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Sp3 controls fibroblast growth factor receptor 4 gene activity during myogenic differentiation
Eric Cavanaugh1, Joseph X DiMario1
1School of Graduate and Postdoctoral Studies and Chicago Medical School, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road, North Chicago, Illinois 60064, United States.
Abstract:
Fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) signaling is a critical component in the regulation of myoblast proliferation and differentiation. The transient FGFR4 gene expression during the transition from proliferating myoblasts to differentiated myotubes indicates that FGFR4 regulates this critical phase of myogenesis. The Specificity Protein (SP) family of transcription factors controls FGFR family member gene activity. We sought to determine if members of the Sp family regulate mouse FGFR4 gene activity during myogenic differentiation. RT-PCR and western blot analysis of FGFR4 mRNA and protein revealed transient expression over 72h, with peak expression between 24 and 36h after addition of differentiation medium to C2C12 myogenic cultures. Sp3 also displayed a transient expression pattern with peak expression occurring after 6h of differentiation. We cloned a 1527bp fragment of the mouse FGFR4 promoter into a luciferase reporter. This FGFR4 promoter contains eight putative Sp binding sites and directed luciferase gene activity comparable to native FGFR4 expression. Overexpression of Sp1 and Sp3 showed that Sp1 repressed FGFR4 gene activity, and Sp3 activated FGFR4 gene activity during myogenic differentiation. Mutational analyses of multiple Sp binding sites within the FGFR4 promoter revealed that three of these sites were transcriptionally active. Electromobility shift assays and chromatin immunoprecipitation of the area containing the activator sites showed that Sp3 bound to this promoter location.
Insights
Specificity Protein 3 (Sp3) activates mouse FGFR4 gene expression during muscle cell differentiation. Sp3 binds to the FGFR4 promoter, regulating this crucial step in myogenesis.
Area of Science:
- Molecular Biology
- Cell Biology
- Developmental Biology
Background:
- Fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) signaling regulates myoblast proliferation and differentiation.
- FGFR4 gene expression is transient during myogenesis, suggesting a regulatory role.
Purpose of the Study:
- To investigate if Specificity Protein (Sp) family members regulate mouse FGFR4 gene activity during myogenic differentiation.
- To identify the role of Sp transcription factors in controlling FGFR4 expression.
Main Methods:
- RT-PCR and Western blot to analyze FGFR4 and Sp3 expression.
- Cloning of the mouse FGFR4 promoter into a luciferase reporter.
- Overexpression studies of Sp1 and Sp3.
- Mutational analysis of Sp binding sites.
- Electromobility shift assays and chromatin immunoprecipitation.
Main Results:
- FGFR4 mRNA and protein showed transient expression during C2C12 cell differentiation (peak 24-36h).
- Sp3 expression was also transient, peaking early (6h) in differentiation.
- Sp1 repressed, while Sp3 activated, FGFR4 gene activity.
- Sp3 was shown to bind to specific active sites on the FGFR4 promoter.
Conclusions:
- Sp3 is a key activator of mouse FGFR4 gene expression during myogenic differentiation.
- Sp3 directly binds to the FGFR4 promoter, mediating its transcriptional regulation.
- These findings elucidate a novel regulatory mechanism in myogenesis involving Sp transcription factors and FGFR4.