Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Proc Natl Acad Sci U S A. 2012 Jul 10;109(28):11336-41. doi: 10.1073/pnas.1208595109. Epub 2012 Jun 25.
By microscopic analysis of fluorescent-labeled GalR, a regulon-specific transcription factor in Escherichia coli, we observed that GalR is present in the cell as aggregates (one to three fluorescent foci per cell) in nongrowing cells. To investigate whether these foci represent GalR-mediated association of some of the GalR specific DNA binding sites (gal operators), we used the chromosome conformation capture (3C) method in vivo. Our 3C data demonstrate that, in stationary phase cells, many of the operators distributed around the chromosome are interacted. By the use of atomic force microscopy, we showed that the observed remote chromosomal interconnections occur by direct interactions between DNA-bound GalR not involving any other factors. Mini plasmid DNA circles with three or five operators positioned at defined loci showed GalR-dependent loops of expected sizes of the intervening DNA segments. Our findings provide unique evidence that a transcription factor participates in organizing the chromosome in a three-dimensional structure. We believe that these chromosomal connections increase local concentration of GalR for coordinating the regulation of widely separated target genes, and organize the chromosome structure in space, thereby likely contributing to chromosome compaction.
通过对大肠杆菌中调控特定转录因子 GalR 的荧光标记进行显微镜分析，我们观察到 GalR 在非生长细胞中以聚集体的形式存在（每个细胞有一到三个荧光焦点）。为了研究这些焦点是否代表 GalR 介导的一些 GalR 特异性 DNA 结合位点（gal 操纵子）的结合，我们在体内使用了染色体构象捕获（3C）方法。我们的 3C 数据表明，在静止期细胞中，分布在染色体周围的许多操纵子相互作用。通过原子力显微镜，我们表明观察到的远程染色体连接是通过直接相互作用发生的，涉及的是 DNA 结合的 GalR，不涉及任何其他因素。带有三个或五个定位在特定位置的操纵子的小型质粒 DNA 环显示出 GalR 依赖性的预期大小的间隔 DNA 片段环。我们的研究结果提供了独特的证据，证明转录因子参与了三维结构中染色体的组织。我们认为这些染色体连接增加了 GalR 的局部浓度，以协调广泛分离的靶基因的调控，并在空间上组织染色体结构，从而可能有助于染色体紧缩。
