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 subunits, 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 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 the two promoters P1 and P2. Transient transfections, chromatin immunoprecipitations, and reverse transcription-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.

[1]  Kristen K. Dang,et al.  Tissue-Specific Expression Patterns of Arabidopsis NF-Y Transcription Factors Suggest Potential for Extensive Combinatorial Complexity1[W][OA] , 2008, Plant Physiology.

[2]  B. Frey,et al.  Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing , 2008, Nature Genetics.

[3]  Marcel H. Schulz,et al.  A Global View of Gene Activity and Alternative Splicing by Deep Sequencing of the Human Transcriptome , 2008, Science.

[4]  Pearlly Yan,et al.  Genome-wide analysis of alternative promoters of human genes using a custom promoter tiling array , 2008, BMC Genomics.

[5]  Michael P. Snyder,et al.  Genome-Wide Occupancy of SREBP1 and Its Partners NFY and SP1 Reveals Novel Functional Roles and Combinatorial Regulation of Distinct Classes of Genes , 2008, PLoS genetics.

[6]  Xiangyin Kong,et al.  Alternative Promoters Influence Alternative Splicing at the Genomic Level , 2008, PloS one.

[7]  Paola Bonizzoni,et al.  ASPicDB: A database resource for alternative splicing analysis , 2008, Bioinform..

[8]  Jacek Majewski,et al.  Genome-wide analysis of transcript isoform variation in humans , 2008, Nature Genetics.

[9]  D. Merico,et al.  The Histone-Like NF-Y Is a Bifunctional Transcription Factor , 2008, Molecular and Cellular Biology.

[10]  Daniele Merico,et al.  A balance between NF-Y and p53 governs the pro- and anti-apoptotic transcriptional response , 2008, Nucleic acids research.

[11]  S. Sinha,et al.  BMC Molecular Biology BioMed Central Research article Reciprocal regulation of p63 by C/EBP delta in human keratinocytes , 2007 .

[12]  Christina Chaivorapol,et al.  Systematic Identification of cis-Regulatory Sequences Active in Mouse and Human Embryonic Stem Cells , 2007, PLoS genetics.

[13]  Zhihua Li,et al.  Regulatory Circuit of Human MicroRNA Biogenesis , 2007, PLoS Comput. Biol..

[14]  Hsien-Da Huang,et al.  RegRNA: an integrated web server for identifying regulatory RNA motifs and elements , 2006, Nucleic Acids Res..

[15]  Giacomo Donati,et al.  Dynamic recruitment of transcription factors and epigenetic changes on the ER stress response gene promoters , 2006, Nucleic acids research.

[16]  R. Mantovani,et al.  Repression of New p53 Targets Revealed by ChIP on Chip Experiments , 2006, Cell cycle.

[17]  K. Nakai,et al.  Diversification of transcriptional modulation: large-scale identification and characterization of putative alternative promoters of human genes. , 2005, Genome research.

[18]  R. Natarajan,et al.  Mapping Global Histone Methylation Patterns in the Coding Regions of Human Genes , 2005, Molecular and Cellular Biology.

[19]  J. Shendure,et al.  Discovering functional transcription-factor combinations in the human cell cycle. , 2005, Genome research.

[20]  D. Lipscombe.  Neuronal proteins custom designed by alternative splicing , 2005, Current Opinion in Neurobiology.

[21]  R. Mantovani,et al.  Direct p53 Transcriptional Repression: In Vivo Analysis of CCAAT-Containing G2/M Promoters , 2005, Molecular and Cellular Biology.

[22]  Pearlly Yan,et al.  Chromatin Immunoprecipitation (ChIP) on Chip Experiments Uncover a Widespread Distribution of NF-Y Binding CCAAT Sites Outside of Core Promoters* , 2005, Journal of Biological Chemistry.

[23]  Keji Zhao,et al.  Active chromatin domains are defined by acetylation islands revealed by genome-wide mapping. , 2005, Genes & development.

[24]  Hiroshi Handa,et al.  NF-Y Is Essential for the Recruitment of RNA Polymerase II and Inducible Transcription of Several CCAAT Box-Containing Genes , 2005, Molecular and Cellular Biology.

[25]  Zengyan Xie,et al.  Asymmetric evolution of duplicate genes encoding the CCAAT-binding factor NF-Y in plant genomes. , 2004, The New phytologist.

[26]  C. Vinson,et al.  Clustering of DNA sequences in human promoters. , 2004, Genome research.

[27]  D. Landsman,et al.  Statistical analysis of over-represented words in human promoter sequences. , 2004, Nucleic acids research.

[28]  J. Deng,et al.  The B subunit of the CCAAT box binding transcription factor complex (CBF/NF-Y) is essential for early mouse development and cell proliferation. , 2003, Cancer research.

[29]  R. Sharan,et al.  Genome-wide in silico identification of transcriptional regulators controlling the cell cycle in human cells. , 2003, Genome research.

[30]  Christophe Romier,et al.  The NF-YB/NF-YC Structure Gives Insight into DNA Binding and Transcription Regulation by CCAAT Factor NF-Y* , 2003, The Journal of Biological Chemistry.

[31]  J. Taub,et al.  Synergistic regulation of human cystathionine-beta-synthase-1b promoter by transcription factors NF-YA isoforms and Sp1. , 2002, Biochimica et biophysica acta.

[32]  Stuart L. Schreiber,et al.  Active genes are tri-methylated at K4 of histone H3 , 2002, Nature.

[33]  Feifei Chen,et al.  Repression of Smad2 and Smad3 transactivating activity by association with a novel splice variant of CCAAT-binding factor C subunit. , 2002, The Biochemical journal.

[34]  D. Moras,et al.  NF-Y Recruitment of TFIID, Multiple Interactions with Histone Fold TAFIIs* , 2002, The Journal of Biological Chemistry.

[35]  R. Mantovani,et al.  The molecular biology of the CCAAT-binding factor NF-Y. , 1999, Gene.

[36]  Carol Imbriano,et al.  Dissection of the NF-Y transcriptional activation potential , 1999, Nucleic Acids Res..

[37]  R. Mantovani,et al.  NF-Y binding to twin CCAAT boxes: role of Q-rich domains and histone fold helices. , 1999, Journal of molecular biology.

[38]  R. Mantovani,et al.  A survey of 178 NF-Y binding CCAAT boxes. , 1998, Nucleic acids research.

[39]  M. Bellorini,et al.  Cloning and expression of human NF-YC. , 1997, Gene.

[40]  R. Roeder,et al.  CCAAT binding NF-Y-TBP interactions: NF-YB and NF-YC require short domains adjacent to their histone fold motifs for association with TBP basic residues. , 1997, Nucleic acids research.

[41]  S. Maity,et al.  Chromosomal assignment and tissue expression of CBF-C/NFY-C, the third subunit of the mammalian CCAAT-binding factor. , 1996, Genomics.

[42]  S. Sinha,et al.  The Transcriptional Activity of the CCAAT-binding Factor CBF Is Mediated by Two Distinct Activation Domains, One in the CBF-B Subunit and the Other in the CBF-C Subunit* , 1996, The Journal of Biological Chemistry.

[43]  S. Maity,et al.  Recombinant rat CBF-C, the third subunit of CBF/NFY, allows formation of a protein-DNA complex with CBF-A and CBF-B and with yeast HAP2 and HAP3. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[44]  C. Benoist,et al.  Intron-exon organization of the NF-Y genes. Tissue-specific splicing modifies an activation domain. , 1992, The Journal of biological chemistry.

[45]  C. Benoist,et al.  A multiplicity of CCAAT box-binding proteins , 1987, Cell.

[46]  V. Rotter,et al.  The promoters of human cell cycle genes integrate signals from two tumor suppressive pathways during cellular transformation , 2005, Molecular systems biology.

[47]  Alexander E. Kel,et al.  Large-scale collection and characterization of promoters of human and mouse genes , 2004, Silico Biol..