Affiliation: Zuckerman Mind Brain Behavior Institute, Columbia University, New York, NY
Keywords: y. posttranslational modifications; c. homeotics
Hox proteins are transcription factors best known for patterning segment identity along the anterior-posterior axis in early embryogenesis. As monomers, Hox proteins exhibit little difference in DNA-binding specificity, however; upon binding their common cofactor Extradenticle (Exd) each Hox paralog gains the DNA-binding specificity necessary to enact segment-specific gene regulation. Hox-Exd binding is facilitated by a conserved motif in Hox that, when bound to an Exd binding pocket, positions a variable “linker” domain in close proximity to the DNA minor groove. The composition of each Hox’s linker domain contributes to paralog-specific Hox-Exd DNA-binding preferences. Recently published work and our own data have found that several Hox proteins can be phosphorylated in their linker domain, leading us to hypothesize that phosphorylation in this domain could influence Hox-Exd DNA-binding preferences. To test this hypothesis, we used genome expansion technology to express and purify Hox proteins with site-specific phosphorylation, and performed the in vitro DNA-binding assay SELEX-seq on phosphorylated and non-phosphorylated Hox-Exd complexes. We observed that for two Hox proteins, phosphorylation induced differences in DNA-binding preferences. Interestingly, phosphorylation of one of these Hox’s promoted sequences that were reduced by phosphorylation for the other, indicating that phosphorylation may act as a mechanism to further differentiate Hox paralogs. To investigate the in vivo function of Hox linker domain phosphorylation, we used genome-engineering to introduce an alanine substitution at one of the predicted Hox phosphorylation sites. Using asymmetric morphogenesis as a model Hox-regulated gene network, we identified one enhancer that, in a transgenic reporter system, exhibited increased expression in a phospho-mutant genetic background. Within that enhancer we identified one conserved Hox-Exd binding site predicted to be more strongly bound by phosphorylated Hox-Exd by our SELEX-seq data. Mutating this site in the context of our transgenic reporter also induced an increase in expression, suggesting that phosphorylation of the linker domain can directly alter gene expression through a phospho-sensitive binding site. These data highlight the capability of post-translational modifications to alter Hox-Exd DNA-binding preferences and that these changes are significant enough to affect the transcriptional activity of Hox-regulated enhancers.