Worm Breeder's Gazette 17(2): 34 (April 1, 2002)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Analysis of cdh-3 upstream regulatory region to find sequences necessary for expression

Stephen T. Sewell, Guojuan Zhang, Helen M. Chamberlin

Department of Molecular Genetics, Ohio State University, Columbus, OH 43210

EGL-38 is a Pax transcription factor important for the development of several organs in C. elegans, including the hindgut. Two genes are known to act downstream of egl-38: lin-48 and cdh-3. Genetic studies indicate that lin-48 and cdh-3 are part of two separate pathways. The two genes also function differently, as LIN-48 is responsible for the specification of cell fates, while CDH-3 coordinates development and epithelial morphogenesis^1,2. Previous studies have shown that lin-48 is a direct target for EGL-38³. To investigate whether cdh-3 might also be a target of EGL-38, we have characterized its upstream regulatory sequence.

In comparison studies between C. elegans and C. briggsae, we found that there are five domains of consensus sequence within a 1000bp region upstream of the start ATG of cdh-3. We chose to focus on these areas, reasoning that these were the most likely spots for protein binding. We constructed clones including different amounts of upstream sequence driving expression of the green fluorescent protein (gfp) and injected them into animals to study their expression.

From this data (Table 1), we found that two regions are important for expression of cdh-3 in the seam cells and hindgut cells. Deletion of the region between 677 and 555 eliminated expression in the seam cells and deletion of the region between 555 and 526 eliminated expression in both seam cells and the cells of the hindgut. To confirm the function of these two regions, we generated point mutations in the reporter gene with the 824bp upstream sequence (Table 2). A clone with a mutation in the more distal site (pSS1) was not expressed in seam cells but maintained hindgut expression. In contrast, clones with a mutation in the more proximal site (pSS20) or in both sites (pSS21) were not expressed in either cell type. This indicates that the proximal element is important for expression in both cell types.

GATA-binding factors have been shown to play a role in seam cell development^4,5, and the distal region contains a block of sequence conserved between C. elegans and C. briggsae that includes a GATA sequence motif. In contrast, although EGL-38 plays a role in cdh-3 hindgut expression, the conserved sequence in the more proximal 555-526 region does not resemble the binding site for Pax proteins. Thus we suspect the relationship between egl-38 and cdh-3 is indirect.

¹Pettit et al., 1996 Development

²Chamberlin et al., 1999 Genetics

³Johnson et al., 2001 Development

⁴Page et al., 1997 Genes and Development

⁵Koh and Rothman, 2001 Development

Table 1

Table 2