Functional characterization of human Homeodomain-interacting protein kinases (HIPKs) inDrosophila melanogasterreveal both conserved functions and differential induction of HOX gene expression

Abstract

Homeodomain-interacting protein kinases (Hipks) are a family of conserved proteins that are necessary for development in both invertebrate and vertebrate organisms. Vertebrates have four paralogues, Hipks 1-4. Mice lacking Hipk1 or Hipk2 are viable, however loss of both is lethal during early embryonic development, with embryos exhibiting homeotic skeletal transformations and incorrect HOX gene expression. While these results suggest Hipks have a role in regulating HOX genes, a regulatory mechanism has not been characterized, and further comparisons of the roles of Hipks in development has not progressed. One challenge with characterizing developmental regulators in vertebrates is the extensive redundancy of genes. For this reason, we used Drosophila melanogaster , which has reduced genetic redundancy, to study the functions of the four human HIPKs (hHIPKs). In D. melanogaster , zygotic loss of the single ortholog dhipk results in lethality with distinct eye and head defects. We used a dhipk mutant background to compare the ability of each hHIPK protein to rescue the phenotypes caused by the loss of dHipk. In these humanized flies, both hHIPK1 and hHIPK2 rescued lethality, while hHIPK3 and hHIPK4 only rescued minor dhipk mutant patterning phenotypes. This evidence for conserved functions of hHIPKs in D. melanogaster directed our efforts to identify and compare the developmental potential of hHIPKs by expressing them in well-defined tissue domains and monitoring changes in phenotypes. We observed unique patterns of homeotic transformations in flies expressing hHIPK1, hHIPK2, or hHIPK3 caused by ectopic induction of Hox proteins. These results were indicative of inhibited Polycomb-group complex (PcG) components, suggesting that hHIPKs play a role in regulating its activity. Furthermore, knockdown of PcG components phenocopied hHIPK and dHipk expression phenotypes. Together, this data shows that hHIPKs function in D. melanogaster, where they appear to have variable ability to inhibit PcG, which may reflect their roles in development.