Chromatin immunoprecipitation (ChIP) on chip experiments uncover a widespread distribution of NF-Y binding CCAAT sites outside of core promoters

J Biol Chem. 2005 Apr 8;280(14):13606-15. doi: 10.1074/jbc.M414039200. Epub 2005 Jan 11.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • CCAAT-Binding Factor / metabolism*
  • Cell Line
  • Chromatin Immunoprecipitation / methods*
  • CpG Islands*
  • Gene Expression Regulation*
  • Humans
  • Molecular Sequence Data
  • Oligonucleotide Array Sequence Analysis / methods*
  • Promoter Regions, Genetic*
  • Reproducibility of Results
  • Transcription, Genetic


  • CCAAT-Binding Factor