Nicotinamide Mononucleotide Promotes Female Germline Stem Cell Proliferation by Activating the H4K16ac-Hmgb1-Fyn-PLD Signaling Pathway through Epigenetic Remodeling [ATAC-seq]

Nicotinamide mononucleotide (NMN), an endogenous nucleotide essential for various physiological processes, has an unclear role and regulatory mechanisms in female germline stem cell (FGSC) development. In this study, we demonstrate that NMN significantly enhances FGSC viability and proliferation. Quantitative acetylation proteomics revealed that NMN markedly increases the acetylation of histone H4 at lysine 16 (H4K16ac). Subsequent chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq) identified high mobility group box 1 (Hmgb1) as a downstream target of H4K16ac, a finding further validated by ChIP-qPCR. Knockdown of Hmgb1 reduced FGSC proliferation by disrupting cell cycle progression, inducing apoptosis, and decreasing chromatin accessibility. High-throughput chromosome conformation capture (Hi-C) analysis showed that Hmgb1 knockdown induced A/B compartment switching, increased the number of topologically associating domains (TADs), and decreased chromatin loop formation in FGSCs. Notably, the chromatin loop at the promoter region of Fyn proto-oncogene (Fyn) disappeared following Hmgb1 knockdown. ChIP-qPCR and dual-luciferase reporter assays further confirmed the interaction between Hmgb1 and the Fyn promoter. Importantly, Fyn overexpression reversed the inhibitory effects of Hmgb1 knockdown on FGSC proliferation. Proteomic analysis suggested this rescue was mediated through the phospholipase D (PLD) signaling pathway, as Fyn overexpression selectively enhanced the phosphorylation of PLD1 at threonine 147 without affecting serine 561. Furthermore, treatment with 5-fluoro-2-indolyldechlorohaloamide, a PLD inhibitor, nullified the pro-proliferative effects of Fyn overexpression. In summary, our findings reveal that NMN promotes FGSC proliferation by activating the H4K16ac-Hmgb1-Fyn-PLD signaling pathway through epigenetic remodeling. These results deepen our understanding of FGSC proliferation and highlight potential therapeutic avenues for advancing FGSC applications in reproductive medicine. Overall design: To investigate the effect of Hmgb1-knockdown on the development of female germline stem cells (FGSCs), we treated FGSCs with Hmgb1-knockdown lentivirus in vitro. Subsequently, we utilized ATAC-seq data from both the control group and the Hmgb1-knockdown group to conduct differential gene expression analysis.

烟酰胺单核苷酸（Nicotinamide mononucleotide, NMN）是一类对多种生理过程至关重要的内源性核苷酸，但其在雌性生殖系干细胞（female germline stem cell, FGSC）发育中的作用与调控机制仍不明确。本研究证实，NMN可显著增强FGSC的活力与增殖能力。定量乙酰化蛋白质组学分析显示，NMN可显著提升组蛋白H4赖氨酸16位的乙酰化水平（H4K16ac）。后续的染色质免疫共沉淀测序（chromatin immunoprecipitation sequencing, ChIP-seq）与RNA测序（RNA sequencing, RNA-seq）鉴定出高迁移率族蛋白B1（high mobility group box 1, Hmgb1）为H4K16ac的下游靶标，该结论经ChIP-qPCR进一步验证。敲低Hmgb1可通过扰乱细胞周期进程、诱导细胞凋亡以及降低染色质开放程度，抑制FGSC的增殖。高通量染色体构象捕获（high-throughput chromosome conformation capture, Hi-C）分析显示，敲低Hmgb1会诱导FGSC发生A/B区室转换，增加拓扑关联结构域（topologically associating domains, TADs）的数量，并减少染色质环的形成。值得注意的是，Fyn原癌基因（Fyn proto-oncogene, Fyn）启动子区域的染色质环在Hmgb1敲低后消失。ChIP-qPCR与双荧光素酶报告基因实验进一步证实了Hmgb1与Fyn启动子的相互作用。值得一提的是，过表达Fyn可逆转Hmgb1敲低对FGSC增殖的抑制效应。蛋白质组学分析提示，该挽救作用通过磷脂酶D（phospholipase D, PLD）信号通路介导：过表达Fyn可选择性增强PLD1苏氨酸147位点的磷酸化，而不影响其丝氨酸561位点的磷酸化。此外，使用PLD抑制剂5-氟-2-吲哚二氯卤代酰胺（5-fluoro-2-indolyldechlorohaloamide）处理，可抵消Fyn过表达的促增殖作用。综上，本研究揭示NMN可通过表观遗传重塑激活H4K16ac-Hmgb1-Fyn-PLD信号通路，进而促进FGSC的增殖。上述结果加深了我们对FGSC增殖机制的理解，并为推动FGSC在生殖医学中的应用提供了潜在治疗靶点与研究方向。总体实验设计：为探究敲低Hmgb1对FGSC发育的影响，本研究体外使用Hmgb1敲低慢病毒处理FGSC。随后，我们利用对照组与Hmgb1敲低组的ATAC-seq数据进行差异基因表达分析。