C. elegans Hox gene
egl-5 is involved in many cell-fate specification events in several tissues in the posterior body region. Jointly with Lisa Girard and Paul Sternberg, we have undertaken an
egl-5 promoter dissection in order to understand how the complex
egl-5 expression pattern is established. During male tail development,
egl-5 is activated in seam cell V6.ppp, which defines the fate of V6.ppp as the precursor of three sensory rays, rays 4, 5, and 6. In an
egl-5 null mutant, V6.ppp adopts the fate of its anterior counterpart V6.pap, which produces rays 2 and 3. Another Hox gene,
mab-5 , activates
egl-5 directly or indirectly. However, MAB-5 is present all through the V6 cell lineage in both hermaphrodites and males. Therefore, the temporally and spatially regulated, sex-specific expression pattern of
egl-5 in V6.ppp remains unaccounted for. To study the mechanism of regulation underlying such a precise expression pattern, we decided to identify the regulatory sequence governing
egl-5 expression in the seam. We've compared the DNA sequences of the 30kb intergenic region upstream of
egl-5 gene in C. elegans and C. briggasae by blasting the two sequences against each other, and found that there are short stretches of noncoding sequences within the
egl-5 promoter region that are well conserved between the two species. We have somewhat arbitrarily numbered the longest of these conserved sequences 1-14 from distal to proximal. To assign the function of these conserved sequences, we've made deletion constructs using a PCR-based method with one of our
egl-5::gfp reporters, EM#285, as template. Based upon the criterion that deletion of a lineage-specific enhancer will inactivate our
egl-5 reporter expression in that lineage, we've roughly mapped the enhancer of rectal epithelial cells to a region containing conserved sequences 7 and 8, and the enhancer of male tail hypodermal cells to a region containing conserved sequences 9 and 10. We've also found that conserved sequences 11 and 12, which lie 3kb upstream of the translation initiation site, are not only necessary for the expression of our reporter in the V6 lineage, but also sufficient to replicate the expression pattern of
egl-5 in the V6 lineage when linked to a D
pes-10::gfp reporter. Sequences 11 and 12 lie right next to each other within a highly conserved 300bp region, which implies that they are likely to share the function of the V6 lineage enhancer. The same region from C. briggasae gives exactly the same expression pattern driving D
pes-10::gfp , further suggesting that the conserved sequences within this region represent the enhancer of the V6 lineage. We've found putative binding sites within the region for several transcription factors including TRA-1 (thanks to M. Sohrmann of the Sanger Center), POP-1 (TCF/LEF1 homolog), MAB-5/CEH-20 (Extradenticle homolog). Now we are making deletions and point mutations to eliminate the binding sites and looking for changes in the expression pattern of the reporter. Surprisingly, when we deleted the putative TRA-1 site, we got ectopic reporter expression in anterior seam cells, ventral cord neurons and head neurons in both male and hermaphrodite. We hypothesize that we have deleted another repressor binding site within or overlapping with this TRA-1 site, and now we are trying to identify this repressor.