The Histone Chaperone Nap1 Promotes Nucleosome Assembly by Eliminating Nonnucleosomal Histone DNA Interactions

Abstract

Summary The organization of the eukaryotic genome into nucleosomes dramatically impacts the regulation of gene expression. The delicate balance between transcription and DNA compaction relies heavily on nucleosome dynamics. Surprisingly, little is known about the free energy required to assemble these large macromolecular complexes and maintain them under physiological conditions. Here we describe the thermodynamic parameters that drive nucleosome formation in vitro. To demonstrate the versatility of our approach, we test the effect of DNA sequence and H3K56 acetylation on nucleosome thermodynamics. Furthermore, our studies reveal the mechanism of action of the histone chaperone Nucleosome Assembly Protein 1 (Nap1). We present evidence for a paradigm in which nucleosome assembly requires the elimination of competing, non-nucleosomal histone-DNA interactions by Nap1. This observation is confirmed in vivo, where deletion of the NAP1 gene in yeast results in a significant increase in atypical histone-DNA complexes, as well as in deregulated transcription activation and repression.

Structural evidence for Nap1‐dependent H2A–H2B deposition and nucleosome assembly

“…Free H2A–H2B preferentially interacted with tetrasomes and only once these were exhausted from the reaction H2A–H2B bound non‐specifically to the free DNA. In accordance with our structure and previously published data (Andrews et al , 2010), yNap1 is suppressing non‐specific DNA binding of H2A–H2B and thereby facilitates specific deposition of H2A–H2B and nucleosome assembly.…”

“…Nap1 deletion only modestly reduces the amounts of nuclear H2A–H2B indicating that there are redundant H2A–H2B transport mechanisms (Mosammaparast et al , 2001). Instead, as previous analysis in a nap1 Δ strain reported significant non‐nucleosomal histone–DNA interaction of H2A–H2B, we favor the model that yNap1 prevents such non‐nucleosomal binding and thereby contributes to H2A–H2B deposition and nucleosome assembly (Andrews et al , 2010). …”

“…How chaperones orchestrate this process remains unclear (Loyola & Almouzni, 2004; De Koning et al , 2007; Elsasser & D'Arcy, 2012). yNap1 is a conserved histone chaperone that is well known to assemble nucleosomes in vitro but if yNap1 contributes to nucleosome assembly, in vivo has remained unknown (Ishimi & Kikuchi, 1991; Walter et al , 1995; Chang et al , 1997; Park et al , 2005; Lorch et al , 2006; Mazurkiewicz et al , 2006; Andrews et al , 2010). Our genomewide mapping of nucleosome positioning in a nap1 Δ strain revealed significantly smaller nucleosomes (subnucleosomes) on average (~120 bp) as compared to those of the wild‐type strain (~150 bp).…”

Partially Assembled Nucleosome Structures at Atomic Detail

“…The nucleosome can disassemble through the reverse reaction that starts with an opening of the (H3$H4) 2 tetramer-H2A$H2B dimers interface, followed by the release of H2A$H2B dimers from the DNA (14,28). It was also shown that H2A$H2B complexes with the DNA are effectively suppressed in vivo, even though the affinity of highly positively charged dimer to the DNA is substantial (29). Some PANS are likely to occur only in specific types of chromatin, e.g., centromeric (30).…”

Principles and practice of nucleosome positioningin vitro

“…This results in mixed populations of histone octamer on both donor and acceptor DNAs. Histone chaperones such as nucleoplasmin (Earnshaw et al 1980) and Nap1 (Ishimi and Kikuchi 1991) have also been shown to act as transfer agents under physiologically relevant salt conditions, and Nap1 has been proposed as route to measure nucleosome affinities (Andrews et al 2010).…”