Chondrocyte differentiation is a critical process for endochondral ossification, which is responsible for long bone development and fracture repair. Considerable progress has been made in understanding the transcriptional control of chondrocyte differentiation; however, epigenetic regulation of chondrocyte differentiation remains to be further studied. NSD1 is a H3K36 (histone H3 at lysine 36) methyltransferase. Here, we showed that mice with Nsd1 deficiency in Prx1⁺ mesenchymal progenitors but not in Col2⁺ chondrocytes showed impaired skeletal growth and fracture healing accompanied by decreased chondrogenic differentiation. Via combined RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) analysis, we identified sex determining region Y box 9 (Sox9), the key transcription factor of chondrogenic differentiation, as a functional target gene of NSD1. Mechanistically, NSD1 regulates Sox9 expression by modulating H3K36me1 and H3K36me2 levels in the Sox9 promoter region, constituting a novel epigenetic regulatory mechanism of chondrogenesis. Moreover, we found that NSD1 can directly activate the expression of hypoxia-inducible factor 1α (HIF1α), which plays a vital role in chondrogenic differentiation through its regulation of Sox9 expression. Collectively, the results of our study reveal crucial roles of NSD1 in regulating chondrogenic differentiation, skeletal growth, and fracture repair and expand our understanding of the function of epigenetic regulation in chondrogenesis and skeletal biology.

软骨细胞分化是软骨内骨化的关键过程，负责长骨发育和骨折修复。在理解软骨细胞分化的转录控制方面取得了相当大的进展；然而，软骨细胞分化的表观遗传调控仍有待进一步研究。NSD1 是 H3K36（赖氨酸 36 处的组蛋白 H3）甲基转移酶。在这里，我们展示了在 Prx1 ⁺间充质祖细胞中具有Nsd1缺陷但在 Col2 ^{+ 中}没有Nsd1缺陷的小鼠软骨细胞显示骨骼生长和骨折愈合受损，并伴有软骨分化减少。通过结合RNA测序（RNA-seq）和染色质免疫沉淀测序（ChIP-seq）分析，我们确定了性别决定区Y框9（Sox9），软骨分化的关键转录因子，作为NSD1的功能靶基因。从机制上讲，NSD1通过调节Sox9启动子区域的H3K36me1 和 H3K36me2 水平来调节Sox9表达，构成了软骨形成的新表观遗传调控机制。此外，我们发现 NSD1 可以直接激活缺氧诱导因子 1α (HIF1α) 的表达，该因子通过调节Sox9表达。总的来说，我们的研究结果揭示了 NSD1 在调节软骨形成分化、骨骼生长和骨折修复中的关键作用，并扩大了我们对表观遗传调控在软骨形成和骨骼生物学中的作用的理解。