Promoter activity and electromobility shift assays localized the site of interaction of EMX2 with the slow MyHC2 gene within a defined binding site located between 3336 and 3326 bp from the 3′ end of the cloned slow MyHC2 DNA containing the transcriptional regulatory region. Using clonally-derived myoblasts stably committed to the formation of fast/slow muscle fibers, they also report the effect of altered EMX2 gene expression on genome-wide gene expression within these myoblasts as indicated by RNA sequencing.
Research concluded that increased EMX2 gene expression in fast/slow myoblasts caused altered gene expression of 1185 genes, indicating that EMX2 plays a central role in the gene expression profile of embryonic myoblasts.
RT-PCR analysis of select genes. The expression of seven genes, analyzed by qRT-PCR, is shown. RNAs were isolated from uninfected myoblasts, myoblasts infected with the CMVGFP lentivirus, and myoblasts infected with the CMVEMX2 lentivirus. Control reactions contained no reverse transcriptase (− RT). Genes analyzed included: Nuclear Factor of Activated T Cells 3 (NFATC3), General Transcription Factor IIH Polypeptide 3 (GTF2H3), GLI family zinc finger 1 (GLI1), Myoferlin (MYOF), Wnt1 Inducible Signaling Pathway 1 (WISP1), Frizzled Class Receptor 7 (FZD7), and Fragile X Mental Retardation 1 (FMR1).