The CCAAT box promoter element and NF-Y, the transcription factor (TF) that binds to it, were among the first cis-elements and trans-acting factors identified; their interplay is required for transcriptional activation of a sizeable number of eukaryotic genes. NF-Y consists of three evolutionarily conserved subunits: a dimer of NF-YB and NF-YC which closely resembles a histone, and the “innovative” NF-YA. In this review, we will provide an update on the functional and biological features that make NF-Y a fundamental link between chromatin and transcription. The last 25 years have witnessed a spectacular increase in our knowledge of how genes are regulated: from the identification of cis-acting sequences in promoters and enhancers, and the biochemical characterization of the corresponding TFs, to the merging of chromatin studies with the investigation of enzymatic machines that regulate epigenetic states. Originally identified and studied in yeast and mammals, NF-Y – also termed CBF and CP1 – is composed of three subunits, NF-YA, NF-YB and NF-YC. The complex recognizes the CCAAT pentanucleotide and specific flanking nucleotides with high specificity (Dorn et al., 1997; <!--RIDCIT0108--> 108. Hatamochi A, Golumbek PT, Van Schaftingen E, de Crombrugghe B. 1988. A CCAAT DNA binding factor consisting of two different components that are both required for DNA binding. J Biol Chem 263:5940–5947. View all references ; <!--RIDCIT0114--> 114. Hooft van Huijsduijnen RA, Bollekens J, Dorn A, Benoist C, Mathis D. 1987. Properties of a CCAAT box-binding protein. Nucleic Acids Res 15:7265–7282. View all references ; <!--RIDCIT0142--> 142. Kim CG, Sheffery M. 1990. Physical characterization of the purified CCAAT transcription factor, alpha-CP1. J Biol Chem 265:13362–13369. View all references ). A compelling set of bioinformatics studies clarified that the NF-Y preferred binding site is one of the most frequent promoter elements ( <!--RIDCIT0234--> 234. Suzuki Y, Tsunoda T, Sese J, Taira H, Mizushima-Sugano J, Hata H, Ota T, Isogai T, Tanaka T, Nakamura Y, Suyama A, Sakaki Y, Morishita S, Okubo K, Sugano S. 2001. Identification and characterization of the potential promoter regions of 1031 kinds of human genes. Genome Res 11:677–684. View all references <!--RIDCIT0235--> 235. Suzuki Y, Yamashita R, Shirota M, Sakakibara Y, Chiba J, Mizushima-Sugano J, Nakai K, Sugano S. 2004. Sequence comparison of human and mouse genes reveals a homologous block structure in the promoter regions. Genome Res 14:1711–1718. View all references ; <!--RIDCIT0063--> 63. Elkon R, Linhart C, Sharan R, Shamir R, Shiloh Y. 2003. Genome-wide in silico identification of transcriptional regulators controlling the cell cycle in human cells. Genome Res 13:773–780. [CrossRef], [PubMed], [Web of Science ®] View all references ; <!--RIDCIT0177--> 177. Mariño-Ramírez L, Spouge JL, Kanga GC, Landsman D. 2004. Statistical analysis of over-represented words in human promoter sequences. Nucleic Acids Res 32:949–958. View all references ; <!--RIDCIT0073--> 73. FitzGerald PC, Shlyakhtenko A, Mir AA, Vinson C. 2004. Clustering of DNA sequences in human promoters. Genome Res 14:1562–1574. [CrossRef], [PubMed], [Web of Science ®] View all references ; <!--RIDCIT0164--> 164. Linhart C, Elkon R, Shiloh Y, Shamir R. 2005. Deciphering transcriptional regulatory elements that encode specific cell cycle phasing by comparative genomics analysis. Cell Cycle 4:1788–1797. [Taylor & Francis Online], [PubMed], [Web of Science ®] View all references ; <!--RIDCIT0287--> 287. Zhu J, Zhang Y, Joe GJ, Pompetti R, Emerson SG. 2005. NF-Ya activates multiple hematopoietic stem cell (HSC) regulatory genes and promotes HSC self-renewal. Proc Natl Acad Sci USA 102:11728–11733. View all references ; <!--RIDCIT0154--> 154. Lee J, Li Z, Brower-Sinning R, John B. 2007. Regulatory circuit of human microRNA biogenesis. PLoS Comput Biol 3:e67. [CrossRef] View all references ; <!--RIDCIT0001--> 1. Abnizova I, Walter K, Te Boekhorst R, Elgar G, Gilks WR. 2007. Statistical information characterization of conserved non-coding elements in vertebrates. J Bioinform Comput Biol 5:533–547. View all references ; <!--RIDCIT0096--> 96. Grskovic M, Chaivorapol C, Gaspar-Maia A, Li H, Ramalho-Santos M. 2007. Systematic identification of cis-regulatory sequences active in mouse and human embryonic stem cells. PLoS Genet 3:e145. [CrossRef] View all references ; <!--RIDCIT0105--> 105. Halperin Y, Linhart C, Ulitsky I, Shamir R. 2009. Allegro: Analyzing expression and sequence in concert to discover regulatory programs. Nucleic Acids Res 37:1566–1579. View all references ; <!--RIDCIT0104--> 104. Häkkinen A, Healy S, Jacobs HT, Ribeiro AS. 2011. Genome wide study of NF-Y type CCAAT boxes in unidirectional and bidirectional promoters in human and mouse. J Theor Biol 281:74–83. View all references ). The same consensus, as determined by mutagenesis and SELEX studies ( <!--RIDCIT0013--> 13. Bi W, Wu L, Coustry F, de Crombrugghe B, Maity SN. 1997. DNA binding specificity of the CCAAT-binding factor CBF/NF-Y. J Biol Chem 272:26562–26572. View all references ), was also retrieved in ChIP-on-chip analysis ( <!--RIDCIT0240--> 240. Testa A, Donati G, Yan P, Romani F, Huang TH, Viganò MA, Mantovani R. 2005. Chromatin immunoprecipitation (ChIP) on chip experiments uncover a widespread distribution of NF-Y binding CCAAT sites outside of core promoters. J Biol Chem 280:13606–13615. View all references ; <!--RIDCIT0034--> 34. Ceribelli M, Alcalay M, Viganò MA, Mantovani R. 2006. Repression of new p53 targets revealed by ChIP on chip experiments. Cell Cycle 5:1102–1110. [Taylor & Francis Online], [PubMed], [Web of Science ®] View all references ; <!--RIDCIT0036--> 36. Ceribelli M, Dolfini D, Merico D, Gatta R, Viganò AM, Pavesi G, Mantovani R. 2008. The histone-like NF-Y is a bifunctional transcription factor. Mol Cell Biol 28:2047–2058. View all references ; <!--RIDCIT0210--> 210. Reed BD, Charos AE, Szekely AM, Weissman SM, Snyder M. 2008. Genome-wide occupancy of SREBP1 and its partners NFY and SP1 reveals novel functional roles and combinatorial regulation of distinct classes of genes. PLoS Genet 4:e1000133. [CrossRef], [PubMed], [Web of Science ®] View all references ). Additional structural features of the CCAAT box – position, orientation, presence of multiple Transcriptional Start Sites – were previously reviewed ( <!--RIDCIT0055--> 55. Dolfini D, Zambelli F, Pavesi G, Mantovani R. 2009. A perspective of promoter architecture from the CCAAT box. Cell Cycle 8:4127–4137. [Taylor & Francis Online] View all references ) and will not be considered in detail here.

The CCAAT box is an important promoter element regulated by NF-Y, a conserved trimer with histone-like features. We describe a new Position Specific Frequency Matrix (PSFM): we derived from 328 NF-...

Regulated gene expression is essential for a proper progression through the cell cycle. The transcription factor NF-Y has a fundamental function in transcriptional regulation of cell cycle genes, particularly of G2/M genes. In order to investigate common and distinct functions of NF-Y subunits in cell cycle regulation, NF-YA, NF-YB and NF-YC have been silenced by shRNAs in HCT116 cells. NF-YA loss led to a delay in S-phase progression, DNA damage and apoptosis: we showed the activation of the replication checkpoint, through the recruitment of Δp53 and of the replication proteins PCNA and Mcm7 to chromatin. Differently, NF-YB depletion impaired cells from exiting G2/M, but did not interfere with S-phase progression. Gene expression analysis of NF-YA and NF-YB inactivated cells highlighted a common set of hit genes, as well as a plethora of uncommon genes, unveiling a different effect of NF-Y subunits loss on NF-Y binding to its target genes. Chromatin extracts and ChIP analysis showed that NF-YA depletion was more effective than NF-YB in hitting NF-Y recruitment to CCAAT-promoters. Our data suggest a critical role of NF-Y expression, highlighting that the lack of the single subunits are differently perceived by the cells, which activate diverse cell cycle blocks and signaling pathways.

NF-Y is a trimeric transcription factor containing H2A/H2B-like subunits, which specifically binds to the CCAAT box, a common eukaryotic promoter element. To gain insights into NF-Y-dependent transcriptional regulation, we assessed its relationships with positive histone marks by chromatin immunoprecipitation-on-chip and correlative-profiling studies. Unbiased identification of binding sites shows that the majority of genes are bound by NF-Y in the promoter and/or within the coding region. Parallel analysis of H3K9-14ac and H3K4me3 sites indicates that NF-Y loci can be divided in two distinct clusters: (i) a large cohort contains H3K9-14ac and H3K4me3 marks and correlates with expression and (ii) a sizeable group is devoid of these marks and is found on transcriptionally silent genes. Within this class, we find that NF-Y binding is associated with negative histone marks, such as H4K20me3 and H3K27me3. NF-Y removal by a dominant negative NF-YA leads to a decrease in the transcription of expressed genes associated with H3K4me3 and H3K9-14ac, while increasing the levels of many inactive genes. These data indicate that NF-Y is embedded in positive as well as in negative methyl histone marks, serving a dual function in transcriptional regulation, as an activator or as a repressor.

NF-Y is a trimeric transcription factor (TF), binding the CCAAT box element, for which several results suggest a pioneering role in activation of transcription. In this work, we integrated 380 ENCODE ChIP-Seq experiments for 154 TFs and cofactors with sequence analysis, protein–protein interactions and RNA profiling data, in order to identify genome-wide regulatory modules resulting from the co-association of NF-Y with other TFs. We identified three main degrees of co-association with NF-Y for sequence-specific TFs. In the most relevant one, we found TFs having a significant overlap with NF-Y in their DNA binding loci, some with a precise spacing of binding sites with respect to the CCAAT box, others (FOS, Sp1/2, RFX5, IRF3, PBX3) mostly lacking their canonical binding site and bound to arrays of well spaced CCAAT boxes. As expected, NF-Y binding also correlates with RNA Pol II General TFs and with subunits of complexes involved in the control of H3K4 methylations. Co-association patterns are confirmed by protein–protein interactions, and correspond to specific functional categorizations and expression level changes of target genes following NF-Y inactivation. These data define genome-wide rules for the organization of NF-Y-centered regulatory modules, supporting a model of distinct categorization and synergy with well defined sets of TFs.