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下的不同表观遗传修饰的全面全基因组图的产生，现在迫切需要超越描述性表观基因组学。重要的是，需要开发功能性的表观基因组学工具来刻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。在这里，我们建议产生新的最先进的可编程DNA结合蛋白，将表观基因组修饰活性准确地运送到基因组中所需的目标位置。通过将转录激活因子样效应子(TALE)蛋白骨架设计的序列特异性DNA结合域(DBD)连接到表观基因组修饰物(EPTALE)，我们将实现高精度的表观基因组工程。我们选择在人类基因组中包含不同功能染色质状态的广泛基因座上编程DNA甲基化(DNAME)变化，包括开放染色质和异染色质。我们将快速构建高度特异的TALL-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)的催化域以从头开始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TALL-DBDS与染色质修饰剂相关联，将使人类基因组中广泛的染色质状态(如增强剂和启动子)中的DNAME状态能够进行位点特异性调节。首先，我们建议设计一个针对IMR90成纤维细胞中不同染色质状态的表面膜，包括活性/非活性增强剂和启动子。联合基因组分析将被用来全面评估表观基因组工程的表观分子的有效性和活性，包括通过CHIP-SEQ、甲基C-SEQ全基因组亚硫酸盐测序和RNA-SEQ鉴定表观分子结合位点。其次，我们将使用可诱导系统在时空上对EMETALEs的表达进行编程，在该系统中，我们可以通过高通量的靶向亚硫酸氢盐测序来动态控制(脉冲和追逐)细胞中DNAME的掺入和删除，从而跟踪靶向DNAME的时空变化。第三，我们介绍了这些表观基因组工程工具在临床上的相关细胞类型的特定应用；诱导DNAME的位点特异性调制以纠正在乳腺癌和诱导的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来评估基因组中任何感兴趣的序列中表观遗传扰动的功能结果，并纠正在疾病或重新编程的细胞中识别的异常表观遗传特征。总体而言，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领域的重大进步。

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.