Caenorhabditis elegans has two muscle tissues, body wall for locomotion and pharynx for feeding. CeMyoD, a C. elegans homologue of MyoD encoded by the gene,
hlh-1 is important but not essential for the body-wall muscle gene expression. One of the enhancer at first intron of
hlh-1, the 1330 sequence, is found to essential for expression of the body wall troponin C gene,
pat-10. To identify the regulatory factor of body-wall muscle genes, and resolve the regulatory mechanism of body-wall muscle gene expression, we isolated LIM-8, a binding factor to CeMyoD by yeast one-hybrid screening. This gene,
lim-8 corresponds to ORF F42G4.3 (
zyx-1) and encodes two isoforms LIM-8a and LIM-8b. LIM-8a has N-terminal proline-rich sequences, nuclear export signal-like sequences and three LIM domains in C-terminus. LIM-8b has only three LIM domains. Three LIM domains of LIM-8a have homology to vertebrate Trip6 and zyxin at 50% and 47% amino acid residues, respectively.
lim-8::gfp reporter genes were expressed in body-wall muscle, vulval muscle, intestinal muscle, sphincter muscle, and marginal cell of pharynx and excretory cells. Knock down of
lim-8(RNAi) treated worms reduced motility to 80% of the wild type. Anti-LIM-8 antiserum was generated in rabbit by injecting gel homogenate containing recombinant NusTag-fused full-length LIM-8a. Using affinity-purified antibody, we observed subcellular localization of LIM-8 in worms by immunostaining. LIM-8 was detected in cytoplasm and nucleus of body-wall muscle cells.
lim-8::gfp expressions in vulval muscle, intestinal muscle, sphincter muscle, marginal cell of pharynx and excretory cell were observed but localization of LIM-8 in these tissues could not be determined. These results suggest that LIM-8 functions in cytoplasm and nucleus in body-wall muscle cells. We also generated anti-CeMyoD antiserum to investigate the interaction between CeMyoD and LIM-8. We are determining which part of LIM-8 contributes to in vitro binding of CeMyoD by protein overlay assay. Co-localization of LIM-8 and CeMyoD will be observed by immunostaining of
lim-8::gfp worms using anti-CeMyoD antibody. Our goal is to find out the gene expression function of LIM-8 in nuclei of body-wall muscle cells.