title: NF-Y recruits Ash2L to impart H3K4 trimethylation on CCAAT promoters.
abstract: 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.
authors: Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, Milano, Italy.
authors: Andrea
authors: A
authors: Fossati
authors: Diletta
authors: D
authors: Dolfini
authors: Giacomo
authors: G
authors: Donati
authors: Roberto
authors: R
authors: Mantovani
mesh_terms: D023081:CCAAT-Binding Factor / Q000235:genetics* / Q000502:physiology*; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000502:physiology*; D055785:Gene Knockdown Techniques; D006657:Histones / Q000378:metabolism*; D006801:Humans; D009687:Nuclear Proteins / Q000502:physiology*; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000502:physiology*
chemical_list: C485622:ASH2L protein, human; D023081:CCAAT-Binding Factor; D004268:DNA-Binding Proteins; D006657:Histones; D009687:Nuclear Proteins; D014157:Transcription Factors