Revisiting chromatin packaging in mouse sperm [CUT&RUN]

Mammalian sperm exhibit an unusual and heavily-compacted genomic packaging state. In addition to its role in organizing the compact and hydrodynamic sperm head, it has been proposed that sperm chromatin architecture helps to program gene expression in the early embryo. Scores of genome-wide surveys in sperm have reported patterns of chromatin accessibility, histone localization, histone modification, and chromosome folding. Here, we revisit these studies in light of recent reports that sperm obtained from the mouse epididymis are contaminated with low levels of cell-free chromatin. In the absence of proper sperm lysis we readily recapitulate multiple prominent genome-wide surveys of sperm chromatin, suggesting that these profiles primarily reflect contaminating cell-free chromatin. Removal of cell-free DNA, along with appropriate lysis conditions, are required to reveal a sperm chromatin state distinct from any yet reported. Using ATAC-Seq to explore relatively accessible genomic loci, we identify a landscape of open loci associated with genes expressed during late spermiogenesis. Histone modification and chromosome folding studies also strongly support the hypothesis that prior studies suffer from contamination, but technical challenges associated with reliably preserving the architecture of the compacted sperm head prevent us from confidently assaying true localization patterns for these epigenetic marks. Together, our studies strongly argue that our knowledge of mammalian chromosome packaging remains largely incomplete, and motivate future efforts to more accurately characterize genome organization in mature sperm. Overall design: Histone modification profiling in mouse sperm samples that are pre-treated with Dnase or DTT using CUTandRUN

哺乳动物精子呈现出一种独特且高度致密的基因组包装状态。除了在构建紧凑且具有流体动力学特性的精子头部中发挥作用外，有研究者提出，精子染色质架构还有助于调控早期胚胎的基因表达程序。
迄今已有数十项针对精子的全基因组研究，报道了染色质开放性、组蛋白定位、组蛋白修饰以及染色体折叠的相关模式。
鉴于近期有报道称从小鼠附睾分离得到的精子样本中存在低水平游离染色质污染，本研究重新审视了此前的相关研究。
若未采用恰当的精子裂解方法，我们可轻易重现多项经典的精子染色质全基因组检测结果，这表明此类检测图谱主要反映的是污染的游离染色质特征。
唯有去除游离DNA并结合优化的裂解条件，才能揭示出此前从未被报道过的真实精子染色质状态。
本研究利用转座酶可及性测序（ATAC-Seq）探究相对开放的基因组位点，鉴定出与精子发生后期表达基因相关的开放染色质区域图谱。
组蛋白修饰与染色体折叠相关研究也有力佐证了此前研究存在污染的假说，但由于难以稳定维持致密精子头部的染色质架构，我们无法可靠检测这些表观遗传标记的真实定位模式。
综上，本研究强烈表明，当前我们对哺乳动物染色体包装的认知仍存在大量空白，并呼吁未来开展更多研究，以精准解析成熟精子的基因组组织状态。
实验整体设计：采用CUTandRUN技术（CUTandRUN），对经脱氧核糖核酸酶（Dnase）或二硫苏糖醇（DTT）预处理的小鼠精子样本进行组蛋白修饰谱分析。