Transcriptional regulation of porcine PABPN1 gene in adipogenesis
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Objective: This study aims to reveal the transcriptional regulatory mechanism and effects of poly(A)-binding protein nuclear 1 (PABPN1) on adipogenesis, along with associated polymorphisms.Methods: Transcription factors were identified using dual-luciferase reporter assay, overexpression techniques site-directed mutagenesis, real-time quantitative polymerase chain reaction (qPCR), electrophoretic mobility shift assays and chromatin immunoprecipitation-qPCR. Preadipocyte differentiation was measured with gain- and loss-of-function, Oil Red O staining and extraction assays. Single nucleotide polymorphisms (SNPs) were identified with direct sequencing of PCR products in the promoter, and the effects of these SNPs on PABPN1 expression were identified with dual-luciferase reporter assay.Results: CCAAT/enhancer-binding protein (C/EBP) α and β regulate PABPN1 expression by directly binding to its promoter. PABPN1 promotes the preadipocyte differentiation in pigs. Three SNPs were identified, with the Haplotype GCC mutation significantly increasing the promoter activity of PABPN1.Conclusion: PABPN1 promotes the preadipocyte differentiation as a downstream gene of C/EBP α and β. Haplotype GCC may serve as a molecular marker for selection of fat traits in pigs.
Abstract
Objective: This study aims to reveal the transcriptional regulatory mechanism and effects of poly(A)-binding protein nuclear 1 (PABPN1) on adipogenesis, along with associated polymorphisms. Methods: Transcription factors were identified using dual-luciferase reporter assay, over expression techniques site-directed mutagenesis, real-time quantitative polymerase chain reaction (qPCR), electrophoretic mobility shift assays and chromatin immunoprecipita tion-qPCR. Preadipocyte differentiation was measured with gain- and loss-of-function, Oil Red O staining and extraction assays. Single nucleotide polymorphisms (SNPs) were identified with direct sequencing of PCR products in the promoter, and the effects of these SNPs on PABPN1 expression were identified with dual-luciferase reporter assay. Results: CCAAT/enhancer-binding protein (C/EBP) α and β regulate PABPN1 expression by directly binding to its promoter. PABPN1 promotes the preadipocyte differentiation in pigs. Three SNPs were identified, with the Haplotype GCC mutation significantly increas ing the promoter activity of PABPN1. Conclusion: PABPN1 promotes the preadipocyte differentiation as a downstream gene of C/EBP α and β. Haplotype GCC may serve as a molecular marker for selection of fat traits in pigs.