C. elegans tbx-2 Gene Expression is Required by Repressive and Activating Mechanisms
Milton, Angenee Cary
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T-box proteins are an ancient and highly conserved family of transcription factors that are essential for the development of all multicellular organisms. Here, we are interested in the genetic regulation and function of the C. elegans T-box gene tbx-2, which is essential for development of the ABa-derived pharyngeal muscles, specification of neural cell fate in the HSN/PHB lineage, adaptation in olfactory neurons, and viability during embryonic and post-embryonic development. To identify mechanisms that transcriptionally regulate tbx-2, we utilized an RNAi-based screen to identify factors regulating tbx-2 expression and identified the Nuclear Factor Y (NF-Y) complex and TBX-2 itself as negative regulators of a tbx-2 transcriptional reporter, Ptbx-2::gfp. We found these factors to be a stage- and tissue-specific repressors of tbx-2. Disruption of NF-Y by RNAi or mutant backgrounds causes ectopic Ptbx-2::gfp expression in the seam cells and intestine of larvae and adults. Mutagenesis of NF-Y binding sites in the tbx-2 promoter indicates repression is direct. Likewise, endogenous tbx-2 mRNA is upregulated in NF-Y mutants. We found a spontaneous mutant nfyb-1(cu13) enhances the lethality of the tbx-2(bx59) hypomorphic mutant, and produces phenotypes distinct from individual mutants, indicating TBX-2 and NF-Y may converge on essential downstream pathways. We conclude NF-Y repression is crucial for normal tbx-2 function in vivo. The second regulatory mechanism we identified involves TBX-2 itself. Using tbx-2(RNAi) and tbx-2 mutants, we found TBX-2 also represses tbx-2::gfp expression in seam cells and intestine of larvae and adults. Additionally, there is strong upregulation in neurons where Ptbx-2::gfp in normally expressed. Endogenous tbx-2 mRNA is significantly upregulated in tbx-2 mutants. Mutations of T-box binding elements in the promoter recapitulate ectopic expression observed in tbx-2 mutants and RNAi, and ChIP analysis strongly suggests these elements are bound by TBX-2 in vivo. TBX-2 function is hypothesized to depend on posttranslational modification by SUMOylation and we find inhibition of SUMOylation similarly leads to ectopic of Ptbx-2::gfp in seam cells and intestine, but we also observed more widespread Ptbx-2::gfp expression throughout hypodermis. This suggests that either the tbx-2 promoter is repressed by other SUMOylation dependent mechanisms, or that decreased SUMOylation leads to stable changes in seam cell nuclei as they fuse with the syncytial hypodermis. Lastly, tbx-2 is expressed in pharyngeal precursors of early embryo where it is required for the specification of ABa-derived pharyngeal muscles. From the tbx-2 promoter, we isolated a pharyngeal enhancer necessary and sufficient for pharyngeal expression. From this enhancer, we identified putative transcription factor binding sites commonly recognized by E2F and Forkhead family proteins and demonstrated these sites are functionally important for enhancer activity, as individual mutations profoundly reduce reporter expression. This work, and current studies, suggests the Forkhead site strongly correlates with the stage and tissue specific binding profile of PHA-4/FoxA in the pharynx. Although the putative E2F site is required for enhancer activity, factors that target this site have not been identified and we suggest it is targeted through a novel pathway.
nuclear factor y
C. elegans pharynx