ABSTRACT
Spn1 is a multifunctional histone chaperone that associates with RNA polymerase II during elongation and is essential for life in eukaryotes. While previous work has elucidated regions of the protein important for its many interactions, it is unknown how these domains contribute to the maintenance of chromatin structure. Here, we employ digestion by micrococcal nuclease followed by single-stranded library preparation and sequencing (MNase-SSP) to characterize chromatin structure in Saccharomyces cerevisiae expressing wild-type or mutants of Spn1. We mapped protections of all sizes genome-wide, and surprisingly, we observed a widespread loss of short fragments over nucleosome-depleted regions (NDRs) at promoters in the Spn1-K192N-containing strain, indicating critical functions of Spn1 in maintaining normal chromatin architecture outside open reading frames. Additionally, there are shifts in DNA protections in the Spn1 mutant expressing strains over open reading frames, which indicate changes in nucleosome and subnucleosome positioning. This was observed in markedly different mutant Spn1 strains, demonstrating that multiple functions of Spn1 are required to maintain proper chromatin structure in open reading frames. Taken together, our results reveal a previously unknown role of Spn1 in the maintenance of NDR architecture and deepen our understanding of Spn1-dependent chromatin maintenance over transcribed regions.
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