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.

项目概要 染色质重塑和基因表达是细胞核中高度动态的过程。的监管 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 染色质结构的变化并控制基因组内外染色质相关因子的循环。 染色质的重塑取决于代谢物（例如 ATP 和 NAD+），代谢物可促进染色质重塑 酶活性。 NMNAT1 是一种代谢酶，主要参与体内 NAD+ 的局部生成。 核。 NMNAT1 被认为提供 NAD+ 池，促进 NAD+ 依赖性染色质重塑 酶，包括 SIRT1 和 PARP1。然而，各种研究表明 NMNAT1 也可能变构 以独立于酶促的方式调节这些酶的活性。尽管如此，NMNAT1 的作用 通过局部 NAD+ 的产生和相互作用来调节染色质重塑和基因表达 染色质重塑酶的定义仍然不明确。在本提案中，我试图定义 NMNAT1 在以下方面的作用： 调节染色质重塑酶活性、染色质结构和基因表达。我假设 NMNAT1 在基因组上的循环产生调节染色质修饰的局部 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 为了解决这个假设，我将 (1) 建立一个 NMNAT1 敲除细胞系统来执行结构功能 在活细胞中进行研究并识别 NMNAT1 耗尽后的表型缺陷。为了验证这个系统，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据的变化，以及 (3) 确定 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 等核代谢酶和代谢产物的产生如何调节染色质结构 和目标基因的基因表达。这些知识将定义关于基因表达和 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. Singer)、叶塞尼娅·塞德诺·塞德诺 接受分子和细胞生物学的多学科培训以及最先进的专业知识 显微镜。这将使 Yessenia 能够成功执行拟议的研究和培训计划， 推进她作为独立科学家的职业生涯。