Functional characterization of human Homeodomain-interacting protein kinases (HIPKs) in Drosophila melanogaster reveal 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. Author summaryThe redundancy of vertebrate genes often makes identifying their functions difficult, and Hipks are no exception. Individually, each of the four vertebrate Hipks are expendable for development, but together they are essential. The reason Hipks are necessary for development is unclear and comparing their developmental functions in a vertebrate model is difficult. However, the invertebrate fruit fly has a single essential dhipk gene that can be effectively removed and replaced with the individual vertebrate orthologs. We used this technique in the fruit fly to compare the developmental capacity of the four human HIPKs (hHIPKs). We found that hHIPK1 and hHIPK2 are each able to rescue the lethality caused by loss of dhipk, while hHIPK3 and hHIPK4 rescue minor patterning defects, but not lethality. We then leveraged the extensive adult phenotypes associated with genetic mutants in the fruit fly to detect altered developmental pathways when hHIPKs are mis-expressed. We found that expression of hHIPKs 1-3 or dhipk each produce phenotypes that mimic loss of function of components of the Polycomb-group complex, which are needed to regulate expression of key developmental transcription factors. We therefore propose that Hipks inhibit Polycomb components in normal development, though details of this interaction remain uncharacterized.