PARP1 (poly-ADP-ribose polymerase) is a protein important for repairing damaged DNA in multicellular animals. PARP1 is involved in many biological processes that monitor DNA integrity, such as cell division, differentiation and cell death. The DNA in every eukaryotic cell is found in the form of a highly compact DNA-protein structure known as chromatin, that is unpacked every time when access to a particular DNA region is needed, and re-packaged immediately after this process, to ensure the fidelity of the process. PARP1 is known to mediate these chromatin rearrangements during the initiation of gene regulation. Several recent studies show that PARP1 also has another, unexpected role in the different process of RNA splicing, i.e., the mechanism that results in the correct formation of the messenger RNAs that direct protein synthesis. This research will provide better understanding of how PARP1 modulates alternative splicing to effect changes in gene regulation. In addition to making the findings widely available to other researchers via established repositories and publication/ presentation of study outcomes, the project will impact the next-generation workforce by engaging graduate, undergraduate, and high school students, particularly students from groups underrepresented in the sciences, in laboratory research in the emerging field of chromatin biology and gene regulation. Participating students will be trained in bioinformatics, large-scale genome analyses, and chromatin biology and provided guidance on science careers. High school students will complete small-scale laboratory projects and write their first computer code for data analyses. During the course of the project, five-minute movie clips on gene regulation will be created and incorporated into an existing biochemistry course.
Strong evidence supports the regulatory role of chromatin in alternative splicing, an integral part of cell differentiation and development, contributing to cell lineage and tissue identity. It is known that the binding of trans-factors (enhancers or silencers) to pre-mRNA-encoded sequence elements is necessary but not sufficient for splicing regulation. The recent discovery in this laboratory that PARP1, a chromatin architectural protein involved in the regulation of transcription initiation, not only bound at transcription start sites of active genes but also bound at internal exons, irrespective of the transcriptional state, suggests different modes of gene regulation by PARP1. Additionally, recent findings showed that depletion of PARP1 or its PARylation activity resulted in specific changes in alternative splicing events, and that in vivo PARP1 bound chromatin, nascent mRNA, and splicing factors. This research will test the hypothesis that PARP1 regulates co-transcriptional splicing in two non-mutually exclusive ways: acting as an adapter molecule to recruit splicing factors to exons and/or to modulate changes to chromatin structure in ways that affect polymerase elongation and kinetics. Large-scale genomics and gene-specific approaches will be utilized to map in fine detail the genetic and biochemical interactions of PARP-1-RNA-chromatin that occur in a cell. Since the same proteins are present in all organisms throughout the animal kingdom, PARP1 likely regulates alternative decisions similarly in simple and complex organisms. Therefore, this problem will be studied in a simple organism that is readily amenable to genetic and molecular analysis, the Drosophila system. Building on the laboratory's history of student training especially minority students, this project will involve local high schools, undergraduates, and graduate students. Additionally, minority students from these schools will be sought for research opportunities in the laboratory. These individuals will be trained in bioinformatics, large-scale genome analyses and chromatin biology. Results from this project will be disseminated to the broader scientific community in the form of presentations at local, national and international meetings. Finally, the results of these studies will be published in scientific journals and presented at scientific meetings.
