Precision engineering of DNA methylation patterns in the human genome

人类基因组 DNA 甲基化模式的精密工程

基本信息

批准号：
8815263
负责人：
PILAR BLANCAFORT
金额：
$ 17.37万
依托单位：
UNIVERSITY OF WESTERN AUSTRALIA
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-30 至 2015-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8815263
关键词：
BRCA1 gene Binding Sites Biological Models Breast Cancer Cell Catalytic Domain Cell Line Cell model Cells ChIP-seq Chromatin Communities Custom DNA DNA Binding Domain DNA Methylation DNA Methyltransferase DNA Modification Methylases DNA-Binding Proteins Defect Development Disease Doxycycline Engineering Enhancers Epigenetic Process Excision Fibroblasts Generations Genome Genomics Health Heterochromatin Human Human Genome Link Maps Memory Methods Modification Molecular Oncogenic Pathogenesis Pattern Physiologic pulse Proteins RNA Sequences Regenerative Medicine Regulation Research Role Site System Technology Testing Transcription Coactivator Tumor Suppressor Genes Vertebral column bisulfite cell type clinical application clinically relevant demethylation design efficacy testing epigenome epigenomics functional outcomes genome-wide improved induced pluripotent stem cell innovation insight interest malignant breast neoplasm novel programs promoter spatiotemporal tool transcriptome sequencing

项目摘要

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.

描述（由申请人提供）：最近一代人在多种细胞类型和疾病状态下各种表观遗传修饰的全面基因组图的综合图现在迫切需要超越描述性表观基因组学。至关重要的是，需要开发功能基因组学工具，以故意和精确改变特定的表观遗传修饰，以便询问表观遗传标记在基因组调节中的作用中的作用。在本文中，我们提出了新型最新的可编程DNA结合蛋白的产生，该蛋白将表观基因组修饰活性精确地归因于基因组中所需的靶基因座。通过将从转录激活剂样效应子（Tale）蛋白主链设计的序列特异性DNA结合结构域（DBD）连接到表观基因组修饰剂（EptiTale），我们将实现高度精确的表观基因组工程。我们选择在人类基因组中进行DNA甲基化（DNAME）的编程变化，其中包括不同功能性染色质状态，包括开放式染色质和异染色质。我们将快速构建能够识别基因组中唯一的〜20 bp序列的高度特异性的故事，并与DNA NOVE DNAME的DNA甲基转移酶3A（DNMT3A）的催化结构域相关，或者是VP64或TET1的TET1或TET1的TET1。我们的具体假设是，将自定义故事 - DBD与染色质修饰符联系起来，将在人类基因组中的广泛染色质状态（例如增强子和启动子）中对DNAME状态的特定地点调节。首先，我们建议设计一个集中的尤其小组，以靶向IMR90成纤维细胞中的不同染色质状态，包括活跃/无活性增强子和启动子。合并的基因组分析将用于全面评估表观基因组工程效应的功效和活性，包括通过chip-seq鉴定地缩合结合位点，甲基二氧化甲基蛋白酶全基因组Bisulfite序列和RNA-Seq。其次，我们将使用诱导系统的时空编程阶段的表达，在该系统中，我们可以通过高通量靶向的双硫酸盐测序来动态地控制细胞中的掺入和删除细胞中的dname和删除。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的特定应用。诱导DNAME的基因座特异性调节，以纠正乳腺癌中发现的异常表观遗传学特征并诱导多能干细胞。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，并纠正患病或重编程细胞中确定的异常表观遗传学特征。总体而言，该项目旨在开发用于表观基因组工程的新型分子工具，这将构成功能基原基学的新生领域的重大进步。