Worm Breeder's Gazette 16(5): 34 (February 1, 2001)
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.
California Institute of Technology, Division of Biology 156-29, Pasadena, Ca 91125
The Hox genes encode a conserved set of transcription factors that are essential for anterior/posterior patterning in many animals and are expressed in a series of consecutive domains along this axis. These genes are almost always expressed in cells that are related by position rather than lineage and mutations in these genes often result in the transformation of one region to an adjacent one. Hox genes are typically found in clusters, with the order of the genes in the cluster colinear with their order of expression along the anterior/posterior axis, except for in C. elegans in which the order of two of the genes is inverted. In C. elegans, the Hox cluster contains a homolog of the anterior group genes, ceh-13, which is most similar to labial in the Drosophila HOM-C complex; two genes homologous to medial-group genes; lin-39 (sex-combs reduced, deformed, proboscipedia); mab-5 (Antennapedia) and a homolog of the posterior group, egl-5 (Abdominal-B)(1). Additionally, two more posterior homologs not contained within the quasi-cluster, nob-1 and php-3, have been characterized (2). The expression of Hox genes in positionally-related groups of cells raises interesting questions at the cis-regulatory level. Functional analysis of Hox gene promoters will help us understand how positional cues are read and interpreted resulting in correct anterior/posterior addressing along the body-length. C. elegans is ideal for such an analysis because fate-specification changes consequential to small perturbations in these promoters can be observed in individual cells, rather than the fields of cells as in many other organisms. To understand how cell-fate is specified in the posterior region of the hermaphrodite by the egl-5 Hox gene, we have undertaken, in a joint project with Yingqi Teng and Scott Emmons, a promoter analysis of this gene. egl-5 is expressed in the hermaphrodite specific neuron (HSN), body wall muscle, posterior lateral microtubule neuron, PVC interneuron, M, V6, the rectal epithelial cells K, F, B, U and the P12 neuroectoblast cell (3). The experiments described here focus on our progress delineating cis-regulatory elements which direct expression in K, F, B, U and P12. egl-5 and its adjacent Hox gene mab-5 are divergently transcribed with approximately 30 kb between them. A gfp translational fusion containing approximately 13 kb of promoter was sufficient to direct expression in K, F, B, U, P12 while a second construct, containing the most proximal 7 kb of promoter was not, suggesting that the 6 kb differential fragment between the two may be sufficient to direct expression in these cell-types. We tested this 6 kb region fused to a heterologous pes-10 basal promoter driving gfp, and found that indeed it was sufficient. We then examined a series of egl-5 promoter truncations, with this 6 kb piece as a starting point and identified a 1.3 kb region within the 6 kb that is sufficient for expression. Within this 1.3 kb fragment we identified a 469 bp sub-fragment sufficient to drive gfp expression in B, but not K, F, U, P12 and a 446 bp piece sufficient to drive expression in K, but not F, B, U or P12. F, U and P12 elements have proven more difficult to define. The 1.3 kb region sufficient to drive gfp expression in K, F, B, U, and P12 contains six sites (denoted here in distal to proximal order as 1-6) in the range of 20-30 bp each which are conserved between C. elegans and the closely related species C. briggsae. There are several notable consensus sites for trans-acting factors within these conserved regions including a binding site for SOX5 and FORKHEAD in site 3. The six sites are arranged in two quasi-clusters, with approximately 300 bp between the clusters. Specific deletions of either 1, 2 and 3 or 4, 5 and 6 abolishes expression in K, F, B, U and P12 suggesting that sequences in both clusters may be important for expression in these regions. One caveat of these types of experiments is that these deletions could alter spacing in the promoter, disturbing enhancer-promoter interactions. However, constructs which specifically deleted 3, 4 and 5, removing a similar number of base pairs as the 1,2,3 and 4,5,6 constructs, and found that that expression in K, F, B, U and P12 was not affected, suggesting that the spacing in this region is not affected by deletions of this magnitude. These conserved regions are being further studied to assess their relative contributions to egl-5 expression in the rectal epithelial cells and P12. The study of the cis-regulation of egl-5 will reveal the natural taxonomy of differential anterior/posterior addressing within C. elegans. 1. Salser, S.J. and Kenyon, C. (1994) Trends in Genetics 10, 159-164 2. Van Auken, K., Weaver, D.C., Edgar, L.G. and Wood, W.B. (2000) Proc. Natl. Acad. Sci. 97(9), 4499-4503 3. Ferreira, H.B., Zhang, Y., Zhao, C. and Emmons, S.W. (1999) Dev. Biol. 207, 215-228