Molecular mechanisms regulating Poly(ADP-ribosyl)ation pathway in living cells

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This project will investigate how the DNA genome is packaged in the cellular nucleus to create alternate gene configurations that either permit or prevent retrieval and use of the genetic information. Understanding the dynamic nature of such DNA packaging may help explain how organisms successfully coordinate normal growth and development in response to ever-changing environmental signals. The project will have broad educational impacts by providing research and training opportunities for high school and undergraduate students, many of whom will be from groups traditionally underrepresented in STEM disciplines. Histone proteins in nucleosome-associated DNA can be reversibly modified by addition of poly(ADP-ribose), so-called PAR, molecules, leading to alterations in chromatin conformation and changes in gene expression. Whereas much as been learned about the action and regulation of the enzyme that adds this modification, PAR polymerase (PARP), not much is yet known about the enzyme, PAR glycohydrolase (PARG), that degrades it. Changes in poly(ADP-ribosyl)ation levels have been attributed to up- and downregulation of PARP activity, with PARG acting constitutively to cleaving PAR at a constant rate. This project challenges that idea and poses the hypothesis that regulation of PARG activity is crucial for tissue- specific and cell cycle-specific differences in poly(ADP-ribosyl)ation rates. Preliminary results suggest that transient phosphorylation of PARG domains may lead to conformational changes in the protein, in turn affecting its activity. Using Drosophila as a model, a combination of in vivo and in vitro studies will be used to detect phosphorylation of the PARG protein and to analyze its nuclear localization and activity. The results are expected to provide new understanding of the role of PARG in regulating steady-state levels of poly(ADP-ribosyl)ation and how this poorly studied histone modification controlls cell-cycle-regulated and tissue-specific gene expression.