Rajendren, Jayashre (2017-12). A TRANSGENIC MODEL FOR IN VIVO GENETIC ANALYSIS: CHARACTERIZING THE TRANSIENTLY-ACTING PROMOTER SWITCH THAT CONTROLS DROSOPHILA SEX DETERMINATION. Doctoral Dissertation. Thesis uri icon

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.
  • 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.

publication date

  • December 2017