Victoria Meller - US grants

For species which use divergent chromosomes (such as X and Y) to determine sex, equalization of sex chromosome gene expression is the most immediate and vital aspect of sexual differentiation. This is termed dosage compensation, and in Drosophila it is achieved by a two- fold up regulation of X-linked genes in the male. The large, non-coding roX1 and roX2 RNAs (RNA on the X) share features suggesting a role in this process, the most striking of which is that both transcripts bind along the length of the male X chromosome. The long term objective of this project is to understand the mechanism by which a chromosome is selectively compensated. Proposed experiments focus on the role of roX RNAs. Initially the genetic interactions between roX1 and roX2, and between these genes and the male-specific lethals (msls), which are required for compensation, will be determined (Specific Aim I). The roX genes are positively regulated by the msls, revealing a previously unknown aspect of the msls function. Regulation of the roX genes will be further explored in Specific Aim II. The products of the msls bind to the male X chromosome as a ribonucleoprotein complex. In Specific Aim III the RNA component of the msls complex, which may include known roX RNAs, will be characterized. Specific Aims III and IV initiate a search for new members of the roX gene family. This information is vital to understanding the function of these RNAs. In contrast to Drosophila, compensation in humans involves inactivation of one of the female X chromosomes. However, both organisms appear to use similar mechanisms, such as histone acetylation, to modify transcription. Additionally, both species selectively coat the modified chromosome with RNA. The large Xist transcript binds to the length of the inactivated X in mammalian females, and is essential for the process of inactivation. These similarities suggest that understanding the mechanism of dosage compensation in Drosophila has relevance to other systems of global transcriptional regulation.

Drosophila melanogaster has highly differentiated sex chromosomes. A similar ratio of X-linked to autosomal gene products must be maintained in XX females and XY males. This is accomplished by an RNA and protein complex that coats the male X chromosome and modulates expression. The roX1 and roX2 (RNA on the X 1 and -2) transcripts ensure X-localization of the associated Male Specific Lethal (MSL) proteins. Elimination of both roX transcripts reduces X-linked gene expression in males. Surprisingly, expression of the entire 4th chromosome is also reduced. Elimination of MLE, one of the MSL proteins, also reduces expression of the male 4th chromosome but has no effect in females. MSL2, another member of the MSL complex, is not necessary for 4th chromosome expression in S2 cells. The notion that the 4th chromosome may be derived from an ancestral X chromosome makes these observations particularly exciting. This research will explore the hypothesis that elements of the X chromosome dosage compensation machinery also regulate the 4th chromosome of D. melanogaster, and constitute a second chromosome-wide epigenetic system in flies. This project will establish the genetic basis of 4th chromosome regulation.

This research will enhance understanding of chromosome targeting and modification systems. It addresses a novel aspect of sex chromosome evolution: how X chromatin becomes autosomal. This project will involve training of graduate and undergraduate students at Wayne State University, which has an exceptionally diverse student population that includes underserved urban and immigrant populations.

DESCRIPTION (provided by applicant): Eukaryotic organisms employ a suite of epigenetic regulatory systems that are essential for genome stability and normal gene expression. Disruptions in epigenetic systems are associated with a wide range of pathologies, including chromosome instability, birth defects, cancer and developmental abnormalities. An intriguing type of epigenetic regulation, X chromosome dosage compensation, adjusts the expression of X-linked genes in one sex to accommodate the different numbers of X chromosomes in males and females. Dosage compensation has been intensively studied in mice, C. elegans and Drosophila. The striking differences in the dosage compensation strategies of these organisms are usually emphasized. However, all three systems rely on chromatin regulatory complexes that are selectively recruited to the X chromosome to modulate expression. These complexes, and their actions on chromatin, have been studied in great detail. But in no case do we understand how X chromatin is identified with the requisite selectivity. Our long-term goal is to understand how an entire chromosome is identified and regulated. The specific hypothesis we will test is that X chromosome recognition in Drosophila involves small RNAs. Our studies reveal that bi-directionally transcribed satellite sequences that are near-exclusive to the X chromosome respond to a potent genetic modifier of compensation. We postulate that the genetic modifier exerts its effect through siRNA. The proposed experiments will determine the role of small RNA pathways and X-linked satellites in dosage compensation. Three specific aims will be pursued to determine if 1) RNAi normally influences X chromatin and dosage compensation, 2) if X-linked satellites are sources and/or targets of RNAi, and 3) if X- linked satellites contribute to the dosage compensation of genes situated nearby. We propose that RNAi acts prior to, or in parallel with previously identified elements, which are modestly enriched on the X chromosome, to ensure exclusive recognition of X chromatin.

Abstract Eukaryotic organisms employ epigenetic regulatory systems that are essential for genome stability and gene expression. Disruptions in these systems are associated with a wide range of pathologies, including chromosome instability, birth defects, cancer and developmental abnormalities. An intriguing type of epigenetic regulation, X chromosome dosage compensation, adjusts the expression of X-linked genes in one sex to accommodate the different numbers of X chromosomes in males and females. Dosage compensation has been intensively studied in mice, C. elegans and Drosophila. The striking differences in the compensation strategies of these organisms are usually emphasized. However, all three systems rely on regulatory complexes that are selectively recruited to X chromatin to modulate expression. These complexes, and their actions on chromatin, have been studied in detail. But in no case do we understand how X chromatin is identified with the requisite selectivity. Our long-term goal is to understand how an entire chromosome is identified and regulated. We have found that the siRNA pathway, and siRNAs from a repetitive element that is near-exclusive to the X, promote recognition of X chromatin. Strikingly, one of these elements, when placed on an autosome, recruits compensation to flanking autosomal genes. These elements are candidate matrix attachment regions and participate in long range interactions. The specific hypothesis we will test is that X chromosome recognition in Drosophila is guided by a chromosome-specific architecture, determined by repetitive elements and modulated by the siRNA pathway. This arrangement facilitates spreading of compensation along the chromosome. We propose that this acts cooperatively with well-studied sites that recruit the dosage compensation complex directly. The proposed experiments will 1) establish the role of the siRNA in modification of chromatin at the X-linked repeats, 2) determine the dependence of matrix attachment and long range interactions on these modifications and 3) test the idea that an X-linked locus containing a key member of the dosage compensation complex, as well as X-specific repeats, acts to coordinate epigenetic modification during compensation.