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，蛋白质编码的从头突变 在所有严重的发育障碍中，有近一半是由地区引起的。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准确信息 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 综合征(CS)是一种罕见的神经发育障碍，其特征是智力残疾、言语迟缓 发育和某些面部畸形。已测序的CS患者具有显性杂合性新生 BAF染色质重塑复合体亚单位的突变：大多数推测为单倍体不足的蛋白- ARID1B的截断变体，以及SMARCE1的少数推测的显性-负错义变体。 传统的分子生物学微扰技术用于机制研究，包括CRISPR/dCas9， RNAi、小分子抑制剂和蛋白质过度表达受到靶外效应、有限的设计、 以及缺乏对时间的控制。针对和调节突触中任何突变基因的通用方法 组成染色质重构体，可以帮助确定许多基因突变的临床意义 开发精准疗法的发育障碍。 化学诱导邻近是一种利用双边小分子共同定位两种蛋白质的策略。 在与生物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 一种将抑制性表观基因组调节子HP1快速和可逆地招募到具有10- 折叠比现有系统更高的位点特异性和精确的时间控制。 这一建议试图扩展我们的假设，即化学诱导的表观基因组编辑可以提供 用于发育障碍机制研究的基因调控的高度位点特异性、动力学控制， 使用从css派生的ipscs作为模型系统。在这项建议中，我们将首先扩大化学- 利用基因组学检测两种激活剂：反式激活剂的特异性的诱导系统 (VPR)和组蛋白甲基转移酶(Ash21)。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，阐明化学诱导的靶上活性与靶外特异性 以全基因组的方式招募表观基因组编辑。我们将最终应用可诱导的表观基因组编辑 为了研究激活ARID1B和抑制SMARCE1Y73C对基因组的精密度和剂量效应， 在css中涉及的两个变体为单倍体不足(ARID1B)或显性负性(SMARCE1Y73C)，在ipscs中。 在这项工作的高潮，我们将不仅开发了一个表观基因组编辑平台 高度特异的基因调控，但也验证了它在定义分子机制方面的用途 与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 在人类遗传学和化学工程的交叉点上，熟练地成为跨学科的研究人员。

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.