Using transgenic Drosophila melanogaster to investigate the functions of human HIPKs reveals their shared and unique roles in development

Homeodomain-interacting protein kinases (HIPKs) are a family of four conserved proteins essential for vertebrate development. However, functional redundancy between the four vertebrate HIPK paralogues has made it difficult to compare their respective functions. Understanding functions of these essential proteins could benefit the fields of biology and medicine, therefore I addressed the gap in knowledge of the four vertebrate HIPK paralogues by studying them in the fruit fly Drosophila melanogaster, where reduced genetic redundancy simplifies our functional assessment. The single Hipk present in the fly allowed for rescue experiments to be performed with human HIPK transgenes that provide new insight into their individual functions not easily assessed in vertebrate models. Furthermore, the abundance of genetic tools available to Drosophila researchers to monitor developmental pathways allowed for unique functional comparisons. I first performed rescue experiments to demonstrate the extent to which each of the human HIPKs can functionally replace Drosophila Hipk for survival and morphological development. I then demonstrated the extent that each human HIPK could modulate developmental pathways, proliferation, and apoptosis, each of which have previously been described for Hipks, but never all together in comparable tissues. Finally, I characterized the severe developmental phenotypes induced by human HIPKs and discovered that they were caused by induction of key developmental regulatory genes, called Hox genes. I found that the resulting homeotic transformation phenotypes were likely associated with the dysregulation of the Trithorax or Polycomb complexes, which act to open and close DNA, respectively, to control the accessibility of tightly regulated genes during development. Investigating this connection found that overexpressing Hipks can sequester components from the Trithorax and Polycomb complexes on polytene chromosomes. However, when staining for endogenous dHipk on chromosomes, dHipk only colocalized with Trithorax complex components. Further experiments expressing dHipk in flies with loss-of-function mutations of Trithorax complex genes show a significant reduction in the phenotypes observed with strong dHipk overexpression. Together, these experiments provide the first direct comparison of all four vertebrate HIPKs, demonstrating their similar and unique activities in developmental pathways, and suggests a potential mechanism for the tumorigenic phenotypes that have been previously described when dHipk is overexpressed.