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

Abstract

AbstractHomeodomain-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 lackingHipk1orHipk2are 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 usedDrosophila melanogaster, which has reduced genetic redundancy, to study the functions of the four human HIPKs (hHIPKs). InD. melanogaster, zygotic loss of the single orthologdhipkresults in lethality with distinct eye and head defects. We used adhipkmutant 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 minordhipkmutant patterning phenotypes. This evidence for conserved functions of hHIPKs inD. melanogasterdirected 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 inD. 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, andHipksare no exception. Individually, each of the four vertebrateHipksare expendable for development, but together they are essential. The reasonHipksare necessary for development is unclear and comparing their developmental functions in a vertebrate model is difficult. However, the invertebrate fruit fly has a single essentialdhipkgene 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 humanHIPKs(hHIPKs). We found thathHIPK1andhHIPK2are each able to rescue the lethality caused by loss ofdhipk, whilehHIPK3andhHIPK4rescue 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 whenhHIPKsare mis-expressed. We found that expression ofhHIPKs 1-3ordhipkeach 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 thatHipksinhibit Polycomb components in normal development, though details of this interaction remain uncharacterized.