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Date of Award
Victoria H. Meller
In humans and fruit flies, males have one X chromosome while females have two. This imbalance in gene dosage is potentially lethal, and the process of dosage compensation corrects it. The MSL (Male Specific Lethal) complex, which is composed of five proteins and one of two functionally redundant long non-coding roX (RNA on the X) RNAs, brings about dosage compensation in Drosophila melanogaster. In fruit fly dosage compensation, all the genes on the single male X chromosome are upregulated approximately twofold, via chromatin modifications, to equalize gene dosage with the two X chromosomes of females. This process calls for highly selective identification of the X chromosome in the nucleus. The current model for X-recognition does not explain how specific binding to the X-chromatin is achieved. It is known however, that the X chromosome assumes a distinct three-dimensional conformation in the male nucleus, and that the siRNA pathway aids in X-recognition.
We hypothesized that repetitive sequences found exclusively on the X chromosome, called the 1.688X repeats, could be a possible source of siRNAs that play a role in specifying X identity, and thus promoting recognition by the MSL complex. To test this hypothesis, I designed a functional test that detects compensation of autosomal chromatin. This required generation of lethal autosomal deficiencies. These were made by recombining parts of the 2nd and Y chromosomes. These translocations can be separated meiotically to generate a duplication and a deficiency.
I then generated a transgene carrying roX1 and 1.6883F (one of the repeat clusters), and integrated it on an autosome. The transgene was designed such that the role of each element could be tested individually. I discovered that all transgenes, including 1.6883F only, recruited the MSL complex to the autosome and increased the expression of nearby genes. When placed opposite the lethal deficiency, the transgenes partially rescued male flies to the pharate stage. Additionally, all transgenes led to a dramatic increase in recovery of male third instar larvae, which was further enhanced by expression of siRNA from 1.6883F. My findings strongly suggest that the 1.688X repeats act in cis to facilitate X-identification and dosage compensation. They also suggest that 1.688X repeats on the X are the targets of the siRNA pathway that contributes to X-recognition.
The close proximity and sequence similarity of the repeat clusters 1.6883F and 1.6884A prompted us to ask if they share a redundant function in generation of the siRNA that promotes X chromosome recognition. We discovered that flies deleted for both repeat clusters did not show an apparent defect in male survival. Thus the specialized role of 1.6883F, and whether it is unique from other 1.688X repeats, remains ambiguous.
Joshi, Sonal Suresh, "A Novel Role For Repetitive Sequences In Recognition Of The Drosophila Melanogaster X Chromosome" (2016). Wayne State University Dissertations. 1547.