Shriners Hospitals for Children - Canada, Montreal, Quebec, Canada.
Department of Orthopedic Surgery, Rush University Medical Center, Chicago, IL, 60612, USA.
Sci Rep. 2020 May 8;10(1):7790. doi: 10.1038/s41598-020-64640-x.
Snorc (Small NOvel Rich in Cartilage) has been identified as a chondrocyte-specific gene in the mouse. Yet little is known about the SNORC protein biochemical properties, and mechanistically how the gene is regulated transcriptionally in a tissue-specific manner. The goals of the present study were to shed light on those important aspects. The chondrocyte nature of Snorc expression was confirmed in mouse and rat tissues, in differentiated (day 7) ATDC5, and in RCS cells where it was constitutive. Topological mapping and biochemical analysis brought experimental evidences that SNORC is a type I protein carrying a chondroitin sulfate (CS) attached to serine 44. The anomalous migration of SNORC on SDS-PAGE was due to its primary polypeptide features, suggesting no additional post-translational modifications apart from the CS glycosaminoglycan. A highly conserved SOX9-binding enhancer located in intron 1 was necessary to drive transcription of Snorc in the mouse, rat, and human. The enhancer was active independently of orientation and whether located in a heterologous promoter or intron. Crispr-mediated inactivation of the enhancer in RCS cells caused reduction of Snorc. Transgenic mice carrying the intronic multimerized enhancer drove high expression of a βGeo reporter in chondrocytes, but not in the hypertrophic zone. Altogether these data confirmed the chondrocyte-specific nature of Snorc and revealed dependency on the intronic enhancer binding of SOX9 for transcription.
Snorc（富含软骨的小 NOVel）已被鉴定为小鼠中的一种软骨细胞特异性基因。然而，人们对 SNORC 蛋白的生化特性知之甚少，也不知道该基因如何以组织特异性的方式进行转录调控。本研究的目的是阐明这些重要方面。在鼠和大鼠组织、分化（第 7 天）的 ATDC5 和 RCS 细胞中证实了 Snorc 表达的软骨细胞特性，在这些细胞中 Snorc 表达是组成型的。拓扑图谱和生化分析提供了实验证据，表明 SNORC 是一种 I 型蛋白，带有附着在丝氨酸 44 上的软骨素硫酸盐 (CS)。SNORC 在 SDS-PAGE 上的异常迁移归因于其一级多肽特征，表明除了 CS 糖胺聚糖外，没有其他翻译后修饰。位于内含子 1 中的高度保守的 SOX9 结合增强子对于在小鼠、大鼠和人中驱动 Snorc 的转录是必需的。该增强子在独立于方向的情况下以及位于异源启动子或内含子中时均具有活性。在 RCS 细胞中，使用 Crispr 介导的增强子失活导致 Snorc 的减少。携带内含子多聚化增强子的转基因小鼠在软骨细胞中驱动βGeo 报告基因的高表达，但在肥大区则不表达。这些数据共同证实了 Snorc 的软骨细胞特异性，并揭示了转录对 SOX9 结合内含子增强子的依赖性。
