High-Speed Atomic Force Microscopy Reveals the Nucleosome Sliding and DNA Unwrapping/Wrapping Dynamics of Tail-less Nucleosomes.

Each nucleosome contains four types of histone proteins, each with a histone tail. These tails are essential for the epigenetic regulation of gene expression through post-translational modifications (PTMs). However, their influence on nucleosome dynamics at the single-molecule level remains undetermined. Here, we employed high-speed atomic force microscopy to visualize nucleosome dynamics in the absence of the N-terminal tail of each histone or all of the N-terminal tails. Loss of all tails stripped 6.7 base pairs of the nucleosome from the histone core, and the DNA entry-exit angle expanded by 18° from that of wild-type nucleosomes. Tail-less nucleosomes, particularly those without H2B and H3 tails, showed a 10-fold increase in dynamics, such as nucleosome sliding and DNA unwrapping/wrapping, within 0.3 s, emphasizing their role in histone-DNA interactions. Our findings illustrate that N-terminal histone tails stabilize the nucleosome structure, suggesting that histone tail PTMs modulate nucleosome dynamics.

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.

Contributions of histone tail clipping and acetylation in nucleosome transcription by RNA polymerase II.

The N-terminal tails of histones protrude from the nucleosome core and are target sites for histone modifications, such as acetylation and methylation. Histone acetylation is considered to enhance transcription in chromatin. However, the contribution of the histone N-terminal tail to the nucleosome transcription by RNA polymerase II (RNAPII) has not been clarified. In the present study, we reconstituted nucleosomes lacking the N-terminal tail of each histone, H2A, H2B, H3 or H4, and performed RNAPII transcription assays. We found that the N-terminal tail of H3, but not H2A, H2B and H4, functions in RNAPII pausing at the SHL(-5) position of the nucleosome. Consistently, the RNAPII transcription assay also revealed that the nucleosome containing N-terminally acetylated H3 drastically alleviates RNAPII pausing at the SHL(-5) position. In addition, the H3 acetylated nucleosome produced increased amounts of the run-off transcript. These results provide important evidence that the H3 N-terminal tail plays a role in RNAPII pausing at the SHL(-5) position of the nucleosome, and its acetylation directly alleviates this nucleosome barrier.

Structural basis for binding diversity of acetyltransferase p300 to the nucleosome.

p300 is a human acetyltransferase that associates with chromatin and mediates vital cellular processes. We now report the cryo-electron microscopy structures of the p300 catalytic core in complex with the nucleosome core particle (NCP). In the most resolved structure, the HAT domain and bromodomain of p300 contact nucleosomal DNA at superhelical locations 2 and 3, and the catalytic site of the HAT domain are positioned near the N-terminal tail of histone H4. Mutations of the p300-DNA interfacial residues of p300 substantially decrease binding to NCP. Three additional classes of p300-NCP complexes show different modes of the p300-NCP complex formation. Our data provide structural details critical to our understanding of the mechanism by which p300 acetylates multiple sites on the nucleosome.

Structural and biochemical analyses of the nucleosome containing Komagataella pastoris histones.

Komagataella pastoris is a methylotrophic yeast that is commonly used as a host cell for protein production. In the present study, we reconstituted the nucleosome with K. pastoris histones and determined the structure of the nucleosome core particle by cryogenic electron microscopy. In the K. pastoris nucleosome, the histones form an octamer and the DNA is left-handedly wrapped around it. Micrococcal nuclease assays revealed that the DNA ends of the K. pastoris nucleosome are somewhat more accessible, as compared with those of the human nucleosome. In vitro transcription assays demonstrated that the K. pastoris nucleosome is transcribed by the K. pastoris RNA polymerase II (RNAPII) more efficiently than the human nucleosome, while the RNAPII pausing positions of the K. pastoris nucleosome are the same as those of the human nucleosome. These results suggested that the DNA end flexibility may enhance the transcription efficiency in the nucleosome but minimally affect the nucleosomal pausing positions of RNAPII.

The msh1 gene is responsible for short life span mutant natural death and functions to maintain mitochondrial DNA integrity.

Wild-type filamentous fungus Neurospora crassa continues to grow its hyphae for a very lengthy period of time (>2 years), whereas mutations at the natural death (nd) locus shorten life span (approximately 20 days). By positional cloning based on heat augmented mutagen sensitivity of the nd strain, we identified a nonsense mutation in the msh1 gene, an eukaryotic homolog of bacterial MutS, and this mutation resulted in encoding non-functional polypeptide. By tagging with GFP, subcellular localization of the MSH1 protein in the mitochondria was observed, and knock out of the msh1 gene caused severe growth deficiency accompanying mitochondrial DNA (mtDNA) aberrations such as large-scale mtDNA deletions and rearrangements as seen in the nd strain. These results suggested that MSH1 may maintain mtDNA integrity. Thus, loss of function compromises mtDNA, leading to the acceleration of cellular aging.