192 Identification of Fos and FosB as Transcriptional Regulators of Bovine Satellite Cell Differentiation

Abstract Transcription factors (TFs) are key regulators of gene expression during cell differentiation. Four TFs including Myf5, MyoD, MyoG and Myf6 have been identified as key myogenic regulatory factors (MYFs) that regulate gene transcription during myogenesis. Satellite cells (SCs) are the myogenic precursor cells in adult skeletal muscle. The objective of this study was to identify additional TFs that control the differentiation of bovine satellite cells. Bovine satellite cells (bSCs) were isolated from 4 crossbred steers and were initially cultured in growth medium for 12 days to expand and then in differentiation medium for 48 hours to differentiate. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) was performed to identify chromatin regions marked with acetylation of histone H3 on lysine 27 (H3K27ac). This ChIP-seq analysis revealed 3,348 and 38,800 H3K27ac-associated chromatin regions in bSCs before and after differentiation, respectively. A motif enrichment analysis of the H3K27ac-marked chromatin regions from the differentiated bSCs indicated the enrichment of binding sites for the 4 MYFs and many other TFs including Fos and FosB. RNA-sequencing revealed the upregulation of Fos and FosB mRNAs in bSCs from growth to differentiation. To verify the roles of Fos and FosB in bSC differentiation, their expressions in bSCs were reduced by siRNA-induced knockdown. Based on qRT-PCR analyses, expressions of MYH2, MYH3, MYOG, and CKM mRNAs, which were selected as markers of muscle cell differentiation, were increased (P < 0.05) in bSCs from growth to differentiation, but the increases in at least three of them were reversed (P < 0.05) by Fos or FosB knockdown. Taken together, these results establish Fos and FosB as transcriptional regulators of bovine satellite cell differentiation.