali J.L. Benoist C. Mathis D. EMBO J. 1992; 11: 3315-3322Crossref PubMed Scopus (165) Google Scholar); NF-YC was polymerase chain reaction-cloned into the PET32b vector (30Bellorini M. Zemzoumi K. Farina A. Berthelsen J. Piaggio G. Mantovani R. Gene. 1997; 193: 119-125Crossref PubMed Scopus (36) Google Scholar). Expression and purification of the recombinant proteins were as described in Mantovani et al. (29Mantovani R. Pessara U. Tronche F. Li X-Y. Knapp A.M. Pasquali J.L. Benoist C. Mathis D. EMBO J. 1992; 11: 3315-3322Crossref PubMed Scopus (165) Google Scholar) and Bellorini et al. (30Bellorini M. Zemzoumi K. Farina A. Berthelsen J. Piaggio G. Mantovani R. Gene. 1997; 193: 119-125Crossref PubMed Scopus (36) Google Scholar), in particular, the three subunits were purified on a Ni2+-agarose column by means of the His tag, either from soluble BL21 LysS bacterial extracts or from renatured inclusion bodies. Native NF-Y protein was purified from CH27 cells according to Kadonaga and Tjian (31Kadonaga J.T. Tjian R. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5889-5893Crossref PubMed Scopus (715) Google Scholar). The COUP-TFII-pGEX-3X expression plasmid was kindly provided by Dr. Paulweber (32Paulweber B. Sandhofer F. Levy-Wilson B. Mol. Cell. Biol. 1993; 13: 1534-1546Crossref PubMed Scopus (48) Google Scholar). To produce glutathione S-transferase (GST)-fused COUP-TFII recombinant protein, BL21 LysS bacteria were transformed, grown in LB at mid-logarithmic phase (0.7–0.8 A at 600 nm) and induced with isopropyl-1-thio-β-d-galactopyranoside (1 mm) for 3 h at 37 °C. Cells were pelleted and frozen at −80 °C; upon thawing, they were resuspended in BC300 (300 mm KCl, 20 mm Hepes pH 7.9, 10% glycerol, 1 mm phenylmethylsulfonyl fluoride), sonicated over a total of 5 min with 30-s pulses, and centrifuged. The supernatant was then processed for purification; soluble bacterial extracts were loaded on a glutathione-Sepharose 4B (Amersham Pharmacia Biotech) column, and then GST-COUP-TFII was eluted at 30 mmglutathione according to standard protocols. For control experiments, GST was removed by proteolytic cleavage with FactorXa (Amersham Pharmacia Biotech). Extracts from COS cells transfected with a COUP-TFII expression vector were provided by Dr. B. Paulweber. Nuclear extract preparation, in vitroincubation of labeled oligonucleotides with nuclear proteins, and electrophoretic analysis (EMSA) were performed according to standard protocols, as previously described in Refs. 23Ronchi A. Bottardi S. Mazzucchelli C. Ottolenghi S. Santoro C. J. Biol. Chem. 1995; 270: 21934-21941Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 25Ronchi A. Berry M. Raguz S. Imam A. Yannoutsos N. Ottolenghi S. Grosveld F. Dillon N. EMBO J. 1996; 15: 143-149Crossref PubMed Scopus (45) Google Scholar, and 28Liberati C. Ronchi A. Lievens P. Ottolenghi S. Mantovani R. J. Biol. Chem. 1998; 273: 16880-16889Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar.32P-Labeled oligonucleotides (0.1–0.5 ng) were incubated for 20 min at 4 °C with the recombinant proteins in a buffer containing 5% glycerol, 50 mm NaCl, 20 mmTris, pH 7.9, 0.5 mm EDTA, 5 mmMgCl2, and 1 mm dithiothreitol. The reaction mix was then run into a 5% polyacrylamide gel (acrylamide/bisacrylamide ratio of 29:1) at 4 °C. The sequence of the oligonucleotides used is described in Table I.Table IWild type and mutated transcription factor binding sites 1. CCCTGAGGGACACAGGTCAGCCTTGACCAATGACTTTTAAGTA (ε2DR) 2. CCCTGAG-GgacCAGCgacGCCTcGACCAATGAgggTTAAGTA (ε2DRmut) 3. CCCTGAG-GgacCAGGgacGCCTTGACCAATGACTTTTAAGTA (ε2DR5′mut) 4. CCCTGAGGGACACAGGTCAGCCTcGACCAATGAgggTTAAGTA (ε2DR3′mut) 5. CCCTGAGGGACACAGGTCAGCCTTGACCAgTGACTTTTAAGTA (ε2DR NF-Ymut) 6. ACTGAACCCTTGACCCCTGCCCT (human apoAI promoter, COUP-TFII consensus oligo) 7. ATTTTTCTGATTGGTTAAAAGT (MHC Ea NF-Y consensus oligo) 8. CTAGGCCTTGCCTTGACCAATAGCCTTGACAAGGCAAACTTGACCAATAGTCTTAGAG (γ promoter, wild type) 9. GCCTTGCCTTGACCAATAGCCTTGACA (γ promoter, distal CCAAT box)10. GCCTTGCCTTaACCAATAGCCTTGACA (γ promoter distal CCAAT box, −117 HPFH)11. GCCTTGCCTTGACCAATAaCCTTGACA (γ promoter, distal CCAAT box, mutant −109)12. GCCTTGCCTTGACCAATAGttTTGACA (γ promoter, distal CCAAT box, double mutant −107/−108)All the oligonucleotides used for gel shift experiments are listed (upper strand only). The CCAAT box is underlined; mutations are in bold lowercase letters. MHC, major histocompatibility complex. Open table in a new tab All the oligonucleotides used for gel shift experiments are listed (upper strand only). The CCAAT box is underlined; mutations are in bold lowercase letters. MHC, major histocompatibility complex. Competition experiments were performed using 20–50-fold molar excess of unlabeled oligonucleotides. The anti NF-Y B subunit antibody was generated by R. Mantovani as described in Ref. 29Mantovani R. Pessara U. Tronche F. Li X-Y. Knapp A.M. Pasquali J.L. Benoist C. Mathis D. EMBO J. 1992; 11: 3315-3322Crossref PubMed Scopus (165) Google Scholar. The anti-ARP-1 T-19 (COUP-TFII) antibody was purchased from Santa Cruz Biotechnology (sc-6578) The ε-globin promoter spanning from nucleotide −220 to nucleotide +18 (23Ronchi A. Bottardi S. Mazzucchelli C. Ottolenghi S. Santoro C. J. Biol. Chem. 1995; 270: 21934-21941Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) was joined by linkers to the HindIII site in the pGL2basic (Promega) luciferase reporter vector. A 46-base pair oligonucleotide corresponding to the erythroid-specific enhancer derived from the human locus control region hypersensitive site II (33Ney P. Sorrentino B.P. McDonagh K.T. Nienhuis A. Genes Dev. 1990; 4: 93-106Crossref Scopus (209) Google Scholar) was inserted in the pGL2 BglII site upstream to the promoter. All the mutant constructs tested were produced by polymerase chain reaction techniques and entirely sequenced. K562 cells were grown in RPMI 1640 medium supplemented with l-glutamine and 5% fetal calf serum. 107 exponentially growing K562 cells were electroporated at 400 V, 960 microfarads with a Bio-Rad apparatus in 0.8 ml of phosphate-buffered saline with 10 μg of plasmid according to Ronchi et al. (23Ronchi A. Bottardi S. Mazzucchelli C. Ottolenghi S. Santoro C. J. Biol. Chem. 1995; 270: 21934-21941Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). To normalize for transfection efficiency, 700 ng of pRL-TK plasmid (Promega, dual luciferase reporter system) were cotransfected in each sample. After 48 h, extracts were prepared, and the double luciferase activity was tested according to the Promega protocol. All experiments were repeated in triplicate with at least three independent plasmid preparations. Previous experiments (23Ronchi A. Bottardi S. Mazzucchelli C. Ottolenghi S. Santoro C. J. Biol. Chem. 1995; 270: 21934-21941Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 26Filipe A. Li Q. Deveaux S. Godin I. Romeo P.H. Stamatoyannopoulos G. Mignotte V. EMBO J. 1999; 18: 687-697Crossref PubMed Scopus (59) Google Scholar, 28Liberati C. Ronchi A. Lievens P. Ottolenghi S. Mantovani R. J. Biol. Chem. 1998; 273: 16880-16889Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) showed that both NF-E3 and (weakly) NF-Y bind to the ε-globin promoter in the CCAAT box region; in addition, the same two factors bind to the CCAAT box region of the γ-globin promoter, although in this case NF-Y binding is much stronger, and NF-E3 binding is weaker with respect to the ε-globin promoter (23Ronchi A. Bottardi S. Mazzucchelli C. Ottolenghi S. Santoro C. J. Biol. Chem. 1995; 270: 21934-21941Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 28Liberati C. Ronchi A. Lievens P. Ottolenghi S. Mantovani R. J. Biol. Chem. 1998; 273: 16880-16889Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Mignotte and co-workers (26Filipe A. Li Q. Deveaux S. Godin I. Romeo P.H. Stamatoyannopoulos G. Mignotte V. EMBO J. 1999; 18: 687-697Crossref PubMed Scopus (59) Google Scholar) recently proposed that COUP-TFII, an orphan nuclear receptor, is part of the NF-E3 complex (26Filipe A. Li Q. Deveaux S. Godin I. Romeo P.H. Stamatoyannopoulos G. Mignotte V. EMBO J. 1999; 18: 687-697Crossref PubMed Scopus (59) Google Scholar). With K562 extracts, NF-E3 runs as a poorly resolved doublet; Fig.1, lane 2, shows that an antibody against COUP-TFII almost completely supershifts the NF-E3 complex formed with an ε-globin promoter oligonucleotide (ε2DR, Table I). In addition, recombinant COUP-TFII obtained by transfection of an expression vector in COS cells, generates on the same oligonucleotide a band that migrates as the slower portion of the NF-E3 band (Fig.1 B). These data confirm that COUP-TFII is at least a component of NF-E3. There are two putative NF-E3/COUP-TFII binding sites on the human ε-globin promoter (26Filipe A. Li Q. Deveaux S. Godin I. Romeo P.H. Stamatoyannopoulos G. Mignotte V. EMBO J. 1999; 18: 687-697Crossref PubMed Scopus (59) Google Scholar); the 3′ site overlaps the NF-Y binding CCAAT box (Fig. 2). To characterize the binding of COUP-TFII to this region, we analyzed by EMSA the interaction of synthetic oligonucleotides with recombinant COUP-TFII obtained as a bacterially produced protein fused to GST at its NH2terminus. Using the ε2DR oligonucleotide, a single band is present at low concentrations of COUP-TFII; at the highest COUP-TFII concentration (lane 4), a smear of slower mobility is observed, suggesting that, if a complex with two COUP-TFII molecules is formed, it is very unstable. NF-Y was previously shown to bind, albeit weakly, to the ε-globin promoter (23Ronchi A. Bottardi S. Mazzucchelli C. Ottolenghi S. Santoro C. J. Biol. Chem. 1995; 270: 21934-21941Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 28Liberati C. Ronchi A. Lievens P. Ottolenghi S. Mantovani R. J. Biol. Chem. 1998; 273: 16880-16889Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). We incubated increasing amounts of the three recombinant A, B, C subunits of NF-Y with the same ε-globin oligonucleotide; at an intermediate concentration of protein, a strong NF-Y band was formed of a mobility slower than the COUP-TFII complex (Fig. 2 A) as expected (23Ronchi A. Bottardi S. Mazzucchelli C. Ottolenghi S. Santoro C. J. Biol. Chem. 1995; 270: 21934-21941Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). To test whether COUP-TFII and NF-Y can bind simultaneously to the same DNA molecule, we then incubated (Fig. 2 A, lanes 8–11) a fixed amount of NF-Y (corresponding to that used in Fig. 2, lane 6) together with increasing amounts of COUP-TFII (i.e. the same amounts as used in lanes 1–4 in the absence of NF-Y). Fig.2 A shows that, at the lowest COUP-TFII concentration, which gives essentially no binding by itself (lane1), most of the NF-Y complex is shifted up to a slower mobility (lane 8), suggesting the simultaneous binding of both proteins on the same DNA molecule; no COUP-TFII band is present, in agreement with the expected result (compare with lane 1). When higher concentrations of COUP-TFII are used, the NF-Y band is progressively shifted to the position of the slower complex (lanes 8–11), and the COUP-TFII single band appears. These results indicate that COUP-TFII, although unable by itself to bind to the ε-globin promoter at low concentrations, readily does so in the presence of NF-Y. A number of control experiments (Fig. 2 B) were also carried out to verify the specificity of the observed bands. In particular, the binding of recombinant COUP-TFII to the ε-globin promoter is competed by an oligonucleotide carrying the human apoAI COUP-TFII binding site (Table I, Refs. 26Filipe A. Li Q. Deveaux S. Godin I. Romeo P.H. Stamatoyannopoulos G. Mignotte V. EMBO J. 1999; 18: 687-697Crossref PubMed Scopus (59) Google Scholar and 32Paulweber B. Sandhofer F. Levy-Wilson B. Mol. Cell. Biol. 1993; 13: 1534-1546Crossref PubMed Scopus (48) Google Scholar, Fig. 2 B, lane 16, and data not shown) but not by an unrelated oligonucleotide (not shown). In addition, an antibody against the YB subunit of NF-Y supershifts both the NF-Y band and the slower complex formed by the addition of an intermediate concentration of COUP-TFII (Fig. 2 B,lanes 13 and 18), confirming the presence of NF-Y in the latter band. Furthermore, both the upper and lower band are competed by an excess of unlabeled NF-Y binding oligonucleotide (major histocompatibility complex Ea NF-Y consensus oligo (Table I, Refs. 23Ronchi A. Bottardi S. Mazzucchelli C. Ottolenghi S. Santoro C. J. Biol. Chem. 1995; 270: 21934-21941Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholarand 29Mantovani R. Pessara U. Tronche F. Li X-Y. Knapp A.M. Pasquali J.L. Benoist C. Mathis D. EMBO J. 1992; 11: 3315-3322Crossref PubMed Scopus (165) Google Scholar, and Fig. 2 B, lane 19), but only the upper band is competed by an unlabeled COUP-TFII binding site (lane 20). Taken together, these experiments indicate that the slowest band observed in the presence of both COUP-TFII and NF-Y is a complex containing both proteins. The upper complex observed in Fig. 2 might either be because of the binding of COUP-TFII and NF-Y to the same DNA molecule or to protein-protein interaction between COUP-TFII and NF-Y before binding. An artifactual effect due to the fused moieties of recombinant COUP-TFII and NF-Y (see "Experimental Procedures" and Refs. 29Mantovani R. Pessara U. Tronche F. Li X-Y. Knapp A.M. Pasquali J.L. Benoist C. Mathis D. EMBO J. 1992; 11: 3315-3322Crossref PubMed Scopus (165) Google Scholar, 30Bellorini M. Zemzoumi K. Farina A. Berthelsen J. Piaggio G. Mantovani R. Gene. 1997; 193: 119-125Crossref PubMed Scopus (36) Google Scholar, and 32Paulweber B. Sandhofer F. Levy-Wilson B. Mol. Cell. Biol. 1993; 13: 1534-1546Crossref PubMed Scopus (48) Google Scholar) was excluded by adding Factor Xa-treated COUP-TFII to a K562 nuclear extract (Fig. 2 C); the NF-Y band (lane 21) was quantitatively shifted to the upper position (lane 22). As a control, COUP-TFII added to a mutant oligonucleotide lacking COUP-TFII sites (ε2DRmut) (see below, Fig. 4) failed to shift the NF-Y band (lane 23). The same results were obtained with native NF-Y purified from CH27 cells (not shown). In addition, we ruled out the possibility of a stable COUP-TFII-NF-Y interaction in the absence of DNA; recombinant GST-COUP-TFII·NF-Y were mixed together and passed onto a Sepharose column carrying immobilized anti-NF-YA antibody. Whereas both COUP-TFII and NF-Y were present in the unbound fraction (as assayed by Western blot with anti-NF-YB, anti-COUP-TFII, and anti-GST antibodies), only NF-Y was retained by the column (data not shown). The experiment shown in Fig. 2 A indicates that COUP-TFII is able to bind to NF-Y-bound ε-globin DNA at concentrations that would not allow significant binding to DNA alone. To provide a quantitative evaluation of this phenomenon, we incubated COUP-TFII at a wide range of concentrations with a fixed amount of DNA in the presence or absence of a given amount of NF-Y. As shown in Fig.3 A, ≈50% of the NF-Y-bound DNA was shifted to the upper band at a concentration of 9 COUP-TFII arbitrary units. In contrast, a similar amount of COUP-TFII band was formed at a much higher COUP-TFII concentration (Fig. 3 A, 60 units, lane 8). This experiment likely underestimates the real difference; in fact, the concentration of the NF-Y-bound DNA is approximately 5-fold lower than that of the total probe. As shown in Fig. 3 B, diminishing the target DNA dramatically decreases binding. For this reason, the COUP-TFII binding experiment was also carried out at lower DNA concentrations (0.3× and 0.15× and data not shown); under these conditions, a 50% shift of the probe because of COUP-TFII binding is observed between 120 and 240 units of protein (Fig. 3 A, right), i.e. at a protein concentration that is 12–25-fold higher than that needed to shift 50% of the NF-Y-bound probe. Furthermore, we assayed the stability of the complexes by incubating the intact ε-globin oligonucleotide with appropriate amounts of COUP-TFII and NF-Y, allowing for the formation of all three complexes. We then added cold competitor oligonucleotides carrying either the COUP-TFII or NF-Y consensus (see Table I). The mixture was kept for various times at 4 °C before loading onto the gel. Fig.3 C shows that the unlabel
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