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.

项目摘要 染色质重塑和基因表达是核中高度动态的过程。调节 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基因组与染色质相关因子的循环。 染色质的重塑取决于代谢产物（例如ATP和NAD+），该代谢物为染色质重塑添加 酶活性。 NMNAT1是一种代谢酶，主要参与NAD+的局部产生 核。 NMNAT1被认为提供了NAD+的池，从而为NAD+依赖性染色质重塑提供 酶，包括SIRT1和PARP1。但是，各种研究表明NMNAT1也可能会变构 以酶促的方式调节这些酶活性。但是，NMNAT1在 通过局部NAD+产生以及与 染色质重塑酶的定义较差。在此提案中，我试图定义NMNAT1在 调节染色质重塑酶的活性，染色质结构和基因表达。我假设这一点 基因组上NMNAT1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。到 解决这个假设，我将（1）建立一个NMNAT1敲除细胞系统以执行结构功能 NMNAT1耗竭后，在活细胞中进行研究并鉴定表型缺陷。为了验证该系统，我将 使用野生型NMNAT1和催化死亡突变体NMNAT1进行救援实验。 （2）检查如何 NMNAT1通过定义来调节染色质重塑酶（例如SIRT1）靶基因的表达 染色质结构，基因表达和基因组占用率的变化，以及（3）建立NMNAT1如何 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NMNAT1和代谢物产生的核代谢酶如何调节染色质结构 和靶基因的基因表达。这些知识将定义有关基因表达和 规定。在博士的指导下。罗伯特·科尔曼（Robert Coleman）和罗伯特·H·辛格（Robert H. 接受分子和细胞生物学的多学科培训，并在最先进的 显微镜。这将使Yessenia能够成功执行拟议的研究和培训计划，并 促进了她作为独立科学家的职业生涯。