Chromosome movements that regulate tissue-specific gene expression

在2015财年，我们完成了以下工作： 1)我们发表了三种不同转录因子介导的IgH基因座染色体构象结构层次的研究。简而言之，我们发现第一水平的折叠是由转录因子CTCF介导的。这种机制将IgH基因座折叠成多个180-250kb的结构域。其次，转录因子Pax5压缩了该基因座VH部分的2Mb。最后，转录因子YY1将IgH基因的5端和3端连接在一起。我们认为，染色质构象的逐步生成可以普遍应用于3D基因组结构的建立。 2)对老年小鼠骨髓PRO-B细胞IgH染色质结构的破坏进行了追踪研究。特别是，我们通过改变组蛋白甲基转移酶Ezh2和转录因子PU.1在原代Pro-B细胞中的水平来测试组蛋白甲基转移酶Ezh2和转录因子PU.1的参与。RNA分析与正在测试的拟议假说一致。荧光原位杂交(FISH)研究正在进行中，以确定这些因子的操纵水平是否会使IGH基因座的构象和功能具有较老的表型。 3)我们与宾夕法尼亚州大学的Michael Atchison博士合作，测试了转录因子YY1在类切换重组(CSR)中的结构作用。FISH研究表明，在体外接受CSR激活的YY1缺陷B细胞中，E和3RR之间的环路中断。通过异位表达YY1逆转录病毒重建环状结构。在这项实验中，我们使用了几个YY1的截短版本来确定这种相互作用所需的蛋白质结构域。

During FY15, we accomplished the following: 1) We published studies of a structural hierarchy of chromosome conformation of the IgH locus mediated by three different transcription factors. Briefly, we found that the first level of folding is mediated by the transcription factor CTCF. This mechanism folds the IgH locus into multiple 180-250 kb domains. Second, the transcription factor Pax5 compacts 2Mb of the VH part of the locus. Finally, the transcription factor YY1 brings together the 5 and 3 ends of the IgH locus. We proposed that stepwise generation of chromatin conformation could apply generally for the establishment of 3D-genome architecture. 2) We followed up studies on the disruption of IgH chromatin structure in bone marrow pro-B cells from aged mice. Specifically, we tested the involvement of the histone methyl transferase Ezh2 and the transcription factor PU.1 by altering levels of these proteins in primary pro-B cells. RNA analyses were consistent with the proposed hypothesis being tested. Fluorescent in situ hybridization (FISH) studies are in progress to determine whether manipulating levels of these factors imparts an older phenotype to IgH locus conformation and function. 3) We collaborated with Dr. Michael Atchison (U. Pennsylvania) to test the structural role of the transcription factor YY1 during class switch recombination (CSR). FISH studies showed disrupted looping between E and the 3RR in YY1-deficient B cells activated to undergo CSR ex vivo. Looping was re-established by ectopic retroviral expression of YY1. We used several truncated versions of YY1 in this assay to identify the domain of the protein required for this interaction.