Chromosome movements that regulate tissue-specific gene expression

在2017财年，我们实现了以下目标： 第一章 继续研究在pro-B细胞系的细胞核中定位荧光RAG 1和RAG 2衍生物。 RAG 2衍生物在RAG 2缺陷型pro-B细胞系中表达。 为了减少内源性RAG 1的背景，我们先前已经将针对WT RAG 1的siRNA转导到表达荧光RAG 1衍生物的细胞中。 为了进一步降低背景效应，我们获得了RAG 1缺陷的pro-B细胞系，其中将进行RAG 1的进一步研究。 （二） 研究了CD 4 + CD 8+（DP）胸腺细胞中IgH位点的构象状态，其中该位点经历部分DH到JH重排。 我们发现，与pro-B细胞相比，DP胸腺细胞中VH基因座远端1 Mb的3D结构被破坏。 这一观察结果与我们早期的提议一致，即远端VH基因座结构需要Pax 5，而Pax 5在DP胸腺细胞中不表达。 在1 Mb结构域内，尽管CTCF结合水平正常，但数百种酶的较小CTCF依赖性结构域也被破坏。 我们使用抗Rad 21 ChIP显示这些位点缺乏Rad 21，为缺乏VH压缩提供了合理的解释。 这些观察结果提出了一种有趣的可能性，即Rad 21募集到基因组中的CTCF结合位点是组织限制性的。 今后将审查这方面的依据。 第三章 在DP细胞中，IgH增强子被部分转录因子占据。 具体地，E47和Els-1在DP细胞中的增强子中不存在，而HEB（E2 A相关的bHLM因子）被发现是增强子。 PU.1是另一种在DP胸腺细胞中不表达的增强子结合蛋白。 相比之下，结构蛋白YY 1的结合在pro-B细胞和DP细胞中相当。 因此，与IgH内含子增强子结合的DNA结合蛋白的星座在pro-B和DP细胞之间是不同的。 这些差异可能导致DP细胞中的部分增强子功能。

During FY17 we accomplished the following: 1) Continued with studies to localize fluorescent RAG1 and RAG2 derivatives in nuclei of pro-B cell lines. RAG2 derivatives were expressed in RAG2-deficient pro-B cell lines. To reduce background of endogenous RAG1 we had previously transduced siRNA against WT RAG1 into cells that expressed fluorescent RAG1 derivatives. To further reduce background effects we obtained a RAG1-deficient pro-B cell line in which further studies of RAG1 will be carried out. 2) Investigated the conformational state of the IgH locus in CD4+CD8+ (DP) thymocytes, in which the locus undergoes partial DH to JH rearrangements. We found that 3D structure of the distal 1Mb of the VH locus was disrupted in DP thymocytes compared to pro-B cells. This observation is consistent with our earlier proposal that distal VH locus structure requires Pax5, which is not expressed in DP thymocytes. Within the 1Mb domain, smaller CTCF-dependent domains of a few hundred kilobases were also disrupted, despite normal levels of CTCF binding. We used anti-Rad21 ChIP to show that these sites lacked Rad21, providing a plausible explanation for lack of VH compaction. These observations raised the interesting possibility that Rad21 recruitment to CTCF-bound sites in the genome is tissue-restricted. The basis for this will be examined in the future. 3) The IgH enhancer was found to be partially occupied by transcription factors in DP cells. Specifically, E47 and Els-1 were absent from the enhancer in DP cells, while HEB (a E2A-related bHLM factor) was found to the enhancer. PU.1 is another enhancer binding protein that is not expressed in DP thymocytes. In contrast, binding of the architectural protein YY1 was comparable in pro-B cells and DP cells. Thus, the constellation of DNA binding proteins that bind to the IgH intronic enhancer is different between pro-B and DP cells. These differences presumably lead to partial enhancer function in DP cells.