Single nucleosome analysis of human cell-cycle CCAAT promoters in vivo : a positive role for KDM1

The CCAAT box is a frequent promoter element, as illustrated by bioinformatic analysis of large sets of promoters, and it is bound by NF-Y which is involved in positive, as well as negative regulation. NF-Y is responsible of the transcriptional regulation of cell-cycle genes: it is an ubiquitous histone-like trimer composed of three subunits, NF-YA, NF-YB, and NF-YC, all necessary for DNA binding. NF-YB and NF-YC contain histone fold motifs (HFM), found in all core histones, whose dimerization is a prerequisite for NF-YA association, that confers to the trimer sequence-specific DNA binding capacity. NF-Y binding is a prerequisite for promoter activation and allows further activators/co-activators buildup. Here, we used a MNase I-based ChIP protocol on homogeneous cell populations to study histone modifications on cell-cycle promoters at a single nucleosome level in vivo. Core regions of active promoters have H3-H4 and are depleted of H2A-H2B, substituted by NF-Y. A complex profile of H3K4 methylations is observed: H3K4me3 increases in active conditions, but significant levels are present on inactive promoters; H3K4me2 is relatively constant. Surprisingly, H3K4me1 best correlates with transcription. Analysis of histone demethylases suggests a positive role of KDM1(LSD1) and KDM5A in transcription, whereas KDM5B is present in repressed states. The use of the KDM1 inhibitor Tranylcipromine confirms that KDM1 is indeed involved in activation of G2/M promoters. NF-Y removal by adenovirus infection has dramatic negative effects on H3K4me3, H3K79me2, H3K36me3, H4K79me3 and leads to the appearance of H4K20me3 and recruitment of KDMs. NF-Y recruits the KDM1 partner CoREST through direct interaction. These data are the first indication that KDM1 partakes in activation of cell-cycle genes and place NF-Y at the heart of regulation of key H3K4 methylations.