Abstract

The Helicase-related protein 3 (Hrp3), an ATP-dependent chromatin remodeling enzyme from the CHD family, is crucial for maintaining global nucleosome occupancy in Schizosaccharomyces pombe (S. pombe). Although the ATPase domain of Hrp3 is essential for chromatin remodeling, the contribution of non-ATPase domains of Hrp3 is still unclear. Here, we investigated the role of non-ATPase domains using in vitro methods. In our study, we expressed and purified recombinant S. pombe histone proteins, reconstituted them into histone octamers, and assembled nucleosome core particles. Using reconstituted nucleosomes and affinity-purified wild type and mutant Hrp3 from S. pombe we created a homogeneous in vitro system to evaluate the ATP hydrolyzing capacity of truncated Hrp3 proteins. We found that all non-ATPase domain deletions (∆chromo, ∆SANT, ∆SLIDE, and ∆coupling region) lead to reduced ATP hydrolyzing activities in vitro with DNA or nucleosome substrates. Only the coupling region deletion showed moderate stimulation of ATPase activity with the nucleosome. Interestingly, affinity-purified Hrp3 showed co-purification with all core histones suggesting a strong association with the nucleosomes in vivo. However, affinity-purified Hrp3 mutant with SANT and coupling regions deletion showed complete loss of interactions with the nucleosomes, while SLIDE and chromodomain deletions reduced Hrp3 interactions with the nucleosomes. Taken together, nucleosome association and ATPase stimulation by DNA or nucleosomes substrate suggest that the enzymatic activity of Hrp3 is fine-tuned by unique contributions of all four non-catalytic domains.

Highlights

  • Chromatin is the fundamental molecular structure for packing DNA into chromosomes

  • We used the reconstituted S. pombe nucleosomes to study the function of non-catalytic domains in the CHD1 homolog Helicase-related protein 3 (Hrp3)

  • We found that the association between Hrp3 and nucleosomes was reduced or abolished by all four domain deletions

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Summary

Introduction

Chromatin is the fundamental molecular structure for packing DNA into chromosomes. In eukaryotic cells, DNA is wrapped around histone octamers, which consist of two molecules of each histone H2A, H2B, H3, and H4 [1]. The human CHD1 enzyme binds to H3K4me through its paired chromo-domains [6]. It is required for maintaining an open chromatin state at active genes [7]. Mutations in its homolog CHD2 has been found to be associated with human cancers, such as chronic lymphoblastic leukemia (CLL) [11]. Both CHD1 and CHD2 have been shown to regulate histone H3/H3.3 occupancy and chromatin accessibility at the transcription start site (TSS) region of their target genes [12].

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