A retrotransposon gag-like-3 gene RTL3 and SOX-9 co-regulate the expression of COL2A1 in chondrocytes. (2nd November 2021)

• Record Type: Journal Article
• Authors: Ball, Hope C.; Ansari, Mohammad Y.; Ahmad, Nashrah; Novak, Kimberly; Haqqi, Tariq M.
• Is Part Of: Connective tissue research. Volume 62:Number 6(2021)
• Page Start: 615
• Page End: 628
• Publication Date: 2021-11-02
• Subjects: ZCCHC5/RTL3 -- gene regulation -- chondrocyte -- chondrogenesis -- extracellular matrix; Connective tissues -- Periodicals; 616.770072
• DOI: 10.1080/03008207.2020.1828380
• Languages: English
• ISSNs: 0300-8207

Abstract

ABSTRACT: Purpose: Transposable elements are known to remodel gene structure and provide a known source of genetic variation. Retrotransposon gag-like-3 (RTL3) is a mammalian retrotransposon-derived transcript ( MART ) whose function in the skeletal tissue is unknown. This study aimed to elucidate the biological significance of RTL3 in chondrogenesis and type-II collagen (COL2A1) gene expression in chondrocytes. Materials and Methods: Expression of RTL3, SOX-9 and COL2A1 mRNAs was determined by TaqMan assays and the protein expression by immunoblotting. RTL3 and Sox-9 depletion in human chondrocytes was achieved using validated siRNAs. An RTL3 mutant (∆RTL3) lacking the zinc finger domain was created using in vitro mutagenesis. Forced expression of RTL3, ∆RTL3, and SOX-9 was achieved using CMV promoter containing expression plasmids. CRISPR-Cas9 was utilized to delete Rtl3 and create a stable ATDC5 Rlt3-/- cell line. Matrix deposition and Col2a1 quantification during chondrogenesis were determined by Alcian blue staining and Sircol™ Soluble Collagen Assay, respectively. Results: RTL3 is not ubiquitously expressed but showed strong expression in cartilage, chondrocytes and synoviocytes but not in muscle, brain, or other tissues analyzed. Loss-of-function and gain-of-function studies demonstrated a critical role of RTL3 in the regulation of SOX-9 and COL2A1 expression and matrix synthesis during chondrogenesis. Both RTL3 and SOX-9 displayed co-regulated expression in chondrocytes. Gene regulatory activity of RTL3 requires the c-terminal CCHC zinc-finger binding domain. Conclusions: Our results identify a novel regulatory mechanism of COL2A1 expression in chondrocytes that may help to further understand the skeletal development and the pathogenesis of diseases with altered COL2A1 expression.