Identification of Cis- and Trans-Acting Factors Regulating the Expression of the T-box Gene Mab-9

Abstract

The T-box gene mab-9 was originally identified in a screen for worms with mating defects; it is now known that correct mab-9 expression is required for the specification of cell fate in the posterior of the worm and for correct nervous system development. Ectopic, or mis-expression of mab-9 is deleterious, hence this gene must be tightly regulated, in both a temporal and a spatial manner. In order to identify upstream regulators of mab-9 expression, we have adopted two strategies that we hope will reveal both cis- and trans-acting factors. In order to identify trans-acting regulators of mab-9 expression, we have adopted a genome-wide RNAi screen of all transcription factors in C. elegans. Using the Ahringer RNAi feeding library, we have established a high-throughput screen to search for genes which, when silenced, lead to an alteration of GFP expression in a strain of worms carrying an integrated mab-9:: GFP reporter. In an initial screen, silencing of 27% of the transcription factors represented in the library resulted in detectable phenotypes, including embryonic lethality, larval arrest and morphological defects. From observation of the progeny of worms exposed to RNAi, we have identified 14 genes which have an altered mab-9::GFP expression pattern. These candidate genes are being screened further to confirm which, if any, play a direct role in mab-9 regulation. A comparison of the C. elegans mab-9 promoter sequence with that of the closest homologue in C. briggsae was used to highlight several regions of close DNA sequence homology (termed Homology Regions 0-5) that may be important in the regulation of mab-9 expression. These sequences have each been deleted from a mab-9::GFP rescuing construct with the aim of ascertaining whether GFP expression and/or rescue of the mutant phenotype is altered. To date, one of the sequences has been assigned a role, being both necessary and sufficient for expression in posterior hypodermal cells. This has enabled a functional role for mab-9 expression to be associated with tail hypodermal cells, distinct from the fate-determining role previously described for mab-9 in male specific blast cells. The upstream factor binding to this sequence is being sought in a candidate-led RNAi screen, based on transcription factor binding site sequences within the Homology Region.