Nucleosomal DNA unwinding pathway through canonical and non-canonical histone disassembly.

The nucleosome including H2A.B, a mammalian-specific H2A variant, plays pivotal roles in spermatogenesis, embryogenesis, and oncogenesis, indicating unique involvement in transcriptional regulation distinct from canonical H2A nucleosomes. Despite its significance, the exact regulatory mechanism remains elusive. This study utilized solid-state nanopores to investigate DNA unwinding dynamics, applying local force between DNA and histones. Comparative analysis of canonical H2A and H2A.B nucleosomes demonstrated that the H2A.B variant required a lower voltage for complete DNA unwinding. Furthermore, synchronization analysis and molecular dynamics simulations indicate that the H2A.B variant rapidly unwinds DNA, causing the H2A-H2B dimer to dissociate from DNA immediately upon disassembly of the histone octamer. In contrast, canonical H2A nucleosomes unwind DNA at a slower rate, suggesting that the H2A-H2B dimer undergoes a state of stacking at the pore. These findings suggest that nucleosomal DNA in the H2A.B nucleosomes undergoes a DNA unwinding process involving histone octamer disassembly distinct from that of canonical H2A nucleosomes, enabling smoother unwinding. The integrated approach of MD simulations and nanopore measurements is expected to evolve into a versatile tool for studying molecular interactions, not only within nucleosomes but also through the forced dissociation of DNA-protein complexes.

Histone chaperone Nap1 dismantles an H2A/H2B dimer from a partially unwrapped nucleosome.

DNA translocases, such as RNA polymerases, inevitably collide with nucleosomes on eukaryotic chromatin. Upon these collisions, histone chaperones are suggested to facilitate nucleosome disassembly and re-assembly. In this study, by performing in vitro transcription assays and molecular simulations, we found that partial unwrapping of a nucleosome by an RNA polymerase dramatically facilitates an H2A/H2B dimer dismantling from the nucleosome by Nucleosome Assembly Protein 1 (Nap1). Furthermore, the results uncovered molecular mechanisms of Nap1 functions in which the highly acidic C-terminal flexible tails of Nap1 contribute to the H2A/H2B binding by associating with the binding interface buried and not accessible to Nap1 globular domains, supporting the penetrating fuzzy binding mechanism seemingly shared across various histone chaperones. These findings have broad implications for the mechanisms by which histone chaperones process nucleosomes upon collisions with translocases in transcription, histone recycling and nucleosomal DNA repair.

The lane-switch mechanism for nucleosome repositioning by DNA translocase.

Translocases such as DNA/RNA polymerases, replicative helicases, and exonucleases are involved in eukaryotic DNA transcription, replication, and repair. Since eukaryotic genomic DNA wraps around histone octamers and forms nucleosomes, translocases inevitably encounter nucleosomes. A previous study has shown that a nucleosome repositions downstream when a translocase collides with the nucleosome. However, the molecular mechanism of the downstream repositioning remains unclear. In this study, we identified the lane-switch mechanism for downstream repositioning with molecular dynamics simulations and validated it with restriction enzyme digestion assays and deep sequencing assays. In this mechanism, after a translocase unwraps nucleosomal DNA up to the site proximal to the dyad, the remaining wrapped DNA switches its binding lane to that vacated by the unwrapping, and the downstream DNA rewraps, completing downstream repositioning. This mechanism may have broad implications for transcription through nucleosomes, histone recycling, and nucleosome remodeling.