Role of Nicotinamide Nucleotide Adenylyltransferase-1 (NMNAT1) in Chromatin Remodeling and Gene Regulation

项目摘要 染色质重塑和基因表达是细胞核中高度动态的过程。调控 这些过程对于维持适当的细胞功能至关重要。染色质重塑酶介导 染色质结构的变化，并控制染色质相关因子在基因组内外的循环。 染色质的重塑取决于代谢物（例如，ATP和NAD+），促进染色质重塑 酶活性NMNAT 1是一种代谢酶，主要参与NAD+在细胞中的局部产生。 原子核NMNAT 1被认为提供NAD+池，其促进NAD+依赖性染色质重塑 包括SIRT 1和PARP 1。然而，各种研究表明，NMNAT 1也可能是变构的， 以不依赖酶的方式调节这些酶的活性。尽管如此，NMNAT 1在 通过局部NAD+产生和与NAD+的相互作用调节染色质重塑和基因表达 染色质重塑酶的定义仍然不清楚。在这一建议中，我试图界定NMNAT的作用， 调节染色质重塑酶活性、染色质结构和基因表达。我假设 NMNAT 1在基因组上的循环产生局部NAD+梯度， 酶活性，影响染色质结构和靶基因表达的快速变化。到 针对这一假设，我将（1）建立一个NMNAT 1敲除细胞系统，以执行结构-功能 在活细胞中的研究，并确定NMNAT 1耗尽后的表型缺陷。为了验证这个系统，我将 用野生型NMNAT 1和催化死亡突变体NMNAT 1进行拯救实验。(2)研究如何 NMNAT 1调节染色质重塑酶（例如，SIRT 1）靶基因， 染色质结构，基因表达和基因组占用的变化，以及（3）建立NMNAT 1 循环动力学调节染色质重塑和转录爆发。这些研究对于 确定核代谢酶如NMNAT 1和代谢产物的产生如何调节染色质结构 和靶基因的基因表达。这些知识将定义关于基因表达的新范式， 调控在罗伯特科尔曼和罗伯特H.歌手Yessenia Cedeno 接受分子和细胞生物学的多学科培训，并结合最先进的专业知识 显微镜这将使Yessenia能够成功地执行拟议的研究和培训计划， 作为一名独立的科学家推进她的职业生涯。

PROJECT SUMMARY Chromatin remodeling and gene expression are highly dynamic processes in the nucleus. The regulation of these processes is crucial to maintain proper cellular functions. Chromatin remodeling enzymes mediate changes in chromatin structure and control the cycling of chromatin-associated factors on and off the genome. The remodeling of chromatin depends on metabolites (e.g., ATP and NAD+), which fuels chromatin remodeling enzymes activity. NMNAT1 is a metabolic enzyme primarily involved in the local production of NAD+ in the nucleus. NMNAT1 is thought to provide pools of NAD+ which fuels NAD+-dependent chromatin remodeling enzymes, including SIRT1 and PARP1. However, various studies suggest that NMNAT1 may also allosterically regulate these enzymes activity in an enzymatic independent manner. Nonetheless, the role of NMNAT1 in regulating chromatin remodeling and gene expression through local NAD+ production and interactions with chromatin remodeling enzymes remains poorly defined. In this proposal, I seek to define the role of NMNAT1 in regulating chromatin remodeling enzymes activity, chromatin structure, and gene expression. I hypothesize that the cycling of NMNAT1 on the genome produces local NAD+ gradients that regulate chromatin-modifying enzyme activity, influencing rapid changes in chromatin structure and gene expression of target genes. To address this hypothesis, I will (1) establish an NMNAT1 knock-out cellular system to perform structure-function studies in live cells and identify phenotypical defects upon depletion of NMNAT1. To validate this system, I will perform rescue experiments with wild-type NMNAT1 and catalytic dead mutant NMNAT1. (2) Examine how NMNAT1 regulates the expression of chromatin remodeling enzymes (e.g., SIRT1) target genes by defining changes in chromatin structure, gene expression and genomic occupancy, and (3) establish how NMNAT1 cycling dynamics regulate chromatin remodelers and transcriptional bursting. These studies will be essential to determine how nuclear metabolic enzymes like NMNAT1 and metabolite production regulate chromatin structure and gene expression of target genes. This knowledge will define new paradigms regarding gene expression and regulation. Under the mentorship of Drs. Robert Coleman and Robert H. Singer, Yessenia Cedeno Cedeno will receive multi-disciplinary training in molecular and cellular biology combined with expertise in state-of-the-art microscopy. This will allow Yessenia to successfully execute the proposed research and training plans and advance her professional career as an independent scientist.