Demethylation and reactivation of the fibrosis-suppressor gene Rasal1 is initiated by R-loop formation which facilitates recruitment of TET3 and GADD45g

Abstract Background Silencing of fibrosis suppressor genes through DNA methylation has been shown to contribute to fibrosis progression in the heart and in other organs. Growth Arrest and DNA Damage 45-(GADD45) and Ten-Eleven-Translocation (TET) proteins are involved in active DNA demethylation, but how they are recruited to the promoter of fibrosis suppressor genes has not yet been understood. Purpose To identify the mechanisms involved in site-specific demethylation of the promoter region of the cardiac fibrosis suppressor gene Rasal1. Methods We previously identified lncRNA Gm15749 located within the Rasal1 promoter region. Using DRIP qRT-PCR, we demonstrated an enrichment of R-loops at the Rasal1 promoter in non-fibrotic mouse cardiac fibroblasts. Such enrichment of R-loops was lost upon TGF-beta-treatment in activated “fibrotic” fibroblasts, and also upon Gm15749 knockdown in untreated non-fibrotic fibroblasts. ChIP qRT-PCR further revealed binding of GADD45g to the Rasal1 promoter and Co-IP experiments confirmed a physical interaction of TET3 and GADD45g. Results Studying the RAS Protein Activator Like 1 (Rasal1), which has been shown to be silenced in fibrosis of the heart due to promoter hypermethylation, we find that the lncRNA Gm15749 within the promoter region of Rasal1 is protective by forming an R-loop at the Rasal1 promoter. Gm15749 mediates the recruitment and binding of GADD45g, which then triggers the demethylation process by recruiting TET3 specifically to the Rasal1 promoter region. During cardiac fibrosis, all three factors; Gm15749, TET3, and GADD45g are significantly downregulated and the protection of Rasal1 is lost due to the failure of the formation of the demethylation complex. Conclusions Our results suggest a complex involving an R-loop forming lncRNA termed Gm15749, which interacts with the demethylation complex consisting of GADD45g and TET3 to perform site-specific demethylation at the Rasal1 promoter. Such R-loop formation may counteract fibrogenesis through reactivation of fibrosis-suppressor genes silenced by hypermethylation. Funding Acknowledgement Type of funding sources: None.