Developmentally Specific Role of the CCAAT Box in Regulation of Human γ-Globin Gene Expression

The CCAAT box is a widespread motif in eukaryotic promoters. In this study we demonstrate that the effects of the CCAAT box on γ-globin gene activation are developmentally distinct. Although this promoter element is essential for high level γ gene expression in adult erythropoiesis, it plays little role in embryonic erythroid cells. The CCAAT mutation in the human γ-globin gene promoter impairs recruitment of TATA-binding protein (TBP), TFIIB, and RNA polymerase II in adult splenic erythroblasts but not in embryonic erythroid cells. We also show that the efficiency of γ gene transcription is correlated with recruitment of TBP on the TATA box but that the level of TBP recruitment is not nuclear factor Y (NF-Y)-dependent. Our data also suggest that it is unlikely that transcriptional stimulation by the CCAAT box is exerted through direct protein-protein interaction between NF-Y and TBP. The CCAAT box is a widespread motif in eukaryotic promoters. In this study we demonstrate that the effects of the CCAAT box on γ-globin gene activation are developmentally distinct. Although this promoter element is essential for high level γ gene expression in adult erythropoiesis, it plays little role in embryonic erythroid cells. The CCAAT mutation in the human γ-globin gene promoter impairs recruitment of TATA-binding protein (TBP), TFIIB, and RNA polymerase II in adult splenic erythroblasts but not in embryonic erythroid cells. We also show that the efficiency of γ gene transcription is correlated with recruitment of TBP on the TATA box but that the level of TBP recruitment is not nuclear factor Y (NF-Y)-dependent. Our data also suggest that it is unlikely that transcriptional stimulation by the CCAAT box is exerted through direct protein-protein interaction between NF-Y and TBP. The CCAAT box is one of the most common promoter elements found in approximately one-third of eukaryotic housekeeping and lineage-specific genes (1Bucher P. J. Mol. Biol. 1990; 212: 563-578Crossref PubMed Scopus (969) Google Scholar). Other common cis elements in the minimal promoter are the TATA and CACCC boxes. The TATA box and the initiator compose the core promoter where the basal transcription apparatus docks. The CCAAT and CACCC boxes compose the proximal promoter that enhances RNA polymerase II (Pol II) 1The abbreviations used are: Pol IIRNA polymerase IINFnuclear factorTBPTATA-binding proteinChIPchromatin immunoprecipitationCBFCCAAT-binding factorCREBcAMP-response element-binding protein.1The abbreviations used are: Pol IIRNA polymerase IINFnuclear factorTBPTATA-binding proteinChIPchromatin immunoprecipitationCBFCCAAT-binding factorCREBcAMP-response element-binding protein. transcription. Because of their widespread distribution and frequent co-existence in eukaryotic promoters, it has been speculated that the co-existence of CACCC and CCAAT boxes facilitates modulation of gene activity (2Mantovani R. Gene (Amst.). 1999; 239: 15-27Crossref PubMed Scopus (673) Google Scholar). RNA polymerase II nuclear factor TATA-binding protein chromatin immunoprecipitation CCAAT-binding factor cAMP-response element-binding protein. RNA polymerase II nuclear factor TATA-binding protein chromatin immunoprecipitation CCAAT-binding factor cAMP-response element-binding protein. There are several examples of CCAAT mutations that cause a decrease in basal or lineage-specific promoter activities. Thus, disruption of the box C (NF-Y binding site) in the albumin gene promoter impairs expression in the liver (3Maire P. Wuarin J. Schibler U. Science. 1989; 244: 343-346Crossref PubMed Scopus (210) Google Scholar). The transcription of farnesyl diphosphate synthase is up-regulated by sterol stimulation in the cell; point mutation in the CCAAT box of the farnesyl diphosphate synthase promoter disrupts sterol-dependent transcription in vivo (4Jackson S.M. Ericsson J. Osborne T.F. Edwards P.A. J. Biol. Chem. 1995; 270: 21445-21448Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Transcription of the gene encoding E2F1, whose promoter contains three CCAAT motifs, is activated at the G1/S boundary; mutation of the proximal CCAAT motif has little effect on basal transcription but reduces transcription severalfold in S phase (5Hsiao K.M. McMahon S.L. Farnham P.J. Genes Dev. 1994; 8: 1526-1537Crossref PubMed Scopus (221) Google Scholar). Each globin gene contains one or two CCAAT boxes (6Efstratiadis A. Posakony J.W. Maniatis T. Lawn R.M. O'Connell C. Spritz R.A. DeRiel J.K. Forget B.G. Weissman S.M. Slightom J.L. Blechl A.E. Smithies O. Baralle F.E. Shoulders C.C. Proudfoot N.J. Cell. 1980; 21: 653-668Abstract Full Text PDF PubMed Scopus (884) Google Scholar). The CCAAT box is one of three prominent boxes found by saturation mutagenesis in the murine β-globin promoter (7Myers R.M. Tilly K. Maniatis T. Science. 1986; 232: 613-618Crossref PubMed Scopus (271) Google Scholar). The CCAAT box of the human β-globin promoter binds CP-1 and NF-E1, and this binding is essential for γ gene expression induced by Me2SO in MEL cells (8deBoer E. Antoniou M. Mignotte V. Wall L. Grosveld F. EMBO J. 1988; 7: 4203-4212Crossref PubMed Scopus (138) Google Scholar). Disruption of the CCAAT box of the human ϵ-globin gene resulted in a 3–4-fold decrease of ϵ expression at the embryonic stage of development in transgenic mice (9Filipe 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 CCAAT box in the human γ-globin promoter has been investigated intensively (10Catala F. deBoer E. Habets G. Grosveld F. Nucleic Acids Res. 1989; 17: 3811-3827Crossref PubMed Scopus (23) Google Scholar, 11Ulrich M.J. Ley T.J. Blood. 1990; 75: 990-999Crossref PubMed Google Scholar, 12Langdon S.D. Kaufman R.E. Blood. 1998; 91: 309-318Crossref PubMed Google Scholar, 13Liberati 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, 14Anagnou N.P. Karlsson S. Moulton A.D. Keller G. Nienhuis A.W. EMBO J. 1986; 5: 121-126Crossref PubMed Scopus (30) Google Scholar, 15Berry M. Grosveld F. Dillon N. Nature. 1992; 358: 499-502Crossref PubMed Scopus (108) Google Scholar, 16Gumucio 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, 17Ronchi 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) (for review, see Ref. 18Stamatoyannopoulos G. Grosveld F. Stamatoyannopoulos G. Majerus P.W. Perlmutter R.M. Varmus H. Molecular Basis of Blood Diseases. 3rd Ed. W. B. Saunders Publishing Company, Philadelphia2001: 135-182Google Scholar) due in part to the fact that several mutations in the CCAAT box region produce the phenotype of hereditary persistence of fetal hemoglobin. The CCAAT box of the human γ-globin promoter has been identified as a positive cis element by various in vitro transcription and transient or stable transfection assays (11Ulrich M.J. Ley T.J. Blood. 1990; 75: 990-999Crossref PubMed Google Scholar, 14Anagnou N.P. Karlsson S. Moulton A.D. Keller G. Nienhuis A.W. EMBO J. 1986; 5: 121-126Crossref PubMed Scopus (30) Google Scholar, 19Mantovani R. Superti-Furga G. Gilman J. Ottolenghi S. Nucleic Acids Res. 1989; 17: 6681-6691Crossref PubMed Scopus (49) Google Scholar, 20Duan Z. Stamatoyannopoulos G. Li Q. Mol. Cell. Biol. 2001; 21: 3083-3095Crossref PubMed Scopus (48) Google Scholar, 21Superti-Furga G. Barberis A. Schaffner G. Busslinger M. EMBO J. 1988; 7: 3099-3107Crossref PubMed Scopus (94) Google Scholar, 22Ottolenghi S. Mantovani R. Nicolis S. Ronchi A. Malgaretti N. Giglioni B. Gilman J. Prog. Clin. Biol. Res. 1989; 316A: 229-236PubMed Google Scholar). Because hereditary persistence of fetal hemoglobin mutations in the CCAAT region activates the silenced γ gene in the adult, it is speculated that the CCAAT-binding proteins may function as repressors (15Berry M. Grosveld F. Dillon N. Nature. 1992; 358: 499-502Crossref PubMed Scopus (108) Google Scholar, 17Ronchi 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, 21Superti-Furga G. Barberis A. Schaffner G. Busslinger M. EMBO J. 1988; 7: 3099-3107Crossref PubMed Scopus (94) Google Scholar). Using gel shift assays several proteins have been characterized as γCCAAT region-binding factors: NF-Y (CP-1, CBF), CCAAT/enhancer-binding protein, CCAAT transcription factor/NF1, CCAAT displacement protein, NF-E3, and GATA1. Of these, only NF-Y has been confirmed as an in vivo γCCAAT-binding factor (20Duan Z. Stamatoyannopoulos G. Li Q. Mol. Cell. Biol. 2001; 21: 3083-3095Crossref PubMed Scopus (48) Google Scholar). CCAAT displacement protein, GATA1, and NF-E3 have been implicated in γ gene silencing. However, Ronchi et al. (17Ronchi 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) have reported that disruption of GATA1 or NF-E3 binding in the context of a 70-kb β-globin locus cosmid could not reactivate γ gene expression in adult transgenic mice, suggesting that these two factors are unlikely to function as γ gene repressors; they also showed that the CCAAT box had little effect on embryonic/fetal globin gene expression (17Ronchi 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). To delineate the in vivo role of the CCAAT box of the γ gene promoter, we produced a transgenic mouse model in which the human γ gene is expressed in high levels during embryonic/fetal development as well as in adult life. The effects of mutations in the γCCAAT box could therefore be monitored at the various developmental stages. Our results showed that the effects of the CCAAT box on γ-globin gene activation are developmentally distinct. Although this promoter element is essential for high level γ gene expression in adult erythropoiesis, it plays little role in embryonic erythroid cells. We also showed that the efficiency of γ gene transcription is correlated with recruitment of TBP on the TATA box but that the level of TBP recruitment is not dependent on recruitment of NF-Y on the CCAAT box. Furthermore, our data suggest that direct protein-protein contacts between NF-Y and TBP in erythroid cells are unlikely. Constructs, Transgenic Mice, RNA, and DNA Analysis—Construct μLCR(-382)Aγ, which contained a μLCR-linked human Aγ gene from -382 (StuI) to +1950 (HindIII), has been described previously (23Stamatoyannopoulos G. Josephson B. Zhang J.W. Li Q. Mol. Cell. Biol. 1993; 13: 7636-7644Crossref PubMed Google Scholar). Plasmid μLCR(-382)Aγ(mut CCAAT) was constructed by replacing the duplicated CCAAT box sequence “ACCAAT” with the sequence “AGATCT” (a BglII site) through PCR-based mutagenesis (20Duan Z. Stamatoyannopoulos G. Li Q. Mol. Cell. Biol. 2001; 21: 3083-3095Crossref PubMed Scopus (48) Google Scholar). Production of transgenic mice, RNase protection assay, and copy number measurement were described previously (24Li Q. Stamatoyannopoulos J.A. Mol. Cell. Biol. 1994; 14: 6087-6096Crossref PubMed Google Scholar). Briefly, the construct μLCR(-382)Aγ(mut CCAAT) was microinjected into mouse eggs, and transgenic founders were identified by slot blot hybridization. Copy number of the transgene in the offspring was determined by Southern hybridization using human genomic DNA as standard and a mouse α-globin probe as loading control. Total RNA was prepared from tissues containing the primitive erythrocytes (day-10 and -12 blood and yolk sac) and tissues containing the definitive erythrocytes (day-12 and -16 fetal liver, day-16 blood, and adult blood). γ-Globin mRNA was detected by RNase protection assay and quantified by a PhosphorImager. To minimize experimental error, samples from individual animals were quantified independently, and multiple measurements were performed with both the RNase protection and Southern hybridization assays. Chromatin Immunoprecipitation (ChIP) Assay—ChIP assays were performed as described previously with minor modifications (25Orlando V. Strutt H. Paro R. Methods (Orlando). 1997; 11: 205-214Crossref Scopus (507) Google Scholar, 26Duan Z.J. Fang X. Rohde A. Han H. Stamatoyannopoulos G. Li Q. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5509-5514Crossref PubMed Scopus (31) Google Scholar). The tissues from the transgenic animals were passed through a 21-gauge needle in 5–10 ml of fresh RPMI 1640 medium and then cross-linked immediately. Protein-DNA cross-linking was performed by incubating cell suspensions with formaldehyde at a final concentration of 1.0% for 10 min at 37 °C with gentle agitation. One spleen, or brain, and six yolk sacs were used/condition. Glycine was added to 0.125 m to quench the reaction. Cells were then collected by centrifugation at 900 × g for 5 min and washed twice with cold phosphate-buffered saline. Nuclei were isolated by incubation in cell lysis buffer (10 mm Tris-HCl (pH 8.0), 10 mm NaCl, 0.2% Nonidet P-40) containing 1× protease inhibitor mixture (Roche Applied Science) for 10 min on ice followed by centrifugation at 600 × g for 5 min. Nuclei were lysed in nuclei lysis buffer (50 mm Tris-HCl (pH 8.0), 10 mm EDTA, 0.5% SDS) with 1× protease inhibitor mixture for 10 min on ice. The lysate was sonicated with 15–20 pulses of 30 s each at 30–40% of maximum power with Fisher Sonic Dismembrator 550 equipped with a to the chromatin to an of The samples were with immunoprecipitation buffer mm Tris-HCl (pH 8.0), m NaCl, mm EDTA, and then for The was with followed by protein The chromatin was with of in a final of ml for at were collected by incubation with of protein for at were washed twice with of immunoprecipitation buffer mm Tris-HCl (pH 8.0), mm NaCl, mm EDTA, and with immunoprecipitation buffer (10 mm Tris-HCl (pH 8.0), m mm EDTA, Nonidet Chromatin DNA was then and for by using the to the The DNA concentration was determined with a of DNA was used as in each in a of 10 Z.J. Fang X. Rohde A. Han H. Stamatoyannopoulos G. Li Q. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5509-5514Crossref PubMed Scopus (31) Google Scholar). and II (Pol II were from was performed on the chromatin samples using the (Roche Applied were performed using the (Roche Applied Science) to the The used were to the sequence of DNA on human and mouse β-globin and by using a for The were and used in this human Aγ promoter and human Aγ CCAAT and human Aγ TATA and mouse gene promoter and and mouse gene promoter and The CCAAT for γ-Globin Gene in but in the effects of CCAAT box on γ gene a CCAAT box mutation was produced in the context of a construct (20Duan Z. Stamatoyannopoulos G. Li Q. Mol. Cell. Biol. 2001; 21: 3083-3095Crossref PubMed Scopus (48) Google the of which is expressed in of development of transgenic mice (23Stamatoyannopoulos G. Josephson B. Zhang J.W. Li Q. Mol. Cell. Biol. 1993; 13: 7636-7644Crossref PubMed Google Scholar). transgenic mouse of the CCAAT transgene were used for RNase protection for γ gene expression were performed on and the were from measurements of individual samples from the To the effects of CCAAT box mutation in embryonic erythropoiesis, we γ gene expression in and blood and yolk The blood and yolk an stage of embryonic the yolk contains of embryonic and the fetal blood is of erythrocytes of yolk a of RNase protection assay, and data from measurements of the assay are in the in the levels of γ mRNA in blood and yolk and in blood samples were the as in the transgenic mice a promoter with the CCAAT box. the CCAAT box mutation has little effect on γ gene expression at the embryonic in with by Ronchi et al. (17Ronchi 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 γ-globin gene expression in embryonic of transgenic mice the construct γ of murine CCAAT) from Ref. from Ref. G. Josephson B. Zhang J.W. Li Q. Mol. Cell. Biol. 1993; 13: 7636-7644Crossref PubMed Google in a To the effect of the CCAAT mutation in definitive erythropoiesis, we γ gene expression in fetal liver, in day-16 blood and fetal liver, and in adult The fetal liver is of definitive erythroblasts and a of embryonic the blood and fetal liver definitive cells and The adult blood contains erythroid cells from in the CCAAT mutation not γ gene expression in the fetal liver, and it resulted in a decrease in the day-16 blood and fetal In γ gene expression in the adult blood of the CCAAT mice was by with the mice, suggesting that an CCAAT box is for γ-globin gene expression in adult γ-globin gene expression in definitive of transgenic mice the construct γ of murine CCAAT) from Ref. from Ref. G. Josephson B. Zhang J.W. Li Q. Mol. Cell. Biol. 1993; 13: 7636-7644Crossref PubMed Google in a The CCAAT for NF-Y to γ-Globin Gene in have that the NF-Y recruitment is with high level γ gene expression in MEL cells with a construct (20Duan Z. Stamatoyannopoulos G. Li Q. Mol. Cell. Biol. 2001; 21: 3083-3095Crossref PubMed Scopus (48) Google Scholar). The show that of mutations to the CCAAT box not the γ gene in embryonic erythroid cells. the NF-Y a role in γ gene expression in embryonic To this we by assay, NF-Y recruitment in embryonic erythroid cells and in adult splenic erythroblasts of and The of splenic cells of animals are adult erythroblasts H. M. J. PubMed Scopus Google Scholar). The DNA by the was by with a of the CCAAT box of the human Aγ In this the murine adult gene promoter was used as an control. The disruption of the CCAAT box the recruitment of NF-Y to in the adult of with the mice Using the we the effect of the CCAAT box mutation on recruitment of NF-Y in blood and yolk In this NF-Y recruitment in the murine embryonic promoter was used as the control. in NF-Y recruitment on the CCAAT γ promoter was to of the in embryonic cells. results suggest that NF-Y recruitment is by the CCAAT γ gene expression a high level in embryonic The of the CCAAT on the of Transcription suggest that the TBP recruitment be for the γ gene expression in MEL cells (20Duan Z. Stamatoyannopoulos G. Li Q. Mol. Cell. Biol. 2001; 21: 3083-3095Crossref PubMed Scopus (48) Google Scholar). we show that in embryonic erythrocytes the CCAAT box mutation not γ gene could these TBP recruitment is dependent on the CCAAT but transcription efficiency is not with the level of TBP recruitment M. E. E. Imam A. Grosveld F. Nucleic Acids Res. 1995; PubMed Scopus Google or transcription efficiency is with TBP but the TBP binding is not dependent on the CCAAT box. To recruitment of TBP is dependent on the CCAAT we the recruitment of TBP in the CCAAT transgenic The DNA by the was by with a of the TATA box of the human Aγ The disruption of the CCAAT box the recruitment of TBP to in the adult with the In disruption of the γCCAAT box had effects on the recruitment of TBP in the embryonic erythrocytes that in embryonic erythrocytes the TBP binding is not correlated with the recruitment of the recruitment of and Pol II to the γ promoter was only in the adult and but not in embryonic cells C and The results suggest that recruitment of TFIIB, and Pol II to the core promoter and recruitment of NF-Y to the CCAAT box are not it is unlikely that a direct interaction between NF-Y and the basal transcription apparatus in embryonic erythroid cells. However, the data not the that a direct in adult erythropoiesis, in γ gene NF-Y with TBP in TBP recruitment and NF-Y recruitment are in adult erythropoiesis, we a assay There is a between the CCAAT and TATA boxes of the γ gene two be detected by of this NF-Y and TBP recruitment be recruitment of NF-Y and recruitment of TBP are through direct protein-protein this in of the TATA box in the chromatin by NF-Y and We performed this using adult splenic erythroblasts of the transgenic mice the The γ gene is expressed in high levels in this the of the CCAAT and TATA be detected in the chromatin by the NF-Y the CCAAT box could be but the TATA box was by only of the In the the TATA could be in the TBP only of the CCAAT was The efficiency of on the cross-linked chromatin was To that the results are we the using which not between the two boxes. In this the TATA and CCAAT were in in the TBP or NF-Y chromatin and results suggest that is direct between NF-Y and TBP in adult erythroid cells. In this study we demonstrate that the effects of the CCAAT box on γ-globin gene activation are developmental Although this promoter element is essential for high level γ gene expression in adult erythropoiesis, it plays little role in embryonic erythroid cells. We also show that the efficiency of γ gene transcription is correlated with recruitment of TBP on the TATA box but that the level of TBP recruitment is not dependent on recruitment of NF-Y on the CCAAT box. Furthermore, our data suggest that it is unlikely that direct protein-protein between NF-Y and TBP in erythroid cells. The developmentally role of the CCAAT box could using transfection assays suggest that the CCAAT box is in gene activation (2Mantovani R. Gene (Amst.). 1999; 239: 15-27Crossref PubMed Scopus (673) Google an in vivo study (17Ronchi 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) that the box is not Our results show that the role of the CCAAT box on γ gene activity is dependent on the stage of in adult erythroid cells the CCAAT box a positive effect on γ gene it has little in embryonic erythroid cells. The of on CCAAT box function using transfection assays have been in cell from definitive the transcriptional of cells most the of adult cells. may performed in cell show a positive role of the CCAAT box in gene the function we in vivo in the erythroid cells of adult transgenic a have the NF-Y enhances gene transcription. has been reported that NF-Y is to with TBP in M. K. L. Mantovani R. Nucleic Acids Res. 1997; PubMed Scopus Google Scholar). the of TBP and the to the of and In to interaction between NF-Y and various factors also was detected in a protein-protein assay M. C. A. B. A. C. L. I. Mantovani R. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). Our results showed that the CCAAT NF-Y it not TBP recruitment in embryonic erythropoiesis, suggesting that TBP recruitment is not by the CCAAT The CCAAT recruitment of NF-Y and TBP in adult However, assays to direct between TBP and on these we that it is unlikely that the γ gene activation is exerted through direct interaction between TBP and We have reported that the CCAAT box mutation resulted in a of chromatin of the γ gene promoter in MEL cells (20Duan Z. Stamatoyannopoulos G. Li Q. Mol. Cell. Biol. 2001; 21: 3083-3095Crossref PubMed Scopus (48) Google and a decrease also was in adult splenic G. and Q. of NF-Y on the CCAAT box is to the by of A. M. N. Mantovani R. Nucleic Acids Res. 1995; PubMed Scopus Google Scholar). In to this direct NF-Y is to to a promoter or R.A. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, V. G. M. W. U. K. Mantovani R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). and S. Mol. Cell. Biol. 1998; 18: PubMed Google Scholar) reported that with resulted in a of expression of the gene and that the CCAAT box of the gene promoter this results were in the promoter in nuclei Q. M. N. N. EMBO J. 1998; 17: PubMed Scopus Google Scholar). suggest that recruitment of NF-Y to the CCAAT box may a promoter that RNA polymerase II transcription. is in with our which suggest that transcription may not be by direct between NF-Y and the basal transcription We and for of this