Biochemical characteristics of the chondrocyte-enriched SNORC protein and its transcriptional regulation by SOX9

Prashant Kumar Jaiswal,Pierre Moffatt,Marie-Hélène Gaumond,Chun-Do Oh,Hideyo Yasuda,Latifa Aljebali

doi:10.1038/s41598-020-64640-x

Abstract

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.

Highlights

Bone development and formation involves a very complex series of events that need to be tightly orchestrated in a spatial and temporal manner[1,2]
The absence of signal in the calvaria indicated that Snorc is expressed in chondrocytes but not in osteoblasts. 5′RACE conducted on mRNA from day 16 differentiated ATDC5 chondrocytes led to the identification of two Snorc transcript variants
The long bones are formed through an endochondral ossification process, whereby a cartilage template is first deposited by chondrocytes and onto which bone further builds[1,2]

Summary

Introduction

Bone development and formation involves a very complex series of events that need to be tightly orchestrated in a spatial and temporal manner[1,2]. A vast number of genes expressed in growth plate chondrocytes that control longitudinal growth have been identified and characterized in detail[3]. These genes encode for many different protein types that are involved in as many different processes controlling gene transcription, cellular proliferation, differentiation, shape, mineralization, adhesion, and signaling. Heinonen et al named the gene Snorc (Small NOvel Rich in Cartilage, aka secondary ossification center associated regulator of chondrocyte maturation) and provided evidence for its expression in cartilages in mice and humans[13]. The aims of the present study were to corroborate the chondrocyte-specific nature of Snorc expression, further explore the biochemical and topological characteristics of the protein, and identify the mechanisms by which it is regulated at the transcriptional level

Objectives

Methods

Results

Conclusion