Linker histone H1.0 loads onto nucleosomes through multiple pathways that are facilitated by histone chaperones

Linker histone H1 is an essential chromatin architectural protein that compacts chromatin into transcriptionally silent regions by interacting with nucleosomal and linker DNA, while rapidly exchanging in vivo. How H1 targets nucleosomes while being dynamic remains unanswered. Using a single-molecule strategy, we investigated human H1.0 interactions with DNA and nucleosomes. H1.0 directly binds nucleosomes and naked DNA with a preference toward nucleosomes. DNA-bound H1.0 exhibited a range of bound lifetimes with both mobile and immobile states, where the mobile H1.0 did not load onto nucleosomes. The histone chaperone Nap1 facilitated H1.0-nucleosome loading by enabling H1.0 loading through DNA sliding, reducing DNA resident times without impacting nucleosome resident times, increasing mobility along DNA, and targeting H1.0 loading onto the nucleosome dyad. These findings reveal linker histones load onto nucleosomes through multiple distinct mechanisms that are facilitated by chaperones to regulate chromatin accessibility.