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.

项目摘要/摘要 最近的外显子组测序工作涉及肌动蛋白相关蛋白(ARP)内发生的变化， ACTL6A和ACTL6B在癌症和神经发育障碍中均有表达。这些高度保守的组织- 特定的蛋白质类似物是多组分分子机器的亚单位，它使用从 从ATP水解到改变染色质结构，从而调节基因表达。ACTL6A和 ACTL6B是已知的重塑染色质结构的四种主要染色质调节剂的亚基：(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)，又称BRG1/BRM相关因子(BAF)，依赖于ATP的染色质重塑 复合体。MSWI/SNF复合体尤其与一系列神经发育有关 在所有癌症中，有超过20%的人患有癌症。然而，尽管有这些耐人寻味的基因研究和数据 提示Arp是适当的染色质调节所必需的，关于Arp的了解很少。 Arps在染色质重塑活动中的生化和全基因组作用。我提出的研究计划旨在 为了进一步了解突变型ACTL6A和ACTL6B在疾病分子机制中的作用 通过确定ARP的功能区以及生化和全基因组活动。AIM 1调查 ACTL6A和ACTL6A参与染色质重塑复合体的功能区和生化活性 与神经发育障碍相关的WT条件和突变环境中的ACTL6B。更具体地说，Sub- 目的比较ACTL6亚基的相互作用组。子目标1B不带偏见地比较蛋白质 WT与突变体ACTL6A/B的相互作用亚目标1C主要研究mSWI/SNF复合体以评估其组蛋白 WT形式和含有与疾病相关的ARP的复合体的结合和酶活性 突变。目的2通过比较WT对照和诱导的ARP在基因组范围内的功能 多能干细胞(IPSCs)被设计成具有与疾病相关的突变。次级目标2 A剖析了 野生型和突变型ipscs的染色质可及性和基因表达谱。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体的基因组靶向 他们。这些目标的成功完成将为了解染色质的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体，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 治疗癌症和智力残疾。Cigall Kadoch博士的研究实验室是进行 在生物化学和功能基因组学的交界处的疾病导向研究在这篇文章中详述 求婚。接受最先进的生化、酶和基因组分析以及专业人员的培训 Kadoch博士提供的关于科学交流、网络、领导力和倡导的发展将允许 让我成为一名有能力的独立科学家。