The histone variant H2A.B plays a crucial role in mammalian spermatogenesis. This study used an in vitro transcription assay and cryo-electron microscopy (cryo-EM) structural analysis to investigate how RNA polymerase II (RNAPII) transcribes nucleosomes containing H2A.B. The transcription assays demonstrated that RNAPII transcribes H2A.B nucleosomes more efficiently than canonical H2A nucleosomes. Cryo-EM structural analysis revealed that one of the H2A.B–H2B dimers dissociates from the nucleosome during transcription. These findings indicate that H2A.B promotes efficient transcription and provide new insights into the function of histone variants in gene expression.

Previous studies of chromatin structure have primarily focused on artificially reconstitutednucleosomes. In contrast, structural analysis of chromatin in the nucleus remainstechnically challenging, and its organization in the native nuclear environment is still poorly understood. In this study, we developed a method using cryo-EM to analyze chromatin from human cells while preserving its higher-order structure. The nucleosome structure derived from human cells closely resembled that of artificially reconstituted nucleosomes. Moreover, cryo-ET analysis of chromatin from human cells revealed not only fiber-like nucleosome arrangements but also irregular clutch-like assemblies. The method established in this study provides a powerful approach for investigating chromatin architecture in the nuclear context.

Barrier-to-autointegration factor (BAF) plays critical roles in recruiting nuclear membrane proteins and Lamin A/C to chromosomes following chromosome segregation, facilitating nuclear envelope reassembly and repair. BAF is also involved in bridging inner nuclear membrane proteins with chromatin. In this study, we report cryo-electron microscopy (cryo-EM) structures of multiple complexes of BAF and the immunoglobulin-fold (IgF) domain of Lamin A/C bound to nucleosomes. One structure reveals the binding of a BAF dimer and Lamin A/C IgF at the nucleosomal dyad, while the other structure shows two additional BAF dimers bridging two nucleosomes. Furthermore, we present the cryo-EM structure of the complex of BAF, Lamin A/C IgF, nucleosomes, and the linker histone H1. This structure suggests that histone H1 promotes BAF-mediated nucleosome bridging. These findings provide structural insights into chromatin organization and nuclear envelope assembly.

RAD51 plays a central role in homologous recombination. This paper reports the structures of the RAD51-nucleosome complex using cryo-electron microscopy. RAD51forms two types of ring structures: one that binds to the nucleosome with linker DNA binding and one that binds to the nucleosome without linker DNA binding. In addition, the RAD51 nucleosome filament structure, which partially peels DNA from the nucleosome, has been observed. The important RAD51 domain for nucleosome binding is the N-terminal lobe domain (NLD), which was confirmed by both in vivo and in vitro experiments. It was also shown that RAD51 incorporates linker DNA into the ring by forming ring structures and that the L1 and L2 loops, which are active centres, binds to the double-strand break sites. RAD51 forms ring structures to accumulate on chromatin and recognize double-strand break sites. It then gradually peels DNA from nucleosomes to form the filament structure which facilitates homologous recombination.

RNA polymerase II proceeds with transcription by pulling off DNA on nucleosomes. In this study, we determined two cryo-EM structures of RNAPII–nucleosome complexes with upstream 'DNA looping'. These structures were different about the length of upstream DNA the forms looping. In addition, we tried the structural analysis of 'DNA looping' with the transcription elongation complex and found that the transcription elongation complex does not form the 'DNAlooping'.