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

摘要：最近的患者外显子组测序数据显示，蛋白质编码区的从头突变 几乎一半的严重发育障碍都是由这些区域造成的。虽然我们可以估计单倍不足或 由于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。一个例子是罕见的神经发育障碍Coffin-Siris 综合征（CSS），一种罕见的神经发育障碍，其特征是智力残疾，言语延迟 发育和某些面部畸形测序的CSS患者具有显性杂合子新发 BAF染色质重塑复合物亚基的突变：一种大多数推定的单倍不足蛋白- ARID 1B的截短变体和SMARCE 1的少数推定显性负义错义变体。 研究机制的传统分子生物学微扰技术，包括CRISPR/dCas 9， RNAi、小分子抑制剂和蛋白质过表达受到脱靶效应、有限设计 缺乏时间控制。一种靶向和调节任何突变基因的通用方法，来自synaptic 染色质重塑的组成部分，可以帮助确定许多突变的临床意义， 开发精确疗法的方向。 化学诱导邻近是一种使用双侧小分子共定位两种蛋白质的策略， 在一个生物学相关的环境中共同感兴趣。为了将这种策略扩展到基因调控，我们开发了 一个初始的系统，以招募一个阻遏性表观基因组调节因子，Hp 1，迅速和可逆的任何位点，10- 倍更高的基因座特异性比现有的系统和精确的时间控制。 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 高度基因座特异性，基因调控的动力学控制，用于发育障碍的机制研究， 使用CSS衍生的iPSC作为模型系统。在这一建议中，我们将首先扩大化学的使用- 诱导系统，通过使用基因组学来检查两种类型的激活剂的特异性： (VPR)和组蛋白甲基转移酶（Ash 2l）。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对脱靶特异性的影响。 以全基因组方式招募表观基因组编辑。我们将最终应用诱导型表观基因组编辑 为了研究对激活ARID 1B和抑制SMARCE 1 Y 73 C的基因组的精确度和剂量效应， 在iPSC中，CSS中涉及的两种变体为单倍不足（ARID 1B）或显性阴性（SMARCE 1 Y 73 C）。 在这项工作的高潮，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，但也验证了其在定义分子机制中的用途， 患者相关的遗传背景。该建议也反映了我的培训目标，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，他拥有丰富的经验。

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.