Mahadevaraju, Sharvani (2015-08). X-Signal amplification by Runt mediated antagonism of Groucho in Drosophila. Doctoral Dissertation.
Cell fate decisions are made in response to small quantitative differences in signal molecules. However the mechanisms by which small differences in protein levels regulate all-or-nothing responses remain mysterious. Sex determination in Drosophila is an example where small, two-fold differences in X-Signaling elements lead to distinct developmental fates. The goal my study is to understand the molecular mechanism that distinguishes the two fold difference in X-signal. The Sex-lethal (Sxl) gene stands on top of the sex determination pathway of Drosophila melanogaster. When active, Sxl triggers female development by regulating downstream target genes, and when inactive, it allows male development by default. The primary sex determination signal is the number of X chromosomes. Four X-linked genes sisA, scute, upd and runt, known generally as X signal elements (XSEs), signal X chromosome dose to Sxl. In XX embryos the two-X dose of XSEs activates the female specific establishment promoter, SxlPe and the protein produced initiates an auto regulatory splicing loop that maintains Sxl expression thereafter. The single dose of XSEs in XY embryos is insufficient to activate Sxlpe, thus no Sxl protein is made and male development follows by default. We previously proposed a 'XSE-signal' amplification mechanism that hinges on the maternally supplied co-repressor Groucho (Gro), a member of the Gro/TLE1 family. In the absence of Gro, SxlPe is expressed in both the sexes in direct proportion to X chromosome dose, meaning there is no signal amplification in the absence of Gro. Antagonism of Gro function in females is a key part of or proposed signal amplification scheme, but the mechanism of damping Gro function were hypothetical. Here I propose and test the hypothesis that the XSE gene runt functions to dampen Gro dependent repression and is thus a key part of signal amplification. Antagonism between Runt and Gro would likely be mediated through Runt's WRPY motif, a tetrapeptide that mediates Runt and Gro interactions throughout the animal kingdom. Studies with a variety of transgenes with modifications in the WRPY motif revealed that, motif is required for Runt to activate Sxl supporting the hypothesis that Runt interferes with Gro repression at SxlPe. My findings also suggest that Runt is distinct from other XSEs as it is not required for initial activation of Sxl but required to maintain Sxl activity in regions of the embryo carrying active Gro. The runt protein normally binds as a heterodimer with either the Big brother (Bgb) or Brother (Bro) proteins. I created Bro and Bgb double mutants to test the hypothesis that neither protein was required for Runt to activate Sxl. Unexpectedly, I found that either Bro or Bgb is required for oogenesis thus I was unable to draw conclusions about a requirement for Bro or Bgb in sex determination. I showed, in contradiction to published studies, that the Bro protein is dispensable for viability and fertility. These findings provide significant knowledge to understand the mechanism by which small differences in protein levels regulate cell fate decisions.