The Role of Nuclear PARPs in Signal-Regulated Transcription

修改后的项目摘要/摘要部分 腺苷二磷酸核糖基化是蛋白质翻译后修饰的可逆过程，导致β-NAD+衍生的腺苷二磷酸核糖单元在多种底物上发生共价连接。它在控制细胞过程中发挥关键作用，如转录调节，这些过程驱动生理结果，如脂肪生成。ADPR化是由聚(ADP-核糖)聚合酶(PARP)家族催化的，包括核酶PARP-1。虽然PARP-1和ADPR化在历史上与DNA修复有关，但现在越来越多的证据支持它们在基因表达调控中的作用。然而，知识方面的关键差距仍然存在。例如，在信号调控的生物系统中，PARP-1介导的基因调控的分子机制尚未完全阐明。此外，位点特异性ADPR化作为组织特异性生理过程的中介体的作用还知之甚少。我们最近的研究已经开始解决这些问题。我们已经证明，PARP-1调节前脂肪细胞和巨噬细胞的转录反应，这些反应(1)与核心组蛋白的位点特异性修饰有关，(2)受信号调节转录因子(TF)控制(例如，C/EBPβ)。 这些研究的长期目标是更好地了解通过PARP-1介导的关键调控蛋白的位点特异性ADPR化来控制信号调控转录的分子、生化和基因组机制，以及这些调控事件在脂肪组织中的下游生理后果。我们的广泛假设是，PARP-1的基因调控活性是通过组蛋白和TF的位点特异性ADPR化来介导的。我们将使用生物化学、分子生物学、细胞生物学、化学生物学、蛋白质组学、基因组学和小鼠遗传学的SET工具的补充，使用综合方法来检验这一假设。此外，我们将测试与PARP-1在脂肪组织中前脂肪细胞和巨噬细胞中组蛋白和C/EBPβADPR化驱动的信号调节基因表达中的作用相关的特定机制假说。我们的具体目标是：(1)探索组蛋白ADPR化在前脂肪细胞和巨噬细胞信号调控基因表达中的作用(目标1)；(2)确定C/EBPβ的动态ADPR化如何调节巨噬细胞的增强子功能和靶基因表达(目标2)；以及(3)确定C/EBPβ的位点特异性ADPR化对脂肪形成的影响(目标3)。总之，这些研究将为PARP-1介导的位点特异性ADPR化在脂肪形成中调控基因的分子机制提供新的见解。考虑到PARP-1在人类疾病中的重要作用，以及它潜在的“可药性”，我们的研究可能会带来利用这些因素作为治疗靶点的新方法。此外，我们的研究有可能揭示新的信息，这些信息将塑造该领域的未来，并可能更广泛地阐明ADPR化的功能。

Modified Project Summary/Abstract Section ADPRylation (ADPRylation) is a reversible of post-translational modification (PTM) of proteins resulting in the covalent attachment of ADP-ribose (ADPR) units derived from β-NAD+ on a variety of substrates. It plays key roles in the control of cellular processes, such as transcriptional regulation, that drive physiological outcomes, such as adipogenesis. ADPRylation is catalyzed by the poly(ADP-ribose) polymerase (PARP) family of enzymes, including the nuclear enzyme PARP-1. Although PARP-1 and ADPRylation have historically been linked to DNA repair, growing evidence now supports their role in the regulation of gene expression. However, key gaps in knowledge remain. For example, the molecular mechanisms underlying PARP-1-mediated gene regulation have not been fully elucidated in signal-regulated biological systems. Moreover, the role of site-specific ADPRylation as a mediator of tissue-specific physiological processes is poorly understood. Our recent studies have begun to address these questions. We have shown that PARP-1 modulates transcriptional responses in preadipocytes and macrophages that are (1) linked to site-specific modification of core histones and (2) controlled by signal-regulated transcription factors (TFs) (e.g., C/EBPβ). The long-term objective of these studies is to achieve a better understanding of the molecular, biochemical, genomic mechanisms underlying the control of signal-regulated transcription by PARP-1-mediated site-specific ADPRylation of key regulatory proteins, as well as the downstream physiological consequences of these regulatory events in adipose tissue. Our broad hypothesis is that the gene regulatory activities of PARP-1 are mediated through site-specific ADPRylation of histones and TFs. We will test this hypothesis using an integrated approach with a complementary of set tools from biochemistry, molecular biology, cell biology, chemical biology, proteomics, genomics, and mouse genetics. In addition, we will test specific mechanistic hypotheses related to the role of PARP-1 in signal-regulated gene expression driven by ADPRylation of histones and C/EBPβ in preadipocytes and macrophages in fat tissue. Our specific aims are to: (1) Explore the role of site-specific histone ADPRylation in signal-regulated gene expression in preadipocytes, as well as macrophages (Aim 1); (2) Determine how dynamic ADPRylation of C/EBPβ regulates enhancer function and target gene expression in macrophages (Aim 2); and (3) Determine the effects of site-specific ADPRylation of C/EBPβ on adipogenesis (Aim 3). Collectively, these studies will provide new insights into the molecular mechanisms of gene regulation by PARP-1-mediated site-specific ADPRylation in adipogenesis. Given the important role of PARP-1 in human disease, as well its potential “drugability,” our studies could lead to new ways to exploit these factors as therapeutic targets. Furthermore, our studies have the potential to reveal new information that will shape the future of the field and may shed light more generally on the functions of ADPRylation.