A Novel Regulatory Mechanism of Type II Collagen Expression via a SOX9-dependent Enhancer in Intron 6

Hideyo Yasuda,Chun-Do Oh,Di Chen,Benoit De Crombrugghe,Jin-Hoi Kim

doi:10.1074/jbc.m116.758425

Abstract

Type II collagen α1 is specific for cartilaginous tissues, and mutations in its gene are associated with skeletal diseases. Its expression has been shown to be dependent on SOX9, a master transcription factor required for chondrogenesis that binds to an enhancer region in intron 1. However, ChIP sequencing revealed that SOX9 does not strongly bind to intron 1, but rather it binds to intron 6 and a site 30 kb upstream of the transcription start site. Here, we aimed to determine the role of the novel SOX9-binding site in intron 6. We prepared reporter constructs that contain a Col2a1 promoter, intron 1 with or without intron 6, and the luciferase gene. Although the reporter constructs were not activated by SOX9 alone, the construct that contained both introns 1 and 6 was activated 5-10-fold by the SOX9/SOX5 or the SOX9/SOX6 combination in transient-transfection assays in 293T cells. This enhancement was also observed in rat chondrosarcoma cells that stably expressed the construct. CRISPR/Cas9-induced deletion of intron 6 in RCS cells revealed that a 10-bp region of intron 6 is necessary both for Col2a1 expression and SOX9 binding. Furthermore, SOX9, but not SOX5, binds to this region as demonstrated in an electrophoretic mobility shift assay, although both SOX9 and SOX5 bind to a larger 325-bp fragment of intron 6 containing this small sequence. These findings suggest a novel mechanism of action of SOX5/6; namely, the SOX9/5/6 combination enhances Col2a1 transcription through a novel enhancer in intron 6 together with the enhancer in intron 1.

Highlights

Type II collagen ␣1 is specific for cartilaginous tissues, and mutations in its gene are associated with skeletal diseases
Its expression has been shown to be dependent on SOX9, a master transcription factor required for chondrogenesis that binds to an enhancer region in intron 1
SOX9 Binds to Intron 6 of the Col2a1 Gene—Based on the reported data, we expected that SOX9 would bind to the enhancer region in intron 1, but the ChIP-Seq data of SOX9 in the Col2a1 gene using rat chondrosarcoma cells (RCS cells) show that there is a strong SOX9-binding site in intron 6 and a weaker binding site in intron 1 (Fig. 1A)

Summary

Introduction

Type II collagen ␣1 is specific for cartilaginous tissues, and mutations in its gene are associated with skeletal diseases. SOX9, but not SOX5, binds to this region as demonstrated in an electrophoretic mobility shift assay, both SOX9 and SOX5 bind to a larger 325-bp fragment of intron 6 containing this small sequence. These findings suggest a novel mechanism of action of SOX5/6; namely, the SOX9/5/6 combination enhances Col2a1 transcription through a novel enhancer in intron 6 together with the enhancer in intron 1. Type II collagen ␣1 is specific for cartilaginous tissues and necessary for the normal embryonic development of the skeleton Mutations in this gene cause achondrogenesis, chondrodysplasia, and a loss of tensile strength of bones [11,12,13]

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Results

Conclusion