Access Type

Open Access Dissertation

Date of Award

January 2014

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Biological Sciences

First Advisor

Victoria H. Meller

Abstract

Over 30% of Drosophila genome is assembled into heterochromatin. Heterochromatin is relatively gene poor, transcriptionally less active and remains condensed during interphase. Previous studies established that roX RNA and some of the Male Specific Lethal (MSL) proteins, all components of the dosage compensation complex, are required for full expression of autosomal heterochromatic genes in male flies but not in females. This was surprising since heterochromatin is generally not thought to be sexually dimorphic. The genetic basis for the regulation of sex-specific heterochromatin was completely unknown.

To determine if roX RNAs localize directly at the heterochromatic regions that they regulate, I generated an MS2-tagged roX1 allele (roX1MS2-6) using a novel gene engineering technique named `Targeted Gene Conversion' (TGC). roX1MS2-6 was used to visualize in vivo roX1 localization in early Drosophila embryos, but subnuclear localization was only detectable on the X chromosome of males after the onset of dosage compensation (3hr AEL).

I then performed genetic screens to determine the signal that dictates differentiation of male and female heterochromatin. I hypothesized that either the sex determination pathway, or direct karyotype sensing, could act as a signal. To determine the signal, I conducted targeted genetic screens using a reporter that responds differently to the loss of roX RNAs in males and females. I found that heterochromatic sex is independent of the female-specific components of the somatic sex determination pathway, as well as the male-limited Y-chromosome and MSL2, a dosage compensation protein that is only present in males. I then explored the possibility that direct sensing of sex chromosome karyotype bypasses the somatic sex determination pathway to determine heterochromatic sex. Examination of various chromatin regulators with known functions in homolog pairing identified Topoisomerase II (Top2) as an essential factor for feminization of XX heterochromatin. Intriguingly, Top2 also binds to a large block of satellite repeats present exclusively on the X chromosome (359bp repeats). I then discovered that deletion of X heterochromatin, which removes one copy of these satellite repeats, masculinizes heterochromatin in XX flies. Simultaneous loss of Top2 and deletion of X heterochromatin enhances masculinization of XX heterochromatin, but has no effect on somatic sexual differentiation. I postulate that the X-exclusive 359 bp heterochromatic satellite repeats and Top2 act together as a mechanism of direct karyotype sensing. This in turn regulates heterochromatin differentiation independent of all known sex determination pathways. My studies thus reveal a novel sex determination signal in Drosophila melanogaster that links fly karyotype to one aspect of sexual differentiation.

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