Recent analyses of the genomes of various higher eukaryotic animals such as mice and humans have shown that an unexpectedly large fraction of transcripts do not encode proteins but rather specify functional RNAs. A growing body of evidence suggests that these non-coding transcripts are involved in a variety of regulatory mechanisms including chromatin organization, and that disruption of these processes can have drastic consequences for the organism. This project focuses on the role of the roX RNAs in dosage compensation in fruit flies, where males have a single X chromosome and females have two X chromosomes, similar to the XX and XY complement present in humans and other mammals. In flies, dosage compensation entails up-regulating the expression of hundreds of unrelated genes present on the X chromosome in order to equalize the quantities of the gene products between the sexes. Thus, it is an excellent model system to understand several aspects of eukaryotic gene regulation by non-coding RNA. The central player in dosage compensation is the MSL (Male Specific Lethal) complex, which includes the non-coding roX1 and roX2 RNAs and acts exclusively on the male X chromosome. A key development in the field was the discovery that the action of the MSL complex on the X chromosme is mediated by histone modification (specifically, acetylation of lysine 16 in histone H4). The goal of the present research is to characterize the molecular mechanism through which the conserved functional domains of the roX RNAs regulate the enzymatic activity of the MSL complex on the male X chromosome to achieve two-fold up-regulation of X-linked genes. By revealing the relationship between the structure and function of the roX RNAs, the project will define new aspects of RNA-protein interactions and their relevance in the context of chromatin remodeling complexes. Importantly, X-chromosome inactivation in mammals is also dependent on a complex that includes a non-coding RNA known as Xist. Although the Xist complex induces gene silencing on the X chromosome, in contrast to the up-regulation promoted by the MSL complex in fruit flies, a shared feature is the initiation of chromatin remodeling accompanied by spreading in cis from a non-coding RNA binding site. Beyond the involvement of epigenetic changes induced by global histone modification, the roles of non-coding RNAs in these processes are not well understood. Thus, the information obtained from studies of roX RNA will lead to enhanced understanding of X-chromosome inactivation in mammals and has the potential to advance general understanding of non-coding RNA-dependent gene regulation in eukaryotes.
Broader impacts: Participants in a minority summer student research program for high school and undergraduate students will be included in the research activities in order to teach them principles and techniques of basic science. To disseminate information about RNA analysis tools including RNA structure prediction, alignmentsand searches, an internet-based facility for public users (http://bioinformatics.njit.edu/rna) will be continuously updated with non-coding RNA (roX) data generated through the project. To ensure open scientific communication, all data, protocols, constructs and transgenic flies developed through the project will be shared with other laboratories and institutions. Members of the research team will participate in multi- and interdisciplinary conferences and present research results obtained from the project. A long-term goal is for the contributions of the research to the field of chromatin organization to be integrated into basic education resources such as textbooks for graduate and undergraduate students.
