Systematic analysis of cis-acting element of a transcription factor regulating C. elegans ray assembly - tbx-2

Abstract

C. elegans male tail consists of nine lateral sensory organs called rays, embedded in a fan structure. The sensory rays go through a rapid developmental process involving multiple steps - cell fate choice, patterning, ray lineage execution, assembly and morphogenesis. From a previous genome-wide RNAi screen for genes acting in ray development, a T-box transcription factor, tbx-2, was identified to be required for the ray assembly process. Mutation of tbx-2 results in a failure of ray constituent cells to assemble into a ray, creating a ray loss phenotype. While tbx-2 mutant displays ray loss in all rays, tbx-2 is expressed in the structural cells of ray 1, 5, and 7 and ray neuronal B of ray 4 only. We are interested in understanding how such expression pattern arises, what factors are regulating tbx-2 expression pattern and ultimately, how a regulatory cascade controlling ray lineage and ray patterning work. Previous study have identified that tbx-2 directly auto-represses its expression in all rays by binding on cis-regulatory elements on its gene. However, this by itself cannot account for the initiation of the expression as well as the generation of expression pattern of the tbx-2 which is restricted to rays 1, 5, 7. Multiple transcription factors were hypothesised to account for the pattern. A systematic mapping of the cis-regulatory elements in combination of phylogenetic mapping would be employed to identify putative transcription factor binding sites (TFBS), which would allow for the identification of candidate regulatory genes that could be tested by genetic analyses. Since ray structural cell is shown to be responsible for ray assembly process (Zhang, Emmons, 1996), I focused my study on identifying the regulatory elements responsible for tbx-2 expression in the ray structural cells. A systematic deletion mapping was carried out on the transcriptional reporter of tbx-2 to identify cis-acting elements that control the expression of tbx-2. A 138bp region region located 3’ to the tbx-2 coding region was identified to be required and sufficient to drive expression in structural cells of ray 1, 5, 7. I identified 2 conserved motifs within this 138bp region that are essential for the expression. In-silico search for TFBS in the motifs identified conserved homeobox protein biding sites (HBS) in each of them with their function verified in mutant analysis. Since HBS were identified in the cis-regulatory element, tbx-2 transcriptional reporter activity was examined in knockdown worms of homeobox genes previously known to affect ray development. Among seven homeobox genes tested, two homeobox genes, unc-62 and ceh-20, were found to be required for the activation of tbx-2 transcription reporter. Knockdown of these 2 genes result in ray loss and ray 6-4 fusion. unc-62 encodes a TALE homeodomain protein known to form heterodimer with PBX (ceh-20). We therefore hypothesize that unc-62 and ceh-20 regulate tbx-2 by forming hetero-dimer. Expression pattern of unc-62 and ceh-20 in male tail was examined. Ultimately we would like to integrate the new information gained in this study into the known regulatory inputs controlling tbx-2 expression such as tbx-2 auto-regulation to elucidate the mechanism behind the generation of tbx-2 expression pattern.

Date of Award	2013
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology