| dc.description.abstract | To be male or female, that is the question. In Drosophila melanogaster, all aspects of
somatic sex determination are under the control of the binary switch gene, Sex lethal (Sxl).
The primary determinant of the activity state of Sxl is the number of X-chromosomes. XX
embryos develop as females, while XY embryos develop as males. Sxl is stably expressed
in females via autoregulatory mRNA splicing that occurs as a consequence of a brief pulse
of transcription from establishment promoter SxlPe. Female-specific expression of SxlPe
requires a two X chromosome dose of the X-signal elements sisA, sc, upd and runt. Males
fail to express SxlPe as they carry a single dose of the X-signal elements (XSEs).
Understanding regulation of SxlPe demanded an advanced quantification tool to monitor
Sxl activity in vivo. The Sxl transgene system thus developed enables the monitoring of
endogenous Sxl activity, both as nascent transcripts and as mature mRNA. The key feature
is that intron sequences are swapped between related species to allow allele-specific
detection, by in situ hybridization, of expression from mutant and wild type transgenes
side-by-side in every nucleus of the embryo. The transgene system is fully functional and
helps exploit classical Drosophila genetics to monitor the biological effects of engineered
Sxl mutations. Using this powerful system, I have characterized the cis interactions of the
X-signal elements Sc/Da, repressor Dpn and also defined the regulatory regions of SxlPe,
to discover the means by which this sensitive promoter switch operates.
In defining the different regulatory units our results validated the previous findings of
Estes et al., using SxlPe lacZ fusions. The 3.0kb enhancer is indeed an equivalent of the
full length endogenous SxlPe enhancer and drives a robust, wild type expression. In
addition, studying the promoter deletions in context of full length Sxl transgenes
confirmed that the minimal, 400bp enhancer that drove low lacZ expression in Estes et al.,
does activate SxlPe and is both necessary and sufficient for SxlPe activation.
In studying how binding site interactions impacted SxlPe regulation, my findings revealed
that the loss of the single canonical E-box, Sc/Da 3 turned the SxlPe switch off, killing
females. This not only led to the discovery of a prominent regulatory site critical for
female-specific regulation but also challenged the previous notion that multiple Sc/Da are
responsible for proper operation of SxlPe switch. Additionally, Sc/Da 3 proved that
canonical E-box sites are important or even more so to SxlPe regulation, compared to the
non-canonical sites.
Mutating repressor Dpn binding sites triggered ectopic SxlPe expression in males and
subsequently male lethality. The non-canonical site 3 had the strongest effect on SxlPe
regulation than the canonical Dpn sites 1 and 2. The surprising finding is that Dpn binding
site mutation is not only capable of initiating SxlPe expression from the mutant bearing
transgene, but also activating the wild-type Sxl from the control transgene in trans. This
phenomenon suggests that transactivation might be a novel approach for the fly to amplify
X dose signal and ensure female specificity. | en |
