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

烟酰胺核苷酸腺苷酸转移酶-1 (NMNAT1) 在染色质重塑和基因调控中的作用

• 批准号: 10607072
• 负责人: Yessenia Cedeno
• 金额: $ 4.77万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10607072
• 关键词: ATAC-seq; Address; Binding; CDKN1A gene; CRISPR/Cas technology; Cell Cycle Arrest; Cell Line; Cell Nucleus; Cell physiology; Cells; Chromatin; Chromatin Fiber; Chromatin Structure; Color; Data; Defect; Disease; Enzymes; Functional disorder; Gene Expression; Gene Expression Regulation; Generations; Genes; Genetic Transcription; Genome; Genomic approach; Genomics; Human Pathology; Impairment; Kinetics; Knock-out; Knowledge; Lead; Ligase; Malignant Neoplasms; Mediating; Mentorship; Metabolic; Metabolism; Microscopy; Modeling; Molecular; Molecular and Cellular Biology; Morphology; Mutation; Nicotinamide-Nucleotide Adenylyltransferase; Nuclear; Phenotype; Power Plants; Process; Production; Publishing; Regulation; Research; Role; SIRT1 gene; Scientist; Series; Structure; System; Training; Work; age related; career; chromatin remodeling; enzyme activity; experimental study; genomic locus; human disease; imaging approach; imaging system; insight; multidisciplinary; mutant; novel; particle; single molecule; transcriptome; transcriptome sequencing

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.

项目总结 染色质重塑和基因表达是细胞核内高度动态的过程。监管 这些过程对于维持正常的细胞功能至关重要。染色质重塑酶介导 染色质结构的变化和控制染色质相关因子在基因组上和基因组外的循环。 染色质的重塑依赖于代谢物(如ATP和NAD+)，而代谢物是染色质重塑的燃料 酶活性。NMNA1是一种代谢酶，主要参与NAD+在体内的局部产生 原子核。NMNA1被认为提供NAD+池，从而促进NAD+依赖的染色质重塑 酶，包括SIRT1和PARP1。然而，各种研究表明，NMNA1也可能是变构的 以不依赖于酶的方式调节这些酶的活性。尽管如此，NMNA1在 通过局部NAD+的产生及其与基因的相互作用调节染色质重塑和基因表达 染色质重塑酶仍然没有明确的定义。在这项提案中，我试图定义NMNA1在 调节染色质重塑酶的活性、染色质结构和基因表达。我假设 基因组上NMNA1的循环产生调节染色质修饰的局部NAD+梯度 酶活性，影响靶基因染色质结构和基因表达的快速变化。至 针对这一假设，我将(1)建立一个NMNA1基因敲除细胞系统来执行结构功能 在活细胞中的研究，并确定NMNA1耗尽时的表型缺陷。为了验证这个系统，我将 用野生型NMNA1和催化死亡突变体NMNA1进行救援实验。(2)研究如何 NMNA1调节染色质重塑酶(如SIRT1)靶基因的表达 染色质结构、基因表达和基因组占有率的变化，以及(3)确定NMNA1是如何 周期动力学调节染色质重构体和转录爆发。这些研究将是必不可少的 确定核代谢酶如NMNA1和代谢物产生如何调节染色质结构 以及靶基因的基因表达。这一知识将定义关于基因表达和 监管。在罗伯特·科尔曼博士和罗伯特·H·辛格博士的指导下，叶塞尼亚·塞德诺·塞德诺将 接受分子和细胞生物学方面的多学科培训，并结合最先进的专业知识 显微镜。这将使Yessenia能够成功执行拟议的研究和培训计划，并 以独立科学家的身份推进她的职业生涯。