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

基本信息

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
项目类别：
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

项目摘要

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：患者的最新外显子序数据显示，蛋白质编码中的从头突变地区几乎驱动了所有严重发育障碍的一半。虽然我们可以估计单倍弹性或这些突变中的显性阴性作用，我们主要缺乏有关分子链的精确信息从突变到发病机理的事件。一个例子是罕见的神经发育障碍棺材丝综合征（CSS），一种以智力障碍为特征的罕见神经发育障碍发展和某些面部异常。测序的CSS患者具有主要的杂合DE。 BAF染色质重塑络合物亚基的突变：大多数假定的单倍蛋白蛋白质 - ARID1B的截断变体，以及Smarce1的少数假定的主导性错义变体。传统的分子生物学扰动技术，以研究包括CRISPR/DCAS9在内的机制 RNAi，小分子抑制剂和蛋白质过表达受到脱靶效应，有限设计的阻碍，缺乏时间控制。从突触中靶向和调节任何突变基因的一般方法染色质重塑的成分可以帮助确定许多突变的临床意义发展精确疗法的发育障碍。化学诱导的接近度是一种使用两侧小分子共定位的策略在与生物学相关的环境中共同兴趣。为了将这种策略扩展到基因调节，我们开发了一种最初的系统，用于募集抑制性基因组调节剂HP1，迅速和可逆地到达具有10-的任何基因座折叠比现有系统更高的基因座特异性和精确的时间控制。该建议旨在扩大我们的假设，即化学诱导的表观基因组编辑可以提供基因调节的高分位特异性动力学控制发育障碍的机理研究，使用CSS来源的IPSC作为模型系统。在此提案中，我们将首先扩展化学上的使用通过使用基因组学检查两种类型的激活剂的特异性：反式激活剂（VPR）和组蛋白甲基转移酶（ASH2L）。为了进一步为未来发展提供一般策略，我们将构建一个生物物理模型，阐明靶向活性，以促进化学诱导的靶向特异性以全基因组的方式招募表观基因组编辑。我们最终将应用可诱导的表观基因组编辑研究对激活ARID1B和抑制Smarce1Y73C的基因组的精度和剂量影响，在IPSC中，有两个与CSS相关的CSS的变体（ARID1B）或显性阴性（SMARCE1Y73C）。以这项工作的高潮，我们不仅将开发出表观基因组编辑平台高度特异性的基因调节，但也验证了其在定义分子机制中的使用与患者有关的遗传环境。提出的提案也反映了我成为我的培训目标在人类遗传学与化学工程的交集中熟练地担任跨学科研究人员。