All 302 neurons in C. elegans arise through asymmetric division of neuroblasts. To investigate the molecular mechanisms underlying this process, we focused on the characterization of
ham-1, a gene implicated in many asymmetric neuroblast divisions during embryogenesis.
ham-1 encodes a 414-amino acid protein that contains a winged-helix motif near the N-terminus, a domain that typically mediates DNA binding. When visualized by immunofluorescence, HAM-1 is found at the cell cortex and is localized asymmetrically in many dividing cells during embryogenesis. However, in transgenic embryos expressing a GFP::HAM-1 fusion protein, GFP fluorescence is also detected in the nucleus. When the same transgenic embryos are stained using either anti-GFP or anti- HAM-1 antibodies only localization to the cell cortex is detected. This result suggests that antibody staining is ineffective at detecting nuclear-localized HAM-1 and may explain our inability to detect endogenous HAM-1 in the nucleus. To identify regions of the protein required for localization and function we performed a deletion analysis. For these experiments, GFP was fused to the N-terminus of HAM-1 and expressed under control of the pan-neural
unc-119 promoter. We confirmed that the full-length fusion protein could rescue specific
ham-1 lineage defects. Our results indicate that the N-terminus is essential for cortical localization. In contrast, sequences near the C-terminus are required for nuclear localization; a GFP fusion to the C-terminal half of HAM-1 resided exclusively in the nucleus. We identified two nuclear localization sequences (NLSs) within the C-terminal half of HAM-1. Mutation of both NLSs eliminated nuclear localization, and significantly impaired
ham-1 function. To test if the loss of function was due to lack of HAM-1 nuclear localization, we fused an SV40 NLS to the N-terminus of GFP::HAM-1 with both NLSs mutated to re-target the protein back to the nucleus. Preliminary results show weak nuclear localization and partial rescue of
ham-1 mutant defects. To determine if the nuclear export of HAM-1 is regulated, we analyzed the localization of GFP::HAM-1 after knocking down
imb-4 (the C. elegans CRM-1 nuclear exportin homologue).
imb-4 RNAi resulted in an increase of GFP::HAM-1 in the nucleus. These results strongly suggest a role for HAM-1 in the nucleus. We propose that the asymmetric localization of HAM-1 at the cell cortex is a mechanism to distribute the protein between daughter cells to mediate a differential transcription program.