Chemical-inducible Epigenome Editors for Allele-specific Gene Regulation in Developmental Disorders

用于发育障碍等位基因特异性基因调控的化学诱导表观基因组编辑器

• 批准号: 10477970
• 负责人: Sai Gourisankar
• 金额: $ 3.95万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-14 至 2023-09-13
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10477970
• 关键词: ASH2L gene; ATAC-seq; Affect; Alleles; Base Pairing; Benchmarking; Binding; Binding Sites; Biochemical; Biological; Biological Models; CRISPR/Cas technology; Cells; ChIP-seq; Chemical Engineering; Chemical Models; Chemicals; Child; Chromatin; Chromatin Remodeling Factor; Clinical Treatment; Clustered Regularly Interspaced Short Palindromic Repeats; Coffin-Siris Syndrome; Complex; DNA Binding; Data; Development; Developmental Therapeutics Program; Disease; Dominant-Negative Mutation; Dose; Event; Face; Future; Gene Activation; Gene Dosage; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Engineering; Genome; Genomics; Goals; Heterochromatin; Human Genetics; In Vitro; Intellectual functioning disability; Kinetics; Knowledge; Mammalian Cell; Measurement; Mediating; Methods; Minority; Missense Mutation; Modeling; Molecular; Molecular Biology; Mus; Mutation; Neurodevelopmental Disorder; Open Reading Frames; Pathogenesis; Patients; Precision therapeutics; Protein Overexpression; Proteins; RNA Interference; Regulation; Repression; Research; Research Personnel; Role; SMARCE1 gene; Side; Small Interfering RNA; Specificity; Speech Delay; Speech Development; Statistical Models; Synapses; System; Techniques; Technology; Testing; Time; Training; Trans-Activators; Variant; Work; antagonist; base; biophysical model; career; chemical genetics; chemical kinetics; chromatin remodeling; clinically significant; comparative; de novo mutation; design; developmental disease; dosage; embryonic stem cell; epigenome; epigenome editing; epigenomics; exome sequencing; experimental study; gene repression; genetic technology; genome-wide; heterochromatin-specific nonhistone chromosomal protein HP-1; histone methyltransferase; induced pluripotent stem cell; inhibitor; insight; interest; loss of function; mutant; nuclease; predictive modeling; protein complex; recruit; small molecule; small molecule inhibitor; stem cell model; therapeutic target; therapy development

ABSTRACT: Recent exome-sequencing data of patients revealed that de novo mutations in protein-coding regions drive almost half of all severe developmental disorders. While we may estimate haploinsufficient or dominant-negative effects from these mutations, we mostly lack precise information on the molecular chain-of- events from mutation to pathogenesis. One example is the rare neurodevelopmental disorder Coffin-Siris Syndrome (CSS), a rare neurodevelopmental disorder characterized by intellectual disability, delayed speech development, and certain facial abnormalities. Sequenced CSS patients have dominant heterozygous de novo mutations in subunits of the BAF chromatin remodeling complex: a majority putative haploinsufficient protein- truncating variants of ARID1B, and a minority putative dominant-negative missense variants of SMARCE1. Traditional molecular biology perturbation techniques to investigate mechanism, including CRISPR/dCas9, RNAi, small-molecule inhibitors, and protein overexpression, are hampered by off-target effects, limited design, and lack of temporal control. A general method to target and regulate any mutant gene, from synaptic component to chromatin remodeler, could help determine the clinical significance of mutations in many developmental disorders towards developing precision therapies. Chemical induced proximity is a strategy to use a two-sided small-molecule to co-localize two proteins of interest together in a biologically-relevant setting. To expand such a strategy to gene regulation, we developed an initial system to recruit a repressive epigenome regulator, Hp1, rapidly and reversibly to any locus with 10- fold higher locus-specificity than existing systems and precise temporal control. This proposal seeks to expand upon our hypothesis that chemical induced epigenome editing can provide highly-locus-specific, kinetic control of gene regulation for mechanistic studies of developmental disorders, using CSS-derived iPSCs as a model system. In this proposal, we will first expand the use of chemically- inducible systems by using genomics to examine the specificity of two types of activators: a transactivator (VPR) and a histone methyltransferase (Ash2l). To further provide a general strategy for future development, we will build a biophysical model clarifying the on-target activity to off-target specificity of chemical induced recruitment of epigenome editors in a genome-wide manner. We will finally apply inducible epigenome editing to study the precision and dosage effects on the genome of activating ARID1B and repressing SMARCE1Y73C, two variants implicated in CSS as haploinsufficient (ARID1B) or dominant-negative (SMARCE1Y73C), in iPSCs. At the culmination of this work, we will have not only developed a platform of epigenome editing for highly-specific gene regulation, but also have validated its use in defining molecular mechanisms in patient-relevant genetic contexts. The proposal presented also reflects my Training Goals of becoming skilled as an interdisciplinary researcher at the intersection of human genetics and chemical engineering.

摘要：最近的患者外显子组测序数据显示，蛋白质编码中存在新生突变。 几乎一半的严重发育障碍都是由地区造成的。虽然我们可以估计单倍体不足或 这些突变的显性负效应，我们大多缺乏关于分子链的精确信息 从突变到发病机制的事件。一个例子是罕见的神经发育障碍 Coffin-Siris 综合症 (CSS)，一种罕见的神经发育障碍，其特征是智力障碍、言语迟缓 发育和某些面部异常。测序 CSS 患者具有显性杂合子 de novo BAF 染色质重塑复合物亚基突变：大多数假定的单倍体不足的蛋白质 - ARID1B 的截短变体，以及 SMARCE1 的少数假定显性失活错义变体。 用于研究机制的传统分子生物学微扰技术，包括 CRISPR/dCas9、 RNAi、小分子抑制剂和蛋白质过度表达受到脱靶效应、有限设计、 以及缺乏时间控制。一种从突触靶向和调节任何突变基因的通用方法 染色质重塑剂的组成部分，可以帮助确定许多突变的临床意义 开发精准疗法的发育障碍。 化学诱导接近是一种利用两侧小分子将两种蛋白质共定位的策略。 对生物学相关的环境共同感兴趣。为了将这种策略扩展到基因调控，我们开发了 一个初始系统，用于招募抑制性表观基因组调节因子 Hp1，快速且可逆地到达任何具有 10- 比现有系统具有更高的位点特异性和精确的时间控制。 该提案旨在扩展我们的假设，即化学诱导的表观基因组编辑可以提供 高度位点特异性的基因调控动力学控制，用于发育障碍的机制研究， 使用 CSS 衍生的 iPSC 作为模型系统。在本提案中，我们将首先扩大化学物质的使用 通过使用基因组学检查两种类型激活剂的特异性来诱导系统：反式激活剂 (VPR) 和组蛋白甲基转移酶 (Ash2l)。为进一步提供未来发展的总体战略， 我们将建立一个生物物理模型，阐明化学诱导的靶向活性与脱靶特异性 以全基因组的方式招募表观基因组编辑器。我们最终将应用诱导表观基因组编辑 研究激活 ARID1B 和抑制 SMARCE1Y73C 对基因组的精度和剂量影响， iPSC 中与 CSS 相关的两个变体为单倍体不足 (ARID1B) 或显性失活 (SMARCE1Y73C)。 在这项工作的顶峰，我们不仅开发了一个表观基因组编辑平台 高度特异性的基因调控，而且还验证了其在定义分子机制中的用途 患者相关的遗传背景。提出的建议也反映了我的培训目标，即成为 作为人类遗传学和化学工程交叉领域的跨学科研究人员。