CRYO-ELECTRON TOMOGRAPHY STUDIES OF EUKARYOTIC CHROMATIN ORGANISATION AT MOLECULAR LEVEL

Abstract

Chromatin is important in regulating many cellular functions. However, the higher-order chromatin structures are in fact, unresolved and debatable. Here, I used cryo-electron tomography to determine the 3-D arrangement of nucleosomes both in vitro and in vivo. In fission yeast Schizosaccharomyces pombe, nucleosomes form irregular clusters in both interphase and mitotic cells. The nucleosomes are mingled with two features: nucleosome-free pockets and megadalton-sized “megacomplexes”. Compared to interphase, the nucleosomes in mitotic chromosomes pack into slightly larger clusters. This uneven chromosome condensation helps explain a longstanding enigma of mitosis: most genes are repressed but a subset is upregulated. Next, I studied the undigested natural chromatin released from cells that do not have evidence of 30-nm fibers in vivo: picoplankton and yeast. In the presence of divalent cations, most of the chromatin from both organisms is compacted into a large mass. Rare irregular 30-nm fibers do form at the periphery of this mass, some of which include face-to-face interactions. In the absence of divalent cations, picoplankton chromatin decondenses into open zigzags. By contrast, yeast chromatin mostly remains compact with looser nucleosome packing. The 3-D configuration of natural chromatin is therefore sensitive to the local environment, but generally nonpermissive of regular motifs.