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J. Biol. Chem., Vol. 280, Issue 14, 13606-13615, April 8, 2005

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Chromatin Immunoprecipitation (ChIP) on Chip Experiments Uncover a Widespread Distribution of NF-Y Binding CCAAT Sites Outside of Core Promoters^*

Anna Testa, Giacomo Donati¶, Pearlly Yan||, Francesca Romani, Tim H.-M. Huang||, M. Alessandra Viganò¶**, and Roberto Mantovani¶

From the Dipartimento di Biologia Animale, Università di Modena e Reggio, Via Campi 213/d, 41100 Modena, Italy, ^¶Dipartimento di Scienze Biomolecolari e Biotecnologie. Università di Milano, Via Celoria 26, 20143 Milano, Italy, and the ^||Division of Human Cancer Genetics, Department of Molecular Virology, Immunology, and Medical Genetics, Comprehensive Cancer Center, Ohio State University, Columbus, Ohio 43210

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.

Received for publication, December 14, 2004 , and in revised form, January 10, 2005.

^* This work was supported by Grants from Fondazione Cariplo (Cassa di Risparmio delle Province Lombarde), Associazione Italiana Ricenca sul Cancro, Fondo Italiano Ricerca di Base (FIRB), and Progetti di Rilevante Interesse Nazionale-Ministero dell Università e della Ricerca Scientifica(to R. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains three additional tables.

Recipient of a Università di Modena fellowship.

^** Recipient of a FIRB "Giovani Ricercatori" contract.

To whom correspondence should be addressed: Dipartimento di Scienze Biomolecolari e Biotecnologie, Via Celoria 26, 20133 Milano, Italy. Tel.: 39-02-50315005; Fax: 39-02-50315044; E-mail: mantor{at}unimi.it.

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