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

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

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BackgroundDifferent 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.

MethodsWe 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 equi jec 7 MLL1 is devoid of the methyltransferase domain and fused to the AF4 gene.ResultsKnock 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 e. cuarenta cerveza 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.ConclusionsThree 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.