Dynamic interplay of Sp1, YY1, and DUX4 in regulating FRG1 transcription with intricate balance

Abstract

Maintaining precise levels of FRG1 is vital. It's over-expression is tied to muscular dystrophy, while reduced levels are linked to tumorigenesis. Despite extensive efforts to characterize FRG1 expression and downstream molecular signaling, a comprehensive understanding of its regulation has remained elusive. This study focused on unravelling the cis -regulatory elements within the FRG1 gene and their interplay. Employing a dual luciferase reporter assay on fragments of the FRG1 promoter upstream of the transcription start site, we observed variations in FRG1 transcription induction. Our in-silico analysis unveiled binding sequences for Sp1 and DUX4 within FRG1 promoter region showing an enhanced luciferase signal. Conversely, we identified a YY1 binding sequence in the FRG1 promoter fragment showing decreased luciferase signal. Confirming these binding sites through site-directed mutagenesis, chromatin immunoprecipitation, and EMSA provided concrete evidence of Sp1, YY1, and DUX4's interaction within the FRG1 promoter. Additionally, interaction between Sp1, YY1, and DUX4 was elucidated using sequential chromatin immunoprecipitation (ChIP re-ChIP) and co-immunoprecipitation assays.

Furthermore, alterations in the expression levels of Sp1, YY1, and DUX4 resulted in parallel changes in FRG1 gene expression. Notably, YY1 exhibited the ability to suppress SP1 or DUX4-mediated FRG1 transcription activation, while Sp1 and DUX4 together could counteract YY1-mediated transcription suppression. Our cell proliferation and colony formation assay underscored the tumorigenic properties of these three transcription factors through the modulation of FRG1 expression levels. The in vitro results were verified in vivo using mouse xenograft model. Leveraging RNA sequencing data from various tissues in the GTEx portal, we established a correlation between FRG1, Sp1, and YY1.

In essence, this study revealed the vital cis-regulatory components residing in the FRG1 promoter. The combined influence of Sp1, YY1, and DUX4 plays a central role in controlling FRG1 expression.