| dc.description.abstract | Cell fate decisions in eukaryotic organisms can be altered in response to small dose changes of transcription factors. Drosophila melanogaster sex determination is such an example. Sex-lethal (Sxl) is the master regulatory gene of fly sex determination. The goal of my work is to understand the molecular mechanism of how Sxl reads and responds to the X chromosome signal. Early establishment Sxl promoter, SxlPe is activated responding to two dose XSEs, but a single XSE dose does not activate SxlPe. Previous efforts to understand sex specific expression of SxlPe relied on Sxl transgenes. Although we learned a lot from the transgene experiments, our knowledge from transgenes was limited because genetic background of the transgene is different from the endogenous Sxl environment. To overcome this limitation, I engineered endogenous Sxl mutant lines by CRISPR/Cas9. The new endogenous Sxl mutants allowed precise quantification of SxlPe expression without the genetic background issue.
Negative regulators such as zygotic deadpan (dpn) and maternal groucho (gro) are the critical element in fly sex determination by establishing X chromosome signal threshold. Analyzing the effect of repressor binding sites showed that all the repressor sites were important for sex specific expression of SxlPe. Mutant repressor sites caused ectopic expression of SxlPe in male embryos. I observed that the non-canonical repressor site, which was expected to be less efficient for repressor Dpn binding, induced strong ectopic SxlPe expression in male embryos. To provide full constitutive activity of SxlPe, I mutated all the three repressor sites. As expected, the mutant allele induced strong ectopic expression of SxlPe. Interestingly, the strong constitutive allele was perfectly countered by loss of sisB.
To assess the direct contribution of transcription activators in sex specific expression of SxlPe, new Sxl alleles with mutant activator binding sites were created. Genetic testing and analysis of the SxlPe expression pattern showed that all the transcription activator binding sites were important. Surprisingly, a non-canonical SisB/Da activator site had a predominating effect in SxlPe expression, suggesting that the activator site may interact with nearby activator sites.
Recently, I inserted epitope tag Llama to N-terminus of endogenous Sxl. The Llama-Sxl allele was ectopically expressed in male embryos which could be attributed to the presence of two SisB/Da activator sites in the tag. Removing these two activator sites eliminated ectopic Sxl expression in male embryo, suggesting that the current balance between transcription activator and repressor binding sites is an evolutionary prerequisite for sex specific expression of SxlPe.
My work showed that all transcription factor sites in the 400bp proximal enhancer are important for sex specific expression of SxlPe, but the contribution of each transcription factor binding site varies in context dependent manner. Future work will require identification of sisA and runt binding sites and characterization of all the transcription factor binding sites. | |
