HMX3 is a homeodomain protein with essential roles in CNS and ear development. Homeodomains are DNA-binding domains and hence homeodomain-containing proteins are usually assumed to be transcription factors. However, intriguingly, our recent data suggest that zebrafish Hmx3a may not require its homeodomain to function, raising the important question of what molecular interactions mediate its effects. To investigate this, we performed a yeast two-hybrid screen and identified 539 potential binding partners of mouse HMX3. Using co-immunoprecipitation, we tested whether a prioritized subset of these interactions are conserved in zebrafish and found that Tle3b, Azin1b, Prmt2, Hmgb1a, and Hmgn3 bind Hmx3a. Next, we tested whether these proteins bind the products of four distinct hmx3a mutant alleles that all lack the homeodomain. Embryos homozygous for two of these alleles develop abnormally and die, whereas zebrafish homozygous for the other two alleles are viable. We found that all four mutations abrogate binding to Prmt2 and Tle3b, whereas Azin1b binding was preserved in all cases. Interestingly, Hmgb1a and Hmgn3 had more affinity for products of the viable mutant alleles. These data shed light on how HMX3/Hmx3a might function at a molecular level and identify new targets for future study in these vital developmental processes.
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