NF-Y recruits Ash2L to impart H3K4 trimethylation on CCAAT promoters

PLoS One. 2011 Mar 21;6(3):e17220. doi: 10.1371/journal.pone.0017220.


Background: Different histone post-translational modifications (PTMs) are crucial in the regulation of chromatin, including methylations of H3 at Lysine 4 by the MLL complex. A relevant issue is how this is causally correlated to the binding of specific transcription factors (TFs) in regulatory regions. NF-Y is a TF that regulates 30% of mammalian promoters containing the widespread CCAAT element. We and others established that the presence of H3K4me3 is dependent upon the binding of NF-Y. Here, we investigate the mechanisms of H3K4me3 deposition by NF-Y.

Methods: We employed Chromatin Immunoprecipitation in cells in which Ash2L and NF-Y subunits were knocked down by RNAi, to monitor the presence of histones PTMs and components of the MLL complex. We performed gene expression profiling of Ash2L-knocked down cells and analyzed the regulated genes. We performed ChIPs in leukemic cells in which MLL1 is devoid of the methyltransferase domain and fused to the AF4 gene.

Results: Knock down of the Ash2L subunit of MLL leads to a decrease in global H3K4me3 with a concomitant increase in H3K79me2. Knock down of NF-Y subunits prevents promoter association of Ash2L, but not MLL1, nor WDR5, and H3K4me3 drops dramatically. Endogenous NF-Y and Ash2L specifically interact in vivo. Analysis of the promoters of Ash2L regulated genes, identified by transcriptional profiling, suggests that a handful TF binding sites are moderately enriched, among which the CCAAT box. Finally, leukemic cells carrying the MLL-AF4 translocation show a decrease of H3K4me3, absence of Ash2L and increase in H3K79me2, while NF-Y binding was not significantly affected.

Conclusions: Three types of conclusions are reached: (i) H3K4 methylation is not absolutely required for NF-Y promoter association. (ii) NF-Y acts upstream of H3K4me3 deposition by recruiting Ash2L. (iii) There is a general cross-talk between H3K4me3 and H3K79me2 which is independent from the presence of MLL oncogenic fusions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CCAAT-Binding Factor / genetics*
  • CCAAT-Binding Factor / physiology*
  • Cell Line
  • Chromatin Immunoprecipitation
  • DNA Methylation*
  • DNA-Binding Proteins / physiology*
  • Gene Knockdown Techniques
  • Histones / metabolism*
  • Humans
  • Nuclear Proteins / physiology*
  • Promoter Regions, Genetic*
  • Reverse Transcriptase Polymerase Chain Reaction
  • Transcription Factors / physiology*


  • ASH2L protein, human
  • CCAAT-Binding Factor
  • DNA-Binding Proteins
  • Histones
  • Nuclear Proteins
  • Transcription Factors