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 H1.0 was largely mobile, diffused over kilobase distances in seconds, but surprisingly did not load onto nucleosomes. Linker histone chaperones significantly 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 the multiple distinct mechanisms linker histones and their chaperones use to interact with nucleosomes to regulate chromatin accessibility.