Elucidating the Functional Role of Post-translational Aminoacylation in Chromatin Regulation

阐明翻译后氨酰化在染色质调节中的功能作用

• 批准号: 10489710
• 负责人: Jose Del Rio Pantoja
• 金额: $ 3.94万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10489710
• 关键词: ACTL6A gene; ACTL6B gene; ATAC-seq; ATP Hydrolysis; ATP phosphohydrolase; Actins; Advocacy; Amino Acids; Aminoacylation; Architecture; Binding; Binding Proteins; Biochemical; Biochemistry; Biological Assay; Biology; Cells; Central Nervous System; ChIP-seq; Chromatin; Chromatin Remodeling Factor; Communication; Complex; Cryoelectron Microscopy; DNA; DNA sequencing; Data; Dedications; Development; Disease; Family; Frequencies; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Generations; Genes; Genetic study; Genome; Genomics; Histone H3; Histones; Human Genome; Intellectual functioning disability; Laboratory Research; Leadership; Link; Malignant Neoplasms; Mass Spectrum Analysis; Molecular; Molecular Machines; Mutation; Neurodevelopmental Disorder; Neuronal Differentiation; Nuclear Proteins; Nucleosomes; Protein Region; Protein Subunits; Proteins; Regulation; Research; Role; SMARCA4 gene; SWI/SNF Family Complex; Scientist; Structure; Testing; Therapeutic Intervention; Tissues; Training; Transferase; Variant; chromatin remodeling; crosslink; developmental disease; differential expression; engineered stem cells; exome sequencing; functional genomics; genome-wide; human disease; induced pluripotent stem cell; insight; mutant; new therapeutic target; novel therapeutics; paralogous gene; protein complex; therapeutic target; transcriptome sequencing

PROJECT SUMMARY/ABSTRACT Recent exome sequencing efforts have implicated alterations that occur within the actin-related proteins (Arps), ACTL6A and ACTL6B, in both cancer and neurodevelopmental disorders. These highly conserved and tissue- specific protein paralogues are subunits of multi-component molecular machines that use the energy derived from ATP hydrolysis to modify chromatin architecture, and hence modulate gene expression. ACTL6A and ACTL6B are subunits of four major chromatin regulators known to remodel the architecture of chromatin: (1) the Tip60/NuA4 histone acetyl transferase, and the (2) INO80, (3) SRCAP, and (4) mammalian SWI/SNF (mSWI/SNF), also called BRG1/BRM Associated Factors (BAF) ATP-dependent chromatin remodeling complexes. The mSWI/SNF complex has been particularly implicated in a range of neurodevelopmental disorders and in over 20% of all cancers. However, despite these intriguing genetic studies as well as data suggesting that Arps are required for proper chromatin regulation, there is very little understanding regarding the biochemical and genome-wide role of Arps in chromatin remodeling activities. My proposed research plan aims to further our understanding of the molecular mechanisms of disease conferred by mutant ACTL6A and ACTL6B by identifying functional regions and the biochemical and genome-wide activities of the Arps. Aim 1 investigates the functional regions and biochemical activities contributed to chromatin remodeling complexes by ACTL6A and ACTL6B in WT conditions and mutant settings linked with neurodevelopmental disorders. More specifically, Sub- aim 1A seeks to compare the interactome of the ACTL6 subunits. Sub-aim 1B unbiasedly compares the protein interactions of WT and mutant ACTL6A/B. Sub-aim 1C focuses on mSWI/SNF complexes to assess their histone binding and enzymatic activities in WT forms and in those complexes bearing Arps with disease-relevant mutations. Aim 2 dissects the genome-wide function of the Arps by comparing a WT control to induced pluripotent stem cells (iPSCs) engineered to have disease-relevant mutations. Sub-aim 2A dissects the chromatin accessibility and gene expression profiles of WT and mutant iPSCs. And, Sub-aim 2B looks at the genome targeting of the mutant and WT ACTL6 subunits and the four major protein complexes known to bind them. Successful completion of these aims will provide new insights regarding the interplay of chromatin remodeling complexes unified by the same family of Arp proteins, define the molecular mechanisms of ACTL6A/B-binding proteins in normal and disease states, and suggest novel targeted therapeutic approaches for cancer and intellectual disabilities. The research laboratory of Dr. Cigall Kadoch is the ideal setting to conduct the disease-oriented research at the interface between biochemistry and functional genomics detailed in this proposal. Training in state-of-the-art biochemical, enzymatic, and genomic assays, as well as the professional development Dr. Kadoch provides on scientific communication, networking, leadership, and advocacy will allow to me to become a capable independent scientist.

项目概要/摘要 最近的外显子组测序工作表明肌动蛋白相关蛋白（Arps）内发生了改变， ACTL6A 和 ACTL6B，在癌症和神经发育障碍中。这些高度保守的组织- 特定的蛋白质旁系同源物是多组分分子机器的亚基，它们使用衍生的能量 通过 ATP 水解来改变染色质结构，从而调节基因表达。 ACTL6A 和 ACTL6B 是已知可重塑染色质结构的四种主要染色质调节因子的亚基：(1) Tip60/NuA4 组蛋白乙酰转移酶，以及 (2) INO80、(3) SRCAP 和 (4) 哺乳动物 SWI/SNF (mSWI/SNF)，也称为 BRG1/BRM 相关因子 (BAF) ATP 依赖性染色质重塑 复合物。 mSWI/SNF 复合体与一系列神经发育相关 疾病和超过 20% 的癌症。然而，尽管有这些有趣的基因研究和数据 表明 Arps 是正确染色质调节所必需的，但人们对 Arps 的了解知之甚少。 Arps 在染色质重塑活动中的生化和全基因组作用。我提出的研究计划的目标 进一步了解突变型 ACTL6A 和 ACTL6B 导致疾病的分子机制 通过识别 Arps 的功能区域以及生化和全基因组活动。目标 1 调查 ACTL6A 促进染色质重塑复合物的功能区域和生化活动 WT 条件和突变环境中的 ACTL6B 与神经发育障碍相关。更具体地说，子 目标 1A 旨在比较 ACTL6 亚基的相互作用组。子目标 1B 无偏见地比较蛋白质 WT 和突变体 ACTL6A/B 的相互作用。子目标 1C 重点关注 mSWI/SNF 复合物以评估其组蛋白 WT 形式以及带有与疾病相关的 Arps 的复合物中的结合和酶活性 突变。目标 2 通过比较 WT 对照和诱导的 Arps 来剖析 Arps 的全基因组功能 多能干细胞（iPSC）经过改造，具有与疾病相关的突变。子目标 2A 剖析 WT 和突变 iPSC 的染色质可及性和基因表达谱。并且，子目标 2B 着眼于 突变体和 WT ACTL6 亚基以及已知结合的四种主要蛋白质复合物的基因组靶向 他们。这些目标的成功完成将为染色质相互作用提供新的见解 由同一 Arp 蛋白家族统一的重塑复合物，定义了 正常和疾病状态下的 ACTL6A/B 结合蛋白，并提出新的靶向治疗方法 用于癌症和智力障碍。 Cigall Kadoch 博士的研究实验室是开展研究的理想场所 生物化学和功能基因组学之间的交叉领域的以疾病为导向的研究详细介绍 提议。最先进的生化、酶学和基因组分析以及专业人士的培训 卡多克博士提供的科学交流、网络、领导力和宣传方面的发展将使 让我成为一名有能力的独立科学家。