Cryo-EM structure and biochemical analyses of the nucleosome containing the cancer-associated histone H3 mutation E97K.

The Lys mutation of the canonical histone H3.1 Glu97 residue (H3E97K) is found in cancer cells. Previous biochemical analyses revealed that the nucleosome containing the H3E97K mutation is extremely unstable as compared to the wild-type nucleosome. However, the mechanism by which the H3E97K mutation causes nucleosome instability has not been clarified yet. In the present study, the cryo-electron microscopy structure of the nucleosome containing the H3E97K mutation revealed that the entry/exit DNA regions of the H3E97K nucleosome are disordered, probably by detachment of the nucleosomal DNA from the H3 N-terminal regions. This may change the intra-molecular amino acid interactions with the replaced H3 Lys97 residue, inducing structural distortion around the mutated position in the nucleosome. Consistent with the nucleosomal DNA end flexibility and the nucleosome instability, the H3E97K mutation exhibited reduced binding of linker histone H1 to the nucleosome, defective activation of PRC2 (the essential methyltransferase for facultative heterochromatin formation) with a poly-nucleosome, and enhanced nucleosome transcription by RNA polymerase II.

Molecular and structural basis of the chromatin remodeling activity by Arabidopsis DDM1.

The histone H2A variant H2A.W occupies transposons and thus prevents access to them in Arabidopsis thaliana. H2A.W is deposited by the chromatin remodeler DDM1, which also promotes the accessibility of chromatin writers to heterochromatin by an unknown mechanism. To shed light on this question, we solve the cryo-EM structures of nucleosomes containing H2A and H2A.W, and the DDM1-H2A.W nucleosome complex. These structures show that the DNA end flexibility of the H2A nucleosome is higher than that of the H2A.W nucleosome. In the DDM1-H2A.W nucleosome complex, DDM1 binds to the N-terminal tail of H4 and the nucleosomal DNA and increases the DNA end flexibility of H2A.W nucleosomes. Based on these biochemical and structural results, we propose that DDM1 counters the low accessibility caused by nucleosomes containing H2A.W to enable the maintenance of repressive epigenetic marks on transposons and prevent their activity.

Cryo-EM structures of RAD51 assembled on nucleosomes containing a DSB site.

RAD51 is the central eukaryotic recombinase required for meiotic recombination and mitotic repair of double-strand DNA breaks (DSBs)1,2. However, the mechanism by which RAD51 functions at DSB sites in chromatin has remained elusive. Here we report the cryo-electron microscopy structures of human RAD51-nucleosome complexes, in which RAD51 forms ring and filament conformations. In the ring forms, the N-terminal lobe domains (NLDs) of RAD51 protomers are aligned on the outside of the RAD51 ring, and directly bind to the nucleosomal DNA. The nucleosomal linker DNA that contains the DSB site is recognized by the L1 and L2 loops-active centres that face the central hole of the RAD51 ring. In the filament form, the nucleosomal DNA is peeled by the RAD51 filament extension, and the NLDs of RAD51 protomers proximal to the nucleosome bind to the remaining nucleosomal DNA and histones. Mutations that affect nucleosome-binding residues of the RAD51 NLD decrease nucleosome binding, but barely affect DNA binding in vitro. Consistently, yeast Rad51 mutants with the corresponding mutations are substantially defective in DNA repair in vivo. These results reveal an unexpected function of the RAD51 NLD, and explain the mechanism by which RAD51 associates with nucleosomes, recognizes DSBs and forms the active filament in chromatin.

Genome-wide mapping and cryo-EM structural analyses of the overlapping tri-nucleosome composed of hexasome-hexasome-octasome moieties.

The nucleosome is a fundamental unit of chromatin in which about 150 base pairs of DNA are wrapped around a histone octamer. The overlapping di-nucleosome has been proposed as a product of chromatin remodeling around the transcription start site, and previously found as a chromatin unit, in which about 250 base pairs of DNA continuously bind to the histone core composed of a hexamer and an octamer. In the present study, our genome-wide analysis of human cells suggests another higher nucleosome stacking structure, the overlapping tri-nucleosome, which wraps about 300-350 base-pairs of DNA in the region downstream of certain transcription start sites of actively transcribed genes. We determine the cryo-electron microscopy (cryo-EM) structure of the overlapping tri-nucleosome, in which three subnucleosome moieties, hexasome, hexasome, and octasome, are associated by short connecting DNA segments. Small angle X-ray scattering and coarse-grained molecular dynamics simulation analyses reveal that the cryo-EM structure of the overlapping tri-nucleosome may reflect its structure in solution. Our findings suggest that nucleosome stacking structures composed of hexasome and octasome moieties may be formed by nucleosome remodeling factors around transcription start sites for gene regulation.