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

描述（由申请人提供）： 由ENCODE项目领导的系统性工作正在进行中，以确定人类基因组中所有功能性DNA元件的特征。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的地方。这一观察结果不能用基因组中位置沿着的简单线性关系来解释，并且我们无法使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个公正的，全基因组的方法，用于识别染色质的相互作用参与转录调控和其他结构和功能的基因组中的角色。我们的方法的主要概念是使用一个专门设计的DNA寡核苷酸序列连接不同的DNA片段，这些DNA片段在基因组中是非线性相关的，但在体内通过蛋白质因素聚集在一起，并根据DNA接头的特征从连接的DNA片段中提取配对末端双标签序列。随后将标签映射到参考基因组序列，从而揭示配对DNA片段之间的关系。这项建议的具体目标是： 1.开发一种无偏倚的全基因组方法，用于表征参与转录调控的长距离染色质相互作用。我们开发了一个用于CIA-PET分析的原型方案，该方案可以从S.粟酒裂殖酵母细胞我们将进一步优化这种方法，简化文库构建、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.使CIA-PET技术适用于哺乳动物基因组，以鉴定参与转录调控的长距离染色质相互作用。我们计划进一步提高CIA-PET的特异性，并增加其能力，以适应哺乳动物基因组全基因组分析的需要。我们将在3个生物系统中验证和证明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）小鼠胚胎干细胞中Nanog和Oct 4介导的转录调控三级网络。c）人乳腺癌细胞中雌激素受体介导的染色质相互作用。 该项目将为转录调控机制提供前所未有的见解，使我们能够更好地了解癌细胞如何发展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.