Department of Cell Biology and Genetics and Center for Biomedical Genetics, Dr. Molewaterplein 50, 3015 GE Rotterdam, The Netherlands.
J Cell Sci. 2012 Oct 1;125(Pt 19):4630-9. doi: 10.1242/jcs.108522. Epub 2012 Jul 5.
The chromatin architecture is constantly changing because of cellular processes such as proliferation, differentiation and changes in the expression profile during gene activation or silencing. Unravelling the changes that occur in the chromatin structure during these processes has been a topic of interest for many years. It is known that gene activation of large gene loci is thought to occur by means of an active looping mechanism. It was also shown for the β-globin locus that the gene promoter interacts with an active chromatin hub by means of an active looping mechanism. This means that the locus changes in three-dimensional (3D) nuclear volume and chromatin shape. As a means of visualizing and measuring these dynamic changes in chromatin structure of the β-globin locus, we used a 3D DNA-FISH method in combination with 3D image acquisition to volume render fluorescent signals into 3D objects. These 3D chromatin structures were geometrically analysed, and results prior to and after gene activation were quantitatively compared. Confocal and super-resolution imaging revealed that the inactive locus occurs in several different conformations. These conformations change in shape and surface structure upon cell differentiation into a more folded and rounded structure that has a substantially smaller size and volume. These physical measurements represent the first non-biochemical evidence that, upon gene activation, an actively transcribing chromatin hub is formed by means of additional chromatin looping.
由于细胞过程，如增殖、分化以及在基因激活或沉默期间表达谱的变化，染色质结构不断变化。多年来，揭示这些过程中染色质结构发生的变化一直是研究的热点。人们知道，大基因座的基因激活被认为是通过活跃的环化机制发生的。β-珠蛋白基因座的研究也表明，基因启动子通过活跃的环化机制与活跃的染色质中心相互作用。这意味着该基因座在三维（3D）核体积和染色质形状上发生了变化。为了可视化和测量β-珠蛋白基因座染色质结构的这些动态变化，我们使用了一种 3D DNA-FISH 方法，结合 3D 图像采集，将荧光信号体渲染成 3D 物体。对这些 3D 染色质结构进行了几何分析，并对基因激活前后的结果进行了定量比较。共聚焦和超分辨率成像揭示了无活性基因座以几种不同的构象存在。这些构象在细胞分化为更折叠和圆形的结构时会发生形状和表面结构的变化，其尺寸和体积显著减小。这些物理测量结果代表了非生化证据，即在基因激活时，通过额外的染色质环化形成了一个活跃转录的染色质中心。
