Chromatin configuration of CCAAT-containing cell cycle promoters

The CCAAT box is a frequent promoter element bound by NF-Y, a trimer with H2A-H2B-like subunits. We developed a MNase I-based ChIP protocol on homogeneous cell populations to study cell-cycle promoters at the single nucleosome level. We analyzed histone acetylations and methylations and the association of enzymatic activities. This thesis presents different novel findings. The first finding was that H3-H4 take part of core promoters under active conditions, with the expected cohort of “positive” modifications, while H2A-H2B are removed and substituted by NF-Y. Through the use of a dominant negative mutant we show also that NF-Y is important for H3K36me3 deposition and for Pol II elongation. The second finding was that H3K4 methylations are highly dynamic and H3K4me1 is a crucial positive mark. Both functional and pharmacological inactivation led to state that KDM1 plays a positive role in transcription of G2/M genes. It requires CoREST, which is recruited on active promoters through direct interactions with NF-Y. Therefore, these preliminary data are the first in vivo indication of a crucial interplay between core histones and “deviant” histone-fold such as NF-Y, leading to fine tuning of histone methylations. The third finding was that NF-Y is not involved in histone acetyl-marks deposition as well as in histone methyl-marks: in fact, histone acetylation status of active cell cycle genes was only slightly perturbed after NF-Y removal. Moreover, this work proposed a special histone acetylation pattern typical of cell cycle gene cluster, characterized by H2BK120ac and H3K9,18,36ac deposited on H3 in repressive conditions. And finally, by in vivo analysis we showed GCN5 and PCAF involvement in the acetyl-marks deposition, and the probable NF-Y dependent recruitment of multisubunit complexes responsible of the chromatin remodelling, like STAGA and ATAC.