Caenorhabditis elegans TBX-2 Directly Regulates Its Own Expression in a Negative Autoregulatory Loop.

Angenee C Milton,Peter G Okkema

doi:10.1534/g3.115.018101

Abstract

T-box genes often exhibit dynamic expression patterns, and their expression levels can be crucial for normal function. Despite the importance of these genes, there is little known about T-box gene regulation. We have focused on the Caenorhabditis elegans gene tbx-2 to understand how T-box gene expression is regulated, and here we demonstrate TBX-2 itself directly represses its own expression in a negative autoregulatory loop. tbx-2 is essential for normal pharyngeal muscle development, and a tbx-2 promoter gfp fusion (Ptbx-2::gfp) is transiently expressed in the pharynx during embryogenesis and in a small number of head neurons in larvae and adults. Reduced tbx-2 function resulted in ectopic Ptbx-2::gfp expression in the seam cells and gut in larvae and adults. Mutation of potential T-box binding sites within the tbx-2 promoter resulted in a similar pattern of ectopic Ptbx-2::gfp expression, and chromatin immunoprecipitation analyses show TBX-2 binds these sites in vivo. This pattern of ectopic Ptbx-2::gfp expression in tbx-2 mutants was very similar to that observed in mutants affecting the NF-Y complex, and our results comparing tbx-2 and nfyb-1 single- and double mutants suggest TBX-2 and NF-Y function in a single pathway to repress the tbx-2 promoter. The tbx-2 promoter is the first direct target identified for TBX-2, and we used it to ask whether SUMOylation is essential for TBX-2 repression. RNAi knockdown of SUMOylation pathway components led to ectopic Ptbx-2::gfp expression in the seam cells and gut. Ectopic Ptbx-2::gfp also was observed in the syncytial hypodermis, suggesting 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. We suggest negative autoregulation is an important mechanism that allows precise control of tbx-2 expression levels and may allow rapid changes in gene expression during development.

Highlights

T-box genes often exhibit dynamic expression patterns, and their expression levels can be crucial for normal function
We previously showed that knockdown of the NF-Y complex results in ectopic Ptbx-2::gfp expression in the gut and lateral hypodermal seam cells (Milton et al 2013)
We found that tbx-2 knockdown results in ectopic Ptbx-2::gfp expression in these tissues

Summary

Introduction

T-box genes often exhibit dynamic expression patterns, and their expression levels can be crucial for normal function. Expression of the endogenous tbx-2 gene and a tbx-2 promoter::gfp reporter is increased in tbx-2 mutants and tbx-2(RNAi) animals, and ectopic tbx-2::gfp expression was found in the gut and seam cells of the lateral hypodermis in these animals This repression was mediated by consensus T-box binding sites within the tbx-2 promoter, and TBX-2 binds these sites in vivo. We observed ectopic Ptbx-2::gfp expression in the seam cells and gut similar to that observed in tbx-2 mutants, but we observed more widespread Ptbx2::gfp expression throughout the hypodermis These results indicate that TBX-2 targets its own promoter in a negative autoregulatory loop, and suggests that TBX-2 and perhaps other factors repress tbx-2 expression in the hypodermis and gut through SUMO-dependent mechanisms

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Results

Conclusion