The 3D-Structures of the Pre-Pro-B and Pro-B Cell Genomes

描述（由申请人提供）：最近的研究提供了大量证据，表明基因组不仅由线性结构组成，而且还折叠成精细的模式，允许由大基因组距离分开的基因组元件之间的相互作用。基因组的精确折叠和拓扑谱仍有待确定。最近的研究提供了对免疫球蛋白重链（Igh）和γ-珠蛋白基因座的3D结构的深入了解。这些数据表明，这些基因座被组织成多个隔室，其特征在于成束的环，与多环亚隔室（MLS）模型一致。我们现在面临的问题是，整个基因组是否像MLS模型所提出的那样折叠。为了解决这个问题，我们最近采用了甲醛交联的方法，允许识别的全基因组网络的相互作用的基因组元件在前B细胞。我们的初步数据表明，原B细胞基因组确实被组织成由MLS模型预测的接头连接的环簇。在此，我们建议继续进行这些研究。我们将深入分析各个隔室，并研究它们是如何组织的，以及它们如何与B系特异性基因表达程序相关。我们将识别和描述染色体内的相互作用。绝缘子和桥接因子将被确定和表征从生物化学和功能的角度。最后，我们建议，以确定有丝分裂染色体的结构，在前原B和原B细胞。总体目标是破译前B和前B细胞染色质纤维形成的构象和轨迹谱，以及染色质结构与功能的关系。 公共卫生相关性：这里描述的建议旨在开发一种新的方法来描述前B和前B细胞中染色质纤维的折叠。该研究将提供物理上的洞察机制，允许增强子和启动子元件以适当的频率相互作用，以调节下游靶基因的表达。这些研究还可能为深入了解基因组不稳定性的机制提供帮助。

DESCRIPTION (provided by applicant): Recent studies have provided substantial evidence that the genome consists not merely of linear structures but rather it is folded into elaborate patterns that permits interactions between genomic elements separated by large genomic distances. The precise folding and the spectrum of topologies of the genome remain to be determined. Recent studies have provided insight into the 3D-architectures of the immunoglobulin heavy chain (Igh) and ¿-globin loci. These data have revealed that these loci are organized into multiple compartments that are characterized by bundles of loops, consistent with the Multiple-Loop-Subcompartment (MLS) Model. We are now faced with the question as to whether the entire genome is folded as proposed by the MLS model. To address this question, we have recently employed a formaldehyde cross-linking approach that permits the identification of a genome-wide network of interacting genomic elements in pro-B cells. Our preliminary data indicates that the pro-B cell genome indeed is organized into clusters of loops that are connected by linkers as predicted by the MLS model. Here we would propose to continue these studies. We would perform an in depth analysis of individual compartments and examine how they are organized and how they relate to B-lineage specific programs of gene expression. We would identify and characterize intra-chromosomal interactions. Insulators and bridging factors would be identified and characterized both from a biochemical and functional perspective. Finally, we propose to determine the structure of mitotic chromosomes in pre-pro-B and pro-B cells. The overall goal would be to decipher the spectrum of conformations and trajectories formed by the pre-pro-B and pro-B cell chromatin fibers and how chromatin structure relates to function. PUBLIC HEALTH RELEVANCE: The proposal described here is aimed at developing a novel approach to describe the folding of the chromatin fiber in pre-pro-B and pro-B cells. The study would provide physical insight into the mechanism that permits enhancer and promoter elements to interact with each other with the appropriate frequencies in order to modulate down- stream target gene expression. The studies may also provide insight into the mechanisms that underpin genomic instability.