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

在之前的授予周期中，我们演示了非编码转录协调染色质 折叠和区隔，以确定T细胞的命运。简单地说，我们鉴定了一个非编码的RNA，名为 胸腺细胞分化因子位于Bcl11b增强子上游20kbp。胸腺- 缺陷小鼠在T细胞发育初期表现出缺陷，并发育成淋巴样细胞 恶性肿瘤。我们发现ThymoD的作用是将Bcl11b增强子从椎板重新定位到 核子内部。重新定位使Bcl11b增强子和启动子进入单环结构域，从而 允许高效的增强子-启动子通信和激活Bcl11b的表达。这一过程 涉及多个步骤：(I)ThymoD基因座和Bcl11b启动子区域上的CTCF占有率， (Ii)激活粘附素依赖环，将增强子和启动子并列成一个环 由ThymoD基因座和Bcl11b启动子区域中的CTCF位点锚定的结构域，(Iii) 将增强子从异色环境重新定位到等色环境以及(Iv)激活 Bcl11b的表达及其对T细胞命运的调控在这里，我们建议继续进行这些研究。我们会 用分子术语描述胸腺激素如何协调T细胞的命运。具体地说，我们将审查 CTCF占位和粘附素依赖的环路是否以及如何协调染色质折叠和 将增强子和启动子区域分隔成一个环状结构域。我们会 在高空时追踪粘连蛋白丢失和恢复过程中的Bcl11b基因座拓扑 决议。我们将研究粘附素介导的环状挤出如何引导分区， 组装Bcl11b环区，诱导T系特异性基因表达程序。已被占用 总之，这些研究将揭示非编码转录是如何诱导环排出的 增强启动子-启动子通信具有很高的特异性，以确定T细胞的命运。

During the previous grant cycle we demonstrated that non-coding transcription orchestrates chromatin folding and compartmentalization to specify T cell fate. Briefly, we identified a non-coding RNA named ThymoD (thymocyte differentiation factor) located 20 kbp upstream of the Bcl11b enhancer. ThymoD- deficient mice displayed a defect at the onset of T cell development and developed lymphoid malignancies. We found ThymoD acts to reposition the Bcl11b enhancer from the lamina into the nuclear interior. The repositioning brings the Bcl11b enhancer and promoter into a single loop domain to permit efficient enhancer-promoter communication and activation of Bcl11b expression. This process involves multiple steps: (i) CTCF occupancy across the ThymoD locus and the Bcl11b promoter region, (ii) activation of cohesin-dependent looping to juxtapose the enhancer and promoter into a single loop domain that is anchored by CTCF sites in the ThymoD locus and the Bcl11b promoter region, (iii) repositioning the enhancer from a heterochromatic to an euchromatic environment and (iv) activation of Bcl11b expression and specification of T cell fate. Here we propose to continue these studies. We would describe in molecular terms how ThymoD orchestrates T cell fate. Specifically, we would examine whether and how CTCF occupancy and cohesin-dependent looping orchestrates chromatin folding and compartmentalization to bring the enhancer and promoter regions into a single loop domain. We would track Bcl11b locus topology during cohesin loss and cohesin recovery at high spatial and temporal resolution. We would examine how cohesin mediated loop extrusion directs compartmentalization, assembles the Bcl11b loop domain and induces a T-lineage specific program of gene expression. Taken together, these studies would reveal how non-coding transcription induced loop extrusion directs enhancer-promoter communication with great specificity to specify T cell fate.