The NF-Y complex negatively regulates Caenorhabditis elegans tbx-2 expression

T-box genes are frequently expressed in dynamic patterns during animal development, but the mechanisms controlling expression of these genes are not well understood. The Caenorhabditis elegans T-box gene tbx-2 is essential for development of the ABa-derived pharyngeal muscles, specification of neural cell fate in the HSN/PHB lineage, and adaptation in olfactory neurons. The tbx-2 expression pattern is complex, and expression has been described in pharyngeal precursors and body wall muscles during embryogenesis, and amphid sensory neurons and pharyngeal neurons in adults. To examine mechanisms regulating tbx-2 gene expression, we performed an RNAi screen of transcription factor genes in strains containing a Ptbx-2::gfp reporter and identified the Nuclear Factor Y (NF-Y) complex as a negative regulator of tbx-2 expression. NF-Y is a heterotrimeric CCAAT-binding complex consisting of A-C subunits, and reduction of the NF-Y subunits nfya-1, nfyb-1, or nfyc-1 by RNAi or using mutants results in ectopic Ptbx-2::gfp expression in hypodermal seam cells and gut. Mutation of two CCAAT-boxes in the tbx-2 promoter results in a similar pattern of ectopic Ptbx-2::gfp expression, suggesting NF-Y directly represses the tbx-2 promoter. tbx-2 mRNA is moderately increased in nfya-1 null mutants, indicating NF-Y represses expression of endogenous tbx-2. Finally we identify and characterize a second-site mutation that enhances lethality of a temperature sensitive tbx-2 mutant and show that this mutation is a deletion in the nfyb-1 gene. Together, these results identify NF-Y as an important regulator of tbx-2 function in vivo.