Super-resolution imaging reveals 3D folding dynamics of the β-globin locus upon gene activation

Abstract

The chromatin architecture is constantly changing due to cellular processes such as cell proliferation, differentiation and changes in the expression profile such as gene activation or silencing. Unraveling 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 active looping mechanism. It was also shown for the β-globin locus that the gene's promotor interacts with an active chromatin hub by means of an active looping mechanism. This predicts that the locus changes in 3D nuclear volume occupation and chromatin shape. In search to visualize and measure 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 analyzed and results prior to and after gene activation were quantitatively compared. Confocal and super-resolution imaging reveal 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.