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

该项目将研究DNA基因组如何包装在细胞核中，以创建允许或阻止检索和使用遗传信息的替代基因配置。了解这种DNA包装的动态性质可能有助于解释生物体如何成功地协调正常的生长和发育，以应对不断变化的环境信号。该项目将通过为高中生和本科生提供研究和培训机会，产生广泛的教育影响，他们中的许多人将来自传统上在STEM学科中代表性较低的群体。核小体相关DNA中的组蛋白可以通过添加所谓的PAR分子来可逆地修饰，导致染色质构象的改变和基因表达的变化。虽然人们对添加这种修饰的酶-PAR聚合酶(PARP)的作用和调节了解得很多，但对降解它的酶-PAR糖水解酶(PARG)却知之甚少。多聚(ADP-核糖基)水平的变化被归因于PARP活性的上调和下调，PARG以恒定的速度对PAR起结构性作用。该项目挑战了这一观点，并提出了一种假设，即PARG活性的调节对于组织特异性和细胞周期特异性的聚(ADP-核糖)转化率的差异至关重要。初步结果表明，PARG结构域的瞬时磷酸化可能导致蛋白质的构象变化，进而影响其活性。以果蝇为模型，将采用体内和体外相结合的研究方法，检测PARG蛋白的磷酸化，并分析其核定位和活性。这些结果有望为PARG在调节多(ADP-核糖基)稳定水平中的作用提供新的理解，以及这种研究较少的组蛋白修饰如何控制细胞周期调节和组织特异性基因表达。

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.