Precision engineering of DNA methylation patterns in the human genome

描述（由申请人提供）：随着最近一代在多种细胞类型和疾病状态中的多种表观遗传修饰的全基因组图谱的出现，现在迫切需要超越描述性表观基因组学。至关重要的是，需要开发功能性表观基因组学工具来故意和精确地改变特定的表观遗传修饰，以询问表观遗传标记在无数细胞类型和模型系统中的基因组调控中的作用。在本文中，我们提出了新的最先进的可编程DNA结合蛋白的产生，其将表观基因组修饰活性精确地运送到基因组中的所需靶基因座。通过将从转录激活因子样效应物（TALE）蛋白骨架工程化的序列特异性DNA结合结构域（DBD）连接至表观基因组修饰物（epiTALE），我们将实现高度精确的表观基因组工程化。我们选择在人类基因组中广泛的基因座上编程DNA甲基化（DNAme）变化，包括不同的功能染色质状态，包括开放染色质和异染色质。我们将快速构建能够识别基因组中独特的~20 bp序列的高度特异性TALE-DBD，并连接到DNA甲基转移酶3a（DNMT 3A）的催化结构域用于从头DNAme，或连接到VP 64或TET 1用于靶向DNA去甲基化。我们的具体假设是，将定制的TALE-DBD与染色质修饰剂连接将使得能够在人类基因组中的广泛的染色质状态（例如增强子和启动子）中对DNAme状态进行位点特异性调节。首先，我们建议设计一组集中的epiTALE，以靶向IMR90成纤维细胞中的不同染色质状态，包括活性/非活性增强子和启动子。将利用组合的基因组分析来全面评估epiTALE用于表观基因组工程的功效和活性，包括通过ChIP-Seq、MethylC-Seq全基因组亚硫酸氢盐测序和RNA-Seq鉴定epiTALE结合位点。其次，我们将使用诱导系统对epiTALE的表达进行时空编程，其中我们可以通过高通量靶向亚硫酸氢盐测序在靶向DNAme的空间和时间变化之后动态控制（"脉冲"和"追逐"）细胞中DNAme的掺入和擦除。第三，我们提出了这些表观基因组工程工具的临床相关细胞类型特异性应用;诱导DNAme的位点特异性调节，以纠正乳腺癌和诱导多能干细胞中发现的异常表观遗传特征。这项研究将为表观基因组学中难以捉摸的问题提供前所未有的见解，例如从给定的成核点传播DNAme和表观遗传记忆的时空动力学。我们的研究将提供创新的分子工具，以评估基因组中任何感兴趣序列的表观遗传扰动的功能结果，并纠正在患病或重编程细胞中发现的异常表观遗传特征。总的来说，该项目旨在为表观基因组工程开发新的分子工具，这将构成功能表观基因组学新兴领域的重大进展。

DESCRIPTION (provided by applicant): With the recent generation of comprehensive genome-wide maps of diverse epigenetic modifications in multiple cell types and disease states there is now a pressing need to move beyond descriptive epigenomics. Crucially, development of functional epigenomics tools to deliberately and precisely change specific epigenetic modifications is required, in order to interrogate the role of epigenetic marks in genome regulation in myriad cell types and model systems. Herein we propose the generation of novel state-of-the-art programmable DNA- binding proteins that ferry epigenome-modifying activity precisely to desired target loci in the genome. By linking a sequence specific DNA-binding domain (DBD) engineered from a Transcription Activator-Like Effector (TALE) protein backbone to an epigenome modifier (epiTALE) we will achieve highly precise epigenome engineering. We have chosen to program DNA methylation (DNAme) changes at a wide range of loci in the human genome encompassing different functional chromatin states, both open chromatin and heterochromatin. We will rapidly construct highly specific TALE-DBDs able to recognize unique ~20 bp sequences in the genome, and linked to either the catalytic domain of DNA methyltransferase 3a (DNMT3A) for de novo DNAme, or VP64 or the TET1 for targeted DNA demethylation. Our specific hypothesis is that linking custom TALE- DBDs to chromatin modifiers will enable site-specific modulation of the DNAme state in a broad range of chromatin states in the human genome, such as enhancers and promoters. First, we propose to design a focused panel of epiTALEs to target different chromatin states in IMR90 fibroblast cells, including active/inactive enhancers and promoters. Combined genomic analyses will be utilized to comprehensively assess the efficacy and activity of epiTALEs for epigenome engineering, including identification of epiTALE binding sites by ChIP-Seq, MethylC-Seq whole-genome bisulfite sequencing, and RNA-Seq. Second, we will spatio-temporally program the expression of epiTALEs using inducible systems, in which we can dynamically control ("pulse" and "chase") the incorporation and the erasure of DNAme in the cells, following the spatial and temporal changes in targeted DNAme by high-throughput targeted bisulfite sequencing. Third, we present clinically relevant cell-type specific applications of these epigenome engineering tools; inducing locus specific modulation of DNAme to correct the aberrant epigenetic signatures found in breast cancer and induced pluripotent stem cells. This study will provide unprecedented insights into elusive questions in epigenomics, such as spreading of DNAme from a given nucleation point and the spatio-temporal dynamics of epigenetic memory. Our research will provide innovative molecular tools to assess the functional outcome of epigenetic perturbation in any sequence of interest in the genome and to correct aberrant epigenetic signatures identified in diseased or reprogrammed cells. Overall, this project aims to develop novel molecular tools for epigenome engineering that will constitute a major advance in the nascent field of functional epigenomics.