Zhou Hong, Liu Yujie, Tian Geng G, Wu Ji
Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (Ministry of Education), Bio-X Institutes, Shanghai Jiao Tong University, Shanghai, 200240, China.
School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, 200240, China.
Cell Biosci. 2025 Apr 17;15(1):48. doi: 10.1186/s13578-025-01387-w.
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.
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.
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.
烟酰胺单核苷酸（NMN）是各种生理过程所必需的内源性核苷酸，在雌性生殖系干细胞（FGSC）发育中的作用和调控机制尚不清楚。
我们证明NMN显著增强FGSC的活力和增殖。定量乙酰化蛋白质组学显示，NMN显著增加组蛋白H4赖氨酸16位点（H4K16ac）的乙酰化。随后的染色质免疫沉淀测序（ChIP-seq）和RNA测序（RNA-seq）确定高迁移率族蛋白B1（Hmgb1）是H4K16ac的下游靶点，这一发现通过ChIP-qPCR得到进一步验证。敲低Hmgb1会破坏细胞周期进程、诱导细胞凋亡并降低染色质可及性，从而减少FGSC增殖。高通量染色体构象捕获（Hi-C）分析表明，敲低Hmgb1会诱导A/B区室转换，增加拓扑相关结构域（TAD）的数量，并减少FGSC中的染色质环形成。值得注意的是，Hmgb1敲低后，原癌基因Fyn（Fyn）启动子区域的染色质环消失。ChIP-qPCR和双荧光素酶报告基因检测进一步证实了Hmgb1与Fyn启动子之间的相互作用。重要的是，Fyn过表达逆转了Hmgb1敲低对FGSC增殖的抑制作用。蛋白质组学分析表明，这种挽救是通过磷脂酶D（PLD）信号通路介导的，因为Fyn过表达选择性地增强了PLD1苏氨酸147位点的磷酸化，而不影响丝氨酸561位点。此外，用PLD抑制剂5-氟-2-吲哚基去氯卤酰胺处理可消除Fyn过表达的促增殖作用。
我们的研究结果表明，NMN通过表观遗传重塑激活H4K16ac-Hmgb1-Fyn-PLD信号通路，促进FGSC增殖。这些结果加深了我们对FGSC增殖的理解，并突出了推进FGSC在生殖医学中应用的潜在治疗途径。
