The 3D-Structure of the Immunoglobulin Heavy Chain Locus

描述（由申请人提供）：本申请中提出的长期研究目标是以高分辨率确定免疫球蛋白重链（Igh）位点的3d结构。Igh基因座被组织成不同的区域，包含多变量（VH）、多样性（DH）、连接（JH）和恒定（CH）编码元件。为了探测Igh基因座的地形，我们最近利用12个基因组标记确定了整个基因座的空间距离分布。这些空间距离分布与计算机模拟的其他染色质排列进行了比较。这一分析预测，Igh基因座被组织成含有由连接物分开的环簇的隔室。然后，我们使用计算几何来确定VH， DH， JH和CH元素的平均相对3d位置。简而言之，数据表明，在B细胞早期发育过程中，整个VH区域（2.5 Mbp）被合并并与DH元件并列，使VH区域能够以相对高且相似的频率遇到DHJH元件。在此，我们建议继续这些研究。我们将以高分辨率描述间期和有丝分裂染色质中的平均Igh轨迹。我们将确定Igh轨迹纤维所采用的构象谱。我们将使用结构照明显微镜来可视化右染色质区域。我们将使用物理方法来描述隔层。我们将使用物理和分子生物学方法来识别环碱基。我们将使用空间距离测量和计算几何来确定Igh位点结构是否是抗原受体位点和真核间期染色质的一般特征。综上所述，这些研究将提供对Igh基因座结构的统计描述，并为抗体多样性如何产生提供机制见解。公共卫生相关性：在以前的研究中，我们使用几何来显示基因位点如何在三维空间中组织。本应用程序中提出的研究应提供深入了解基因组结构如何允许对各种入侵病原体产生免疫反应。

DESCRIPTION (provided by applicant): The long-range goals of the research proposed in this application are to determine at high resolution the 3D-structure of the immunoglobulin heavy chain (Igh) locus. The Igh locus is organized into distinct regions that contain multiple variable (VH), diversity (DH), joining (JH) and constant (CH) coding elements. To probe the topography of the Igh locus, we have recently determined the spatial distance distributions using 12 genomic markers that spanned the entire locus. These spatial distance distributions were compared to computer simulations of alternative chromatin arrangements. This analysis predicted that the Igh locus is organized into compartments containing clusters of loops separated by linkers. We then used computational geometry to determine the mean relative 3D-positions of the VH, DH, JH and CH elements. Briefly, the data showed that during early B cell development, the entire repertoire of VH regions (2.5 Mbp) is merged and juxtaposed to the DH elements, allowing the VH regions to encounter DHJH elements with relatively high and similar frequencies. Here we propose to continue these studies. We would describe at high resolution the average Igh locus trajectories in interphase and mitotic chromatin. We would determine the spectrum of conformations adopted by the Igh locus fiber. We would use structured illumination microscopy to visualize the Igh chromatin territories. We would characterize compartments using physical approaches. We would identify loop bases using both physical and molecular biological approaches. We would use spatial distance measurements and computational geometry to determine whether the Igh locus structure is a general feature of antigen receptor loci and eukaryotic interphase chromatin. Taken together, these studies would provide a statistical description of Igh locus structure and provide mechanistic insight into how antibody diversity is generated. PUBLIC HEALTH RELEVANCE: In previous studies we have used geometry to show how a genetic locus is organized in 3D space. The studies proposed in this application should provide insight into how the structure of the genome permits the generation of an immune response to a wide variety of invading pathogens.