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，以确定人类基因组中所有功能DNA元件的特征。我们和其他人产生的越来越多的数据表明，很大一部分假定的调控元件定位在远离基因编码区的地方。这一观察结果不能用基因组位置的简单线性关系来解释，我们也无法使用现有技术解决功能DNA元素之间的非线性相互作用。我们的目标是开发一种无偏见的全基因组方法来识别参与转录调控和基因组中其他结构和功能作用的染色质相互作用。该方法的基本思想是使用一个特殊设计的DNA寡核苷酸序列将基因组中不同的DNA片段连接在一起，这些DNA片段在基因组中是非线性相关的，但在体内被蛋白质因素紧密地连接在一起，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双标签序列。然后将标签映射到参考基因组序列，从而揭示成对的DNA片段之间的关系。这项建议的具体目标是： 1.建立一种无偏倚的全基因组方法，用于鉴定参与转录调控的染色质长程相互作用。我们开发了一个用于CIA-PET分析的原型协议，该协议可以从S.pombe细胞中非线性相关DNA相互作用的连接物连接的DNA片段中提取双标签。我们将进一步优化这一方法，并简化文库建设、测序和数据分析的整个过程。此外，我们将使用各种可用的低通量技术严格验证CIA-PET数据。 2.将CIA-PET技术应用于哺乳动物基因组，用于鉴定参与转录调控的染色质长程相互作用。我们计划进一步提高CIA-PET的特异性，并增加其容量，以适应哺乳动物基因组全基因组分析的需要。我们将在三个生物系统中验证和演示CIA-PET方法的实用性：a)小鼠红系细胞中血红蛋白基因和调控元件之间的染色质相互作用。B)由Nanog和Oct4介导的小鼠胚胎干细胞转录调控的三级网络。C)雌激素受体介导的人乳腺癌细胞染色质相互作用。这个项目将提供对转录调控机制的前所未有的见解，使我们能够更好地了解癌细胞是如何发展的，以及干细胞如何保持多能性。

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.