Chromatin Immunoprecipitation (ChIP) on Chip Experiments Uncover a Widespread Distribution of NF-Y Binding CCAAT Sites Outside of Core Promoters
2005; Elsevier BV; Volume: 280; Issue: 14 Linguagem: Inglês
10.1074/jbc.m414039200
ISSN1083-351X
AutoresAnna Testa, Giacomo Donati, Pearlly S. Yan, Francesca Romani, Tim H.M. Huang, M Viganò, Roberto Mantovani,
Tópico(s)NF-κB Signaling Pathways
ResumoThe CCAAT box is a prototypical promoter element, almost invariably found between –60 and –100 upstream of the major transcription start site. It is bound and activated by the histone fold trimer NF-Y. We performed chromatin immunoprecipitation (ChIP) on chip experiments on two different CpG islands arrays using chromatin from hepatic HepG2 and pre-B cell leukemia NALM-6 cell lines, with different protocols of probe preparation and labeling. We analyzed and classified 239 known or predicted targets; we validated several by conventional ChIPs with anti-YB and anti-YC antibodies, in vitro EMSAs, and ChIP scanning. The importance of NF-Y binding for gene expression was verified by the use of a dominant negative NF-YA mutant. All but four genes are new NF-Y targets, falling into different functional categories. This analysis reinforces the notion that NF-Y is an important regulator of cell growth, and novel unexpected findings emerged from this unbiased approach. (i) A remarkable proportion of NF-Y targets, 40%, are complex transcriptional units composed of divergent, convergent, and tandem promoters. (ii) 40–50% of NF-Y sites are not in core promoters but are in introns or at distant 3′ or 5′ locations. The abundance of "unorthodox" CCAAT positions highlights an unexpected complexity of the NF-Y-mediated transcriptional network. The CCAAT box is a prototypical promoter element, almost invariably found between –60 and –100 upstream of the major transcription start site. It is bound and activated by the histone fold trimer NF-Y. We performed chromatin immunoprecipitation (ChIP) on chip experiments on two different CpG islands arrays using chromatin from hepatic HepG2 and pre-B cell leukemia NALM-6 cell lines, with different protocols of probe preparation and labeling. We analyzed and classified 239 known or predicted targets; we validated several by conventional ChIPs with anti-YB and anti-YC antibodies, in vitro EMSAs, and ChIP scanning. The importance of NF-Y binding for gene expression was verified by the use of a dominant negative NF-YA mutant. All but four genes are new NF-Y targets, falling into different functional categories. This analysis reinforces the notion that NF-Y is an important regulator of cell growth, and novel unexpected findings emerged from this unbiased approach. (i) A remarkable proportion of NF-Y targets, 40%, are complex transcriptional units composed of divergent, convergent, and tandem promoters. (ii) 40–50% of NF-Y sites are not in core promoters but are in introns or at distant 3′ or 5′ locations. The abundance of "unorthodox" CCAAT positions highlights an unexpected complexity of the NF-Y-mediated transcriptional network. The CCAAT box is a DNA element that controls transcriptional initiation in eukaryotic promoters; recent bioinformatic studies unambiguously identify it as one of the most widespread. The analysis on 1031 human promoters isolated through unbiased determination of mRNA start sites suggested that the CCAAT box or its reverse ATTGG is present in as many as 67% of promoters (1.Suzuki Y. Tsunoda T. Sese J. Taira H. Mizushima-Sugano J. Hata H. Ota T. Isogai T. Tanaka T. Nakamura Y. Suyama A. Sakaki Y. Morishita S. Okubo K. Sugano S. Genome Res. 2001; 11: 677-684Crossref PubMed Scopus (204) Google Scholar). A statistical, unbiased analysis of random octanucleotides on a large 13,000-promoter data set confirmed that the CCAAT is second only to the Sp1-binding GC box in terms of abundance, despite the fact that the percentage of CCAAT promoters was inferior, 7.5% (2.FitzGerald P.C. Shlyakhtenko A. Mir A.A. Vinson C. Genome Res. 2004; 14: 1562-1574Crossref PubMed Scopus (172) Google Scholar). Furthermore, analysis of cell cycle-regulated genes identified the CCAAT box as specifically present in promoters of G2/M genes (3.Elkon R. Linhart C. Sharan R. Shamir R. Shiloh Y. Genome Res. 2003; 13: 773-780Crossref PubMed Scopus (262) Google Scholar). Most importantly, specific flanking nucleotides emerging from these studies matched specifically the consensus of the NF-Y transcription factor. A combination of EMSAs and transfections with highly diagnostic dominant negative vectors implicated NF-Y as the CCAAT activator (4.Mantovani R. Gene. 1999; 239: 15-27Crossref PubMed Scopus (673) Google Scholar). It is composed of three subunits, NF-YA, NF-YB, and NF-YC, all necessary for sequence-specific binding to a G/A, G/A, C, C, A, A, T, C/G, A/G, G/C consensus. NF-YB and NF-YC contain evolutionarily conserved histone fold motifs common to all core histones, mediating dimerization, a feature strictly required for NF-YA association and sequence-specific DNA binding (5.Maity S.N. de Crombrugghe B. Trends Biochem. Sci. 1998; 23: 174-178Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar, 6.Romier C. Cocchiarella F. Mantovani R. Moras D. J. Biol. Chem. 2003; 278: 1336-1345Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). In essentially all cases described so far, the binding of the trimer is important or essential for transcriptional regulation (7.Mantovani R. Nucleic Acids Res. 1998; 26: 1135-1143Crossref PubMed Scopus (442) Google Scholar). NF-Y is considered as a general promoter organizer: thanks to its histone-like nature, it presets chromatin structure locally (8.Li Q. Herrler M. Landsberger N. Kaludov N. Ogryzko V.V. Nakatani Y. Wolffe A.P. EMBO J. 1998; 17: 6300-6315Crossref PubMed Scopus (177) Google Scholar), interfacing well with nucleosomes (9.Coustry F. Hu Q. de Crombrugghe B. Maity S.N. J. Biol. Chem. 2001; 276: 40621-40630Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), it helps the binding of neighboring factors (reviewed in Refs. 4.Mantovani R. Gene. 1999; 239: 15-27Crossref PubMed Scopus (673) Google Scholar and 5.Maity S.N. de Crombrugghe B. Trends Biochem. Sci. 1998; 23: 174-178Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar) and attracts coactivators, such as p300/CREB-binding protein (8.Li Q. Herrler M. Landsberger N. Kaludov N. Ogryzko V.V. Nakatani Y. Wolffe A.P. EMBO J. 1998; 17: 6300-6315Crossref PubMed Scopus (177) Google Scholar, 10.Caretti G. Salsi V. Vecchi C. Imbriano C. Mantovani R. J. Biol. Chem. 2003; 278: 30435-30440Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). The location of the CCAAT box is far from random, being positioned between –60 and –100 in the vast majority of the promoters analyzed. In general, our knowledge of the anatomy of NF-Y-binding sites in terms of flanking sequences, position with respect to transcriptional start sites, and promoter context (6.Romier C. Cocchiarella F. Mantovani R. Moras D. J. Biol. Chem. 2003; 278: 1336-1345Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 11.Dorn A. Bollekens J. Staub A. Benoist C. Mathis D. Cell. 1987; 50: 863-872Abstract Full Text PDF PubMed Scopus (471) Google Scholar, 12.Liang S.G. Maity S.N. J. Biol. Chem. 1998; 273: 31590-31598Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) enables us to make predictions as to whether a gene will be regulated by NF-Y. Chromatin Immunoprecipitation (ChIP) 1The abbreviations used are: ChIP, chromatin immunoprecipitation; Pipes, 1,4-piperazinediethanesulfonic acid; PMSF, phenylmethylsulfonyl fluoride; IP, immunoprecipitation; CTU, complex transcriptional unit; EMSA, electrophoretic mobility shift assay; CREB, cAMP-response element-binding protein. 1The abbreviations used are: ChIP, chromatin immunoprecipitation; Pipes, 1,4-piperazinediethanesulfonic acid; PMSF, phenylmethylsulfonyl fluoride; IP, immunoprecipitation; CTU, complex transcriptional unit; EMSA, electrophoretic mobility shift assay; CREB, cAMP-response element-binding protein. experiments determined that NF-Y is bound in vivo before gene activation (10.Caretti G. Salsi V. Vecchi C. Imbriano C. Mantovani R. J. Biol. Chem. 2003; 278: 30435-30440Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 11.Dorn A. Bollekens J. Staub A. Benoist C. Mathis D. Cell. 1987; 50: 863-872Abstract Full Text PDF PubMed Scopus (471) Google Scholar, 12.Liang S.G. Maity S.N. J. Biol. Chem. 1998; 273: 31590-31598Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 13.Bennett M.K. Osborne T.F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6340-6344Crossref PubMed Scopus (100) Google Scholar); NF-Y is bound to a transcribing cyclin B1 promoter during mitosis in HeLa cells (14.Sciortino S. Gurtner A. Manni I. Fontemaggi G. Dey A. Sacchi A. Ozato K. Piaggio G. EMBO Rep. 2001; 2: 1018-1023Crossref PubMed Scopus (50) Google Scholar). Indeed, binding to cell cycle-regulated promoters is not constitutive but is time-regulated, being found before activation and displaced when promoters are repressed (10.Caretti G. Salsi V. Vecchi C. Imbriano C. Mantovani R. J. Biol. Chem. 2003; 278: 30435-30440Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Furthermore, conditional knock-out experiments of CBF-B (NF-YA) unambiguously determined that the protein is required for cell proliferation of mouse embryo fibroblasts and mouse development (15.Bhattacharya A. Deng J.M. Zhang Z. Behringer R. de Crombrugghe B. Maity S.N. Cancer Res. 2003; 63: 8167-8172PubMed Google Scholar). The analysis of 130 mammalian CCAAT-containing promoters suggests a prevalence in genes that are active in a tissue- or development-specific way and in inducible genes, either by external stimuli or during the cell cycle (7.Mantovani R. Nucleic Acids Res. 1998; 26: 1135-1143Crossref PubMed Scopus (442) Google Scholar). Whereas this is certainly informative, very little information exists as to the binding to other regions. Finding all genes targeted by a particular transcription factor is crucial to reconstruct its transcriptional network. To expand our knowledge of NF-Y binding in vivo, a valuable approach is to use DNA derived from ChIPs to probe microarrays. DNA arrays have been developed in which clones derived from a CpG island library have been spotted (16.Cross S.H. Charlton J.A. Nan X. Bird A.P. Nat. Genet. 1994; 6: 236-244Crossref PubMed Scopus (393) Google Scholar); CpG islands have long been known to be associated to regulatory elements in promoters (17.Ioshikhes I.P. Zhang M.Q. Nat. Genet. 2000; 26: 61-63Crossref PubMed Scopus (219) Google Scholar) and also elsewhere in the genome. They are believed to be mainly associated to "housekeeping" genes (i.e. genes active in all cells), albeit at different levels (reviewed in Ref. 18.Antequera F. Cell. Mol. Life Sci. 2003; 60: 1647-1658Crossref PubMed Scopus (336) Google Scholar). To gain a wider understanding of the NF-Y transcriptional circuitry, we took a high throughput genomic approach by screening with anti-YB chromatin-immunoprecipitated DNA two CpG island arrays. The procedure for ChIP was essentially as described previously (10.Caretti G. Salsi V. Vecchi C. Imbriano C. Mantovani R. J. Biol. Chem. 2003; 278: 30435-30440Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) with some modifications. Rabbit polyclonal anti-YB and anti-YC antibodies were derived by purification of the corresponding sera on affinity columns containing purified recombinant NF-YB or NF-YC linked to CnBr-Sepharose (Sigma). Nalm-6 and HepG2 cells, grown in RPMI, supplemented with 10% fetal calf serum, 2 mm l-glutamine, 50 μm β-mercaptoethanol, were treated by adding formaldehyde directly to tissue culture medium to a final concentration of 1% and incubated for 10 min at room temperature. Approximately 5 × 106 cells were used for each immunoprecipitation. Cross-linking reactions were stopped by the addition of phosphate-buffered saline-glycine to a final concentration of 0.125 m. Cells were washed twice with ice-cold phosphate-buffered saline, scraped, and centrifuged at 2000 rpm for 2 min. Cells were then resuspended in cell lysis buffer (5 mm Pipes, pH 8.0, 85 mm KCl, and 0.5% Nonidet P-40) containing protease inhibitors (100 ng/ml aprotinin and 100 ng/ml leupeptin) and 0.5 mm PMSF and kept on ice for 15 min. Cells were homogenized using a Dounce homogenizer (B pestel) several times, and the resultant homogenates were centrifuged at 5000 rpm for 5 min at 4 °C to pellet the nuclei. The pellets were resuspended in nuclei lysis buffer (50 mm Tris-HCl, pH 8.1, 10 mm EDTA, 0.1% SDS, and 0.5% deoxycholic acid) containing protease inhibitors and PMSF and kept on ice for 20 min. The nuclear lysates were sonicated on ice to an average chromatin length of 2–2.5 kb and then centrifuged at 12,000 rpm for 10 min at 4 °C. The supernatants were incubated in IP buffer (50 mm Tris-HCl, pH 8.1, 10 mm EDTA, 0.1% SDS, 0.5% deoxycholic acid, and 500 mm LiCl) containing protease inhibitors and PMSF, with Protein G-agarose (KPL) for 2 h at 4 °C in rotation. After removal of Protein G-agarose, the precleared lysates were used as soluble chromatin for ChIP. Chromatin was incubated at 4 °C overnight with 4 μg of anti-NF-YB or anti-NF-YC antibodies. No antibody and anti-FLAG (Sigma) control samples were included. Immunoprecipitates were recovered by incubation for 2 h at 4 °C with Protein G-agarose previously precleared in IP buffer (1 μg/μl bovine serum albumin, 1 μg/μl salmon testis DNA, protease inhibitors, and PMSF). To perform a second immunoprecipitation, 30 μl of elution buffer (50 mm NaHCO3, 1% SDS) were added, and the recovered material was diluted with 270 μl of IP buffer. 2 μgof the second antibody were added and incubated at 4 °C overnight. The recovery proceeded as in the first IP reaction. Reversal of formaldehyde cross-linking, RNase A, and Proteinase K treatments were performed as previously described (19.Caretti G. Cocchiarella F. Sidoli C. Villard J. Peretti M. Reith W. Mantovani R. J. Mol. Biol. 2000; 302: 539-552Crossref PubMed Scopus (39) Google Scholar). Data validation was performed with conventional ChIPs (10.Caretti G. Salsi V. Vecchi C. Imbriano C. Mantovani R. J. Biol. Chem. 2003; 278: 30435-30440Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), with chromatin of 0.8 kb and with anti-YB as well as anti-YC purified polyclonal antibodies. The sequence of PCR primers used to analyze the genes reported in Fig. 2 are shown in Supplemental Table I. DNAs from 20–30 individual ChIPs were used to generate a probe for array screening. Immunoprecipitated chromatin was used as template for random priming reactions in the presence of 10 mm amino allyl-UTP (Sigma catalog no. A-0410) using the BioPrime DNA labeling system (Invitrogen). The DNAs were desalted and concentrated with a Microcon YM30 filter column (Millipore Corp.) and then lyophilized. After resuspension in water, amino allyl-dUTP-labeled chromatin was coupled with Cy5 dye (Amersham Biosciences) solubilized in 0.1 m sodium bicarbonate, pH 9.0, for 1 h in the dark. After the addition of 0.1 m sodium acetate, pH 5.2, DNAs were purified with QIAquick columns (Qiagen) and lyophilized. The generation of amplicons from individual ChIPs was performed following the protocols of LM-PCR described in Refs. 20.Oberley M.J. Inman D.R. Farnham P.J. J. Biol. Chem. 2003; 278: 42466-42476Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar and 21.Oberley M.J. Tsao J. Yau P. Farnham P.J. Methods Enzymol. 2004; 376: 315-334Crossref PubMed Scopus (79) Google Scholar. Briefly, two unidirectional linkers (oligonucleotide JW102, 5′-GCGGTGACCCGGGAGATCTGAATTC-3′; oligonucleotide JW103, 5′-GAATTCAGATC-3′) were annealed and ligated to the chromatin IPs, previously blunted by T4 DNA polymerase. The first amplicons were generated by PCR (one cycle at 55 °C for 2 min, 72 °C for 5 min, 95 °C for 2 min, followed by 15 cycles at 95 °C for 30 min, 55 °C for min, 72 °C for 1 min, and a final extension of 4 min at 72 °C). The reaction was purified using the Qiaquick PCR purification kit (Qiagen) or the GFX PCR purification kit (Amersham Biosciences) according to the manufacturer's instructions. One-tenth of these initial reactions were used to generate more amplicons, using the same PCR program for a subsequent 30 cycles. After purification of these last rounds of amplification, the DNA was quantified and examined by gene-specific PCR to ensure that the initial enrichment was maintained. 5 μg of amplicons for α-NF-YB, α-FLAG, and input DNA (subjected to the same number of PCR manipulations as the IPs) were labeled using the LabelIT Cy5/Cy3 nucleic acid labeling kit (Mirus), following the manufacturer's instructions, with a reagent/DNA ratio of 2.5 for Cy5 (IPs) and 1.5 for Cy3 (input). 7776 CpG Array—The development of the 7776 CpG island array was described previously (21.Oberley M.J. Tsao J. Yau P. Farnham P.J. Methods Enzymol. 2004; 376: 315-334Crossref PubMed Scopus (79) Google Scholar, 22.Caretti G. Motta M.C. Mantovani R. Mol. Cell. Biol. 1999; 19: 8591-8603Crossref PubMed Scopus (58) Google Scholar, 23.Bi W. Wu L. Coustry F. de Crombrugghe B. Maity S.N. J. Biol. Chem. 1997; 272: 26562-26572Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Prior to hybridization, spotted CpG island slides were incubated with a solution of 3× SSC, 0.25% SDS, and 1.5 μg/μl salmon testis DNA under a glass coverslip at 37 °C for 30 min to block nonspecific binding. Slides were washed twice with water and dried for 5 min at 600 rpm in a centrifuge. Labeled DNAs were added to hybridization buffer (0.25 m NaPO4, 4.5% SDS, 1 mm EDTA, and 1× SSC), denatured at 95 °C for 2 min, cooled to 60 °C, and dropped onto slides placed in prewarmed hybridization chambers. Incubation was performed at 60 °C overnight. After hybridization, the slides were washed successively at 50 °C with 1× SSC, 0.1% SDS at room temperature with 1× SSC (0.1%) and at room temperature with 0.2 SSC for 5 min each and then dried. Hybridized slides were scanned with the GenePix 4000A scanner (Axon), and the acquired images were analyzed with the software GenePix Pro, Version 3.0. A global normalization factor was determined for each replica, evaluating the anti-NF-YB ChIP Cy5/control ChIP Cy5 ratio relative to control repetitive elements. Data were normalized prior to comparison. After normalization, positive loci were defined by hybridization intensities at least 2 times greater than that of control. 12K Array—The Cy5- and Cy3-labeled DNA were each resuspended in 10 μl of 1 μg/μl Cot-1 DNA (Invitrogen) and mixed together in order to have the same amount of input Cy3-labeled DNA for each IP Cy5-labeled DNA. The hybridization solution was then added to a final composition of 43% formamide, 4.3× SSPE, 0.42% SDS, 42 μg of salmon sperm DNA, 0.2 μg of tRNA, heated for 2 min at 95 °C and cooled down to 37 °C over 30 min. 95 μl of each mixture solution was applied to two human CpG 12K slides (University Health Network, The Microarray Center, Toronto, Canada) and hybridized at 37 °C for >18 h. The slides were prehybridized for 1 h at42 °C with 25% formamide, 5× SSC, 0.1% SDS, and 10 μg/μl bovine serum albumin. The slides were washed at room temperature for 5 min twice in 2× SSC, 0.1% SDS; once in 1× SSC, 0.1% SDS; and one final time in 0.1× SSC; dried; and immediately scanned using a ScanArray 4000 scanner (Packard). The hybridized microarrays were analyzed using the Quantarray microarray analysis software (Packard). Features of poor intensity ( 2-fold in the YB samples were further analyzed. Two independent experiments were performed, each consisting of one α-NF-YB IP and one control α-FLAG IP slide, normalized to the same input DNA, and the commonly enriched spots were considered. Positive clones were sequenced and mapped with BLAT. The presence of CCAAT sequences were searched for 2 kb on the flanking of the 7776 CpG island array and 500 bp on the 12K array, annotated in individual files corresponding to the genomic loci identified. The criteria for classifications are described below. Mouse orthologs were retrieved using BLAT. The annotated genes were classified according to functional categories, and the classification was compared with those performed on the MYC and E2F4 targets. HepG2 cells were infected with control green fluorescent protein, wild type NF-YA, or dominant negative YAm29 adenovirus. 2Imbriano, C., Gurtner, A., Cocchiarella, F., Di Agostino, S., Basile, V., Gostissa, M., Dobbelstein, M., Del Sal, G., Piaggio G., and Mantovani, R. (2005) Mol Cell Biol., in press Adenovirus vectors to express NF-YA or the YAm29 dominant negative mutant were generated using AdEasy, using HindIII and XbaI from the corresponding pcDNA3-based vectors, and introduced into the same sites of the shuttle vector pAdTrack-CMV. This plasmid was recombined with the vector pAdEasy1, followed by treatment with PacI and transfection into an E1-complementing cell line. We infected exponentially growing cells for 7 h in the absence of serum. Fetal calf serum was then added, and cells were incubated for 48 h. RNA was extracted using an RNA-Easy kit (Qiagen), according to the manufacturer's protocol. For cDNA synthesis, 4 μg of RNA were used with the M-MLV-RT kit (Invitrogen). Semiquantitative PCR analysis was performed with oligonucleotides detailed in Supplemental Table II. EMSA analyses of Fig. 3 were performed under standard NF-Y conditions (6.Romier C. Cocchiarella F. Mantovani R. Moras D. J. Biol. Chem. 2003; 278: 1336-1345Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 11.Dorn A. Bollekens J. Staub A. Benoist C. Mathis D. Cell. 1987; 50: 863-872Abstract Full Text PDF PubMed Scopus (471) Google Scholar, 22.Caretti G. Motta M.C. Mantovani R. Mol. Cell. Biol. 1999; 19: 8591-8603Crossref PubMed Scopus (58) Google Scholar, 23.Bi W. Wu L. Coustry F. de Crombrugghe B. Maity S.N. J. Biol. Chem. 1997; 272: 26562-26572Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), with anti-YB supershift antibodies and recombinant NF-Y and the indicated oligonucleotides. 32P-Labeled oligonucleotides were incubated in 20 mm Tris-HCl, pH 7.8, 50 mm NaCl, 1 mm dithiothreitol, 3% glycerol, 5 mm MgCl2 for 30 min at 20 °C with 5 ng of recombinant NF-Y trimer or with 5 μg of HepG2 nuclear extracts together with 200 ng of poly(dI-dC) (Sigma). The samples were loaded on a 4.5% polyacrylamide gel, run for 2 h, dried, and exposed. To produce recombinant NF-Y, Escherichia coli BL21 DE3LysS was induced at an A600 value of 0.6 by the addition of isopropyl-β-d-thiogalactopyranoside to a final concentration of 1 mm for 3 h. Bacterial pellets were resuspended and sonicated in sonication buffer (150 mm KCl, 20 mm Tris-HCl, pH 7.8, 0.05% Nonidet P-40, 0.1 mm EDTA, 5 mm 2-mercaptoethanol, 1 mm PMSF (Sigma), and protein inhibitors) and centrifuged at 23,000 × g in a Beckman SW 27Ti rotor for 30 min at 4 °C. The inclusion bodies pellet was resuspended in sonication buffer, sonicated, and centrifuged again. Inclusion bodies were finally resuspended in 6 m guanidium chloride, 20 mm sodium acetate (pH 5.2), 5 mm 2-mercaptoethanol, and 1 mm PMSF. The three subunits were mixed to a final concentration of 0.5 mg/ml and dialyzed against a 100-fold excess of BC300 (300 mm KCl, 20 mm Tris-HCl, pH 7.8, 0.05% Nonidet P-40, 5 mm 2-mercaptoethanol, 1 mm PMSF); glycerol concentration was adjusted to 20%, and proteins were loaded on a nickel-nitrilotriacetic acid-agarose column, washed with BC300, and eluted with 0.25 m imidazole. The proteins were finally dialyzed against BC100, the purity being routinely >80%. Our goal was to identify novel targets of NF-Y in an unbiased way. The combination of chromatin immunoprecipitation with microarray analysis was performed in yeast (24.Ren B. Robert F. Wyrick J.J. Aparicio O. Jennings E.G. Simon I. Zeitlinger J. Schreiber J. Hannett N. Kanin E. Volkert T.L. Wilson C.J. Bell S.P. Young R.A. Science. 2000; 290: 2306-2309Crossref PubMed Scopus (1566) Google Scholar, 25.Iyer V.R. Horak C.E. Scafe C.S. Botstein D. Snyder M. Brown P.O. Nature. 2001; 409: 533-538Crossref PubMed Scopus (869) Google Scholar, 26.Lieb J.D. Liu X. Botstein D. Brown P.O. Nat. Genet. 2001; 28: 327-334Crossref PubMed Scopus (545) Google Scholar) and humans (20.Oberley M.J. Inman D.R. Farnham P.J. J. Biol. Chem. 2003; 278: 42466-42476Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 27.Weinmann A.S. Yan P.S. Oberley M.J. Huang T.H. Farnham P.J. Genes Dev. 2002; 16: 235-244Crossref PubMed Scopus (388) Google Scholar, 28.Ren B. Cam H. Takahashi Y. Volkert T. 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For the 7776 array, we prepared several sequential ChIPs with chromatin from the liver HepG2 cell line, with a highly specific anti-NF-YB antibody, and, in parallel, control ChIPs with a commercially available anti-FLAG control. The chromatin used in this procedure was larger (∼2–2.5 kb) than the one used in conventional ChIPs (0.5–1 kb). Because of the modifications of our routine ChIPs with extended chromatin, we first verified whether immunoprecipitated DNAs were indeed enriched in NF-Y-targeted fragments. We used oligonucleotides amplifying several CCAAT-containing promoters in semiquantitative PCRs. Fig. 1A shows that essentially all of the promoters tested were clearly positive in the anti-YB ChIP, compared with the FLAG and no antibody controls: the liver-specific genes αGA, MVK, OAT, and mATP synthase and the ubiquitous HnRNPA1, NP95, PPP1R7, HMGB2, ABL, CDC25A, β-actin, and OGG1. Note that only the last two genes were previously known to be regulated by NF-Y (31.Danilition S.L. Frederickson R.M. Taylor C.Y. Miyamoto N.G. Nucleic Acids Res. 1991; 19: 6913-6922Crossref PubMed Scopus (46) Google Scholar, 32.Gowri P.M. Yu J.H. Shaufl A. Sperling M.A. Menon R.K. Mol. Cell. Biol. 2003; 23: 815-825Crossref PubMed Scopus (30) Google Scholar), whereas all of the others were derived from a CCAAT-containing promoter data set. 3A. Testa and R. Mantovani, manuscript in preparation. In parallel ChIP analysis, CCAAT-less promoters, p107, α-tubulin, RPS19, and YBL1, were negative (Fig. 1A, lower panel). For the 12K hybridization, we took a different approach, by PCR-amplifying chromatin from Nalm-6 cells after ligation of linker DNA. The advantage is that a very limited amount of ChIP material is required to yield enough DNA for hybridization. We also checked that the successive rounds of PCR amplifications would not decrease the enrichment of bona fide NF-Y targets in the amplicons. Indeed, Fig. 1B shows that the NF-YA promoter amplicon is no less, and in fact probably more, enriched in the final LM-PCR chromatin compared with the initial starting material. Therefore, we conclude that both of these procedures yield sufficiently enriched DNA for further genomic analysis. Results of the 7776 Array—We used DNAs from 20–30 individual ChIPs to generate probes for the 7776 array screening. We identified at least 230 spots, in which the corrected signal obtained with the NF-YB chromatin was at least 2-fold higher than the anti-FLAG signal. We sequenced all positive clones and derived their chromosomal localizations. A positive clone will indicate that a bound NF-Y site lies somewhere within 2.5 kb of the CpG island. The genomic sequences surrounding the CpG island were therefore scrutinized for the presence of CCAAT sequences for a length of 2 kb on either sides. Table I shows a list of the positive clones. Several criteria helped us to classify them as follows.Table IClassification of NF-Y-targeted genes Open table in a new tab Flanking sequences are essential for high affinity NF-Y binding both at the 5′ and 3′ of the pentanucleotide, with a variation of 2 logs in Kd in vitro, between high and low affinity sites (for details, see Refs. 6.Romier C. Cocchiarella F. Mantovani R. Moras D. J. Biol. Chem. 2003; 278: 1336-1345Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 7.Mantovani R. Nucleic Acids Res. 1998; 26: 1135-1143Crossref PubMed Scopus (442) Google Scholar, 11.Dorn A. Bollekens J. Staub A. Benoist C. Mathis D. Cell. 1987; 50: 863-872Abstract Full Text PDF PubMed Scopus (471) Google Scholar, and 23.Bi W. Wu L. Coustry F. de Crombrugghe B. Maity S.N. J. Biol. Chem. 1997; 272: 26562-26572Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). In essence, functional low affinity CCAAT boxes are rare and mostly found in proximity of high affinity ones. We classified as high affinity those NF-Y sites having optimal sequences both at the 5′ and at
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