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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.

We demonstrate two synthetic single-cell systems that can be used to better understand how the acquisition of an orphan gene can affect complex phenotypes. The Arabidopsis orphan gene, Qua-Quine Starch ( QQS ) has been identified as a regulator of carbon (C) and nitrogen (N) partitioning across multiple plant species. QQS modulates this important biotechnological trait by replacing NF-YB (Nuclear Factor Y, subunit B) in its interaction with NF-YC. In this study, we expand on these prior findings by developing Chlamydomonas reinhardtii and Saccharomyces cerevisiae strains, to refactor the functional interactions between QQS and NF-Y subunits to affect modulations in C and N allocation. Expression of QQS in C. reinhardtii modulates C ( i.e. , starch) and N ( i.e. , protein) allocation by affecting interactions between NF-YC and NF-YB subunits. Studies in S. cerevisiae revealed similar functional interactions between QQS and the NF-YC homolog (HAP5), modulating C ( i.e. , glycogen) and N ( i.e. , protein) allocation. However, in S. cerevisiae both the NF-YA (HAP2) and NF-YB (HAP3) homologs appear to have redundant functions to enable QQS and HAP5 to affect C and N allocation. The genetically tractable systems that developed herein exhibit the plasticity to modulate highly complex phenotypes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Wang, Tonsager, Zheng, Wang, Stessman, Fang, Stenback, Campbell, Tanvir, Zhang, Cothron, Wan, Meng, Spalding, Nikolau and Li.)