Whole Genome Chromatin Interaction Analysis using Pair-End-diTagging (CIA-PET)

描述（由申请人提供）：由ENCODE项目领导的系统工作正在进行中，以表征人类基因组中所有功能DNA元素。我们自己和其他人产生的越来越多的数据表明，大部分假定的调控元件位于远离基因编码区域的地方。这种观察结果不能用基因组位置的简单线性关系来解释，而且我们无法利用现有技术解决功能DNA元件之间的非线性相互作用。我们的目标是开发一种无偏倚的全基因组方法，用于鉴定基因组中转录调控和其他结构和功能作用中涉及的染色质相互作用。我们的方法的主要概念是使用特殊设计的DNA寡核苷酸序列连接基因组中非线性相关但在体内被蛋白质因子聚集在一起的不同DNA片段，并根据DNA连接物的特征从连接的DNA片段中提取成对的末端标记序列。接下来是将标签映射到参考基因组序列，从而揭示成对DNA片段之间的关系。这项建议的具体目标是：

DESCRIPTION (provided by applicant): Systematic efforts led by the ENCODE project are underway to characterize all functional DNA elements in the human genome. A growing amount of data generated by ourselves and others has shown that a large portion of the putative regulatory elements are localized far away from gene coding regions. This observation cannot be explained by a simple linear relationship of locations along the genome, and we are unable to address non-linear interactions between functional DNA elements using current technologies. Our goal is to develop an unbiased, whole genome approach for the identification of chromatin interactions involved in transcriptional regulation and other structural and functional roles in the genome. The principal concept of our approach is to use a specially designed DNA oligonucleotide sequence to link different DNA fragments that are non-linearly related in the genome but brought together in close spatial proximity by protein factors in vivo, and extract paired end ditag sequences from the ligated DNA fragments based on the features of the DNA linker. This is followed by mapping the tags to the reference genome sequence, hence revealing the relationship between the paired DNA fragments. The specific aims of this proposal are: 1. To develop an unbiased, whole genome approach for the characterization of long-range chromatin interactions involved in transcription regulation. We developed a prototype protocol for CIA-PET analysis that can extract ditags from the linker-ligated DNA fragments of non-linearly related DNA interactions in S. pombe cells. We will further optimize this methodology and streamline the entire process of library construction, sequencing, and data analysis. In addition, we will rigorously validate the CIA-PET data using a variety of available low-throughput technologies. 2. To adapt CIA-PET technology to mammalian genomes for the identification of long range chromatin interactions involved in transcription regulation. We plan to further improve the specificity of CIA-PET and increase its capacity to suit the need for whole genome analysis in mammalian genomes. We will validate and demonstrate the utility of CIA-PET method in 3 biological systems: a) Chromatin interactions between hemoglobin genes and regulatory elements in mouse erythroid cells. b) Tertiary networks of transcription regulation mediated by Nanog and Oct4 in mouse embryonic stem cells. c) Estrogen receptor mediated chromatin interactions in human breast cancer cells. This project will provide unprecedented insight into the mechanisms of transcription regulations allowing us to better understand how cancer cells develop and how stem cells retain pluripotency.