NF-YC complexity is generated by dual promoters and alternative splicing

The CCAAT box is a DNA element present in the majority of human promoters, bound by the trimeric NF-Y, composed of NF-YA, NF-YB and NF-YC subunits. We describe and characterize novel isoforms of one of the two histone-like subunit, NF-YC. The locus generates a minimum of four splicing products, mainly located within the Q-rich activation domain. The abundance of each isoform is cell dependent: only one major NF-YC isoform is present in a given cell type. The the 37 and 50 kDa isoforms are mutually exclusive and preferential pairings with NF-YA isoforms possess different transcriptional activities, with specific combinations being more active on selected promoters. The transcriptional regulation of the NF-YC locus is also complex, and mRNAs arise from two promoters: P1 and P2. Transient transfections, ChIPs and RT-PCRs indicate that P1 has a robust, housekeeping activity; P2 possesses a lower basal activity, but it is induced in response to DNA damage, in a p53 dependent way. Alternative promoter usage directly affects NF-YC splicing, with the 50 kDa transcript being excluded from P2. Specific functional inactivation of the 37 kDa isoform affects the basal levels of G1/S blocking and pro-apoptotic genes, but not G2/M promoters. In summary, our data highlight an unexpected degree of complexity and regulation of the NF-YC gene, demonstrating the existence of a discrete cohort of NF-Y trimer subtypes resulting from the functional diversification of Q-rich transactivating subunits and a specific role of the 37 kDa isoform in suppression of the DNA-damage response under growing conditions