THE HISTONE CHAPERONE SPN1 PRESERVES SUBNUCLEOSOMAL STRUCTURES AT PROMOTERS AND NUCLEOSOME POSITIONING IN OPEN READING FRAMES

Spn1 is a multifunctional histone chaperone 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 yeast expressing wild-type or mutants of Spn1. We mapped nucleosome and subnucleosomal protections genome-wide, and surprisingly, we observed a genome-wide loss of subnucleosomal protection over nucleosome-depleted regions (NDRs) in the Spn1-K192N-containing strain, indicating critical functions of Spn1 in maintaining normal chromatin architecture in promoter regions. Additionally, alterations in nucleosome and hexasome positioning were observed in markedly different mutant Spn1 strains, demonstrating that multiple functions of Spn1 are required to maintain proper chromatin structure in open reading frames. Moreover, the impact of these functions correlated with gene expression and length. 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. MNase-SSP of following yeast strains: WT, spn1 K192N, aaand spn1 141-305 at 32U and 128U of MNase. Spike-in normalized RNA-seq of following yeast strains: WT, spn1 K192N, aaand spn1 141-305

Spn1是一种在真核生物生命活动中不可或缺的多功能组蛋白伴侣（histone chaperone）。此前的研究已阐明该蛋白参与诸多相互作用的关键结构区域，但目前尚不清楚这些结构域如何影响染色质结构的维持。本研究采用微球菌核酸酶（micrococcal nuclease, MNase）消化后进行单链文库制备与测序（MNase-SSP）的实验策略，对表达野生型或突变型Spn1的酵母菌株的染色质结构进行表征。我们在全基因组范围内绘制了核小体与亚核小体的保护区域；令人意外的是，在携带Spn1-K192N突变的菌株中，我们观察到核小体缺失区域（nucleosome-depleted regions, NDRs）的全基因组亚核小体保护信号丢失，这表明Spn1在维持启动子区域的正常染色质架构中发挥关键作用。此外，在两类差异显著的Spn1突变菌株中，我们观测到核小体与六聚核小体的定位发生改变，证实Spn1的多项功能对于维持开放阅读框（open reading frames）内的正常染色质结构是必需的。进一步分析发现，这些功能的影响程度与基因表达水平及基因长度密切相关。综上，本研究揭示了Spn1在维持NDR架构中此前未被报道的作用，并加深了我们对转录区域内依赖Spn1的染色质维持机制的理解。本研究包含以下酵母菌株的MNase-SSP测序数据：野生型（WT）、spn1 K192N、spn1 141-305，实验设置了32U与128U的微球菌核酸酶处理条件。同时包含以下酵母菌株的spike-in 归一化RNA测序（spike-in normalized RNA-seq）数据：野生型（WT）、spn1 K192N、spn1 141-305