| In Drosophila, activation of the gene Sex-lethal (Sxl) in the pre-cellular embryo leads to female development. At least four genes on the X-chromosome work in a dose-dependent fashion to activate Sxl. In diplo-X, but not haplo-X animals, initial Sxl activation leads to an autoregulatory feedback loop that ensures Sxl expression thereafter. The primary factors that initiate Sxl expression are the X-linked signaling elements (XSEs), which activate the Sxl establishment promoter, SxlPe. Maternally deposited factors also play a role in SxlPe activation and their functions are intertwined with those of the XSEs.;The most recent XSE identified is unpaired, which codes for the ligand of the Drosophila JAK/STAT signaling cascade. Using several null alleles and deficiencies that uncover upd, I examined the role of upd in the activation of the SxlPe. Elimination of upd causes misregulation of Sxl in a spatially restricted portion of the embryo. Removal of the maternal contributions of the Drosophila JAK, hopscotch , and STAT, Stat92E, have more severe effects than removal of upd alone. This suggests that other ligands may be present in the early embryo. We eliminated as candidates the upd -like genes, upd2 and upd3, as haploid embryos carrying deficiencies for these genes behaved identically as upd null mutants. Stat92E was shown to act at SxlPe through three consensus STAT binding sites, as mutations in these sites reduced expression of a SxlPe-lacZ reporter. Remarkably, analysis of hop and Stat92E deficient embryos reveals that removal of activated STAT92E does not affect initial Sxl activation as had been expected. Instead, transcription of Sxl initiates normally but cannot be maintained during its peak period of expression in the absence of JAK/STAT function. Preliminary evidence is presented that the XSE runt acts similarly to STAT92E being required for the maintenance of SxlPe rather than for its initial activation. My results suggest that a two-step mechanism is used to achieve full production from SxlPe. This calls into question both existing models for the activation of SxlPe and of XSE function. Specifically I suggest that XSEs function in two related, but mechanistically distinct steps, activation, and maintenance to ensure an effective sex-specific response to X-chromosome dose.;In this thesis, I describe and analyze a lethal, maternal effect interaction between genes in the 11E region of the X-chromosome and mutation in XSEs. A candidate gene, brahma associated protein 60 kDa ( bap60), from the 11E interval, was identified and a null bap60 allele, bap601, was generated. bap601 caused lethality in both sexes that could be rescued by over expression of wild-type bap60. Further genetic tests revealed that bap601 had the same maternal effect seen with the larger deficiencies. BAP60 was shown to have DNA binding activity, and to interact genetically with other members of the Brahma complex, an ATP-dependent chromatin remodeling complex. BAP60 interacts with the XSEs sisA and sc in vitro and in vivo. However, removal of maternal bap60 revealed that it had little effect on the activation of Sxl, suggesting that the BAP60-SISA or SC interaction is non-specific. |
