Precision engineering of DNA methylation patterns in the human genome

描述（由申请人提供）：随着最近一代的多种细胞类型和疾病状态的各种表观遗传修饰的全面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，以故意和精确地改变特定的表观遗传修饰，以便在无数细胞类型和模型系统中询问表观遗传标记在基因组调控中的作用。在此，我们提出了新一代的最先进的可编程DNA结合蛋白，将表观基因组修饰活性精确地传递到基因组中所需的目标位点。通过将转录激活子样效应子（TALE）蛋白骨架工程的序列特异性dna结合域（DBD）与表观基因组修饰子（epiTALE）连接，我们将实现高度精确的表观基因组工程。我们选择对人类基因组中包含不同功能染色质状态（包括开放染色质和异染色质）的广泛位点的DNA甲基化（DNAme）变化进行编程。我们将快速构建高度特异性的tale - dbd，能够识别基因组中独特的~ 20bp序列，并连接到DNA甲基转移酶3a （DNMT3A）的催化结构域（用于从头DNAme）或VP64或TET1（用于靶向DNA去甲基化）。我们的具体假设是，将定制的TALE- dbd与染色质修饰子连接起来，将能够在人类基因组中广泛的染色质状态（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epitale靶向IMR90成纤维细胞中不同的染色质状态，包括活性/非活性增强子和启动子。综合基因组分析将用于综合评估epiTALE在表观基因组工程中的功效和活性，包括通过ChIP-Seq、MethylC-Seq全基因组亚硫酸盐测序和RNA-Seq鉴定epiTALE结合位点。其次，我们将使用诱导系统对epiTALEs的表达进行时空编程，在该系统中，我们可以动态控制（“脉冲”和“追逐”）DNAme在细胞中的掺入和擦除，通过高通量靶向亚硫酸盐测序来跟踪目标DNAme的时空变化。第三，我们介绍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；诱导基因座特异性调节DNAme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dna从给定的成核点扩散和表观遗传记忆的时空动态。我们的研究将提供创新的分子工具来评估表观遗传扰动在基因组中任何感兴趣的序列中的功能结果，并纠正在患病或重编程细胞中发现的异常表观遗传特征。总体而言，本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.