The nucleosome assembly protein 1: elucidation of the histone binding preference and the mechanism of self-association

Abstract

The nucleosome assembly protein 1 (NAP1) is a member of the histone chaperone family of proteins, and is thought to function in the deposition of the histone (H2A-H2B) dimer complex onto DNA during chromatin assembly. However, NAP1 is also involved in the nuclear import of (H2A-H2B), the regulation of cell cycle events involving the onset of DNA replication, and the transient disruption of intact nucleosomes that facilitates transcription within a chromatin context. Though NAP1 is a common laboratory 'tool' used to assemble nucleosomes onto plasmid DNA in order to study chromatin transcription in vitro, there remains great uncertainty as to the mechanism with which yeast NAP1 assembles both the (H3-H4)2 tetramer and the (H2A-H2B) dimer onto plasmid DNA. In this study, direct binding assays and native gel electrophoresis were used to determine that, in contrast to finding in past investigations, yNAP1 shows a clear preference for the tetramer over the dimer. Further, using select deletion mutants of yNAP1 and histones devoid of their N-terminal tails, it was determined that the mechanism for the preference lies in the binding of yNAP1 to the N-terminal tails of histones H3 and H4, but not H2A or H2B. Finally, it was shown that the most acidic portion of yNAP1, previously shown to be dispensable for chromatin assembly, appears to be a non-specific binding platform for basic proteins. Native gel electrophoresis of yNAP1 revealed more than one band, thus it was hypothesized that yNAP1 self-associates. The self-association of yNAP1 was studied using biochemical, hydrodynamic and thermodynamic methods. yNAP1 was revealed to be an obligate dimer, in that it likely does not exist in the monomeric state at physiological conditions or concentrations. The monomeric form of yNAP1 was only revealed in the presence of moderate levels of denaturant. Further, we observe that yNAP1 oligomerizes into tetramers and hexamers, and this oligomerization is regulated by counteracting the high electrostatic charge on yNAP1 with high ionic strength. The dimerization of yNAP1 likely plays a role in chromatin assembly, as the histone complexes also exhibit a two-fold symmetry.