BMAL1 and CLOCK proteins directly bind to the core enhancer of the Myod1 promoter

Abstract

The molecular clock regulating circadian rhythms is a network of transcriptional‐translational feedback loops that drive rhythmic expression of genes over a 24‐h period. BMAL1 and CLOCK are two of the main transcription factors that regulate the molecular clock mechanism. Myod1 (myogenic differentiation 1) was identified as having a circadian pattern of expression in adult skeletal muscle. The primary objective of this study was to determine whether CLOCK and BMAL1 bind the Myod1 promoter in a temporal manner. Mice were entrained for 2 weeks in 12 hour light /12 hour dark and then hindlimb muscles were collected every 4 hours for 24 hours. The chromatin immuno‐precipitation (ChIP) assay with nuclear extracts from mouse muscle was performed. We tested binding of BMAL1, CLOCK, and RNA Polymerase to the core enhancer (CE) and distal regulatory region (DRR) of the Myod1 promoter. Results from these studies determined that BMAL1 and CLOCK did bind to the Myod1 promoter but only to the upstream core enhancer CE and not the DRR. Binding of BMAL1 and CLOCK at the CE was also found to oscillate over the course of the circadian cycle. We conclude that Myod1 is a direct clock controlled gene with BMAL1 and CLOCK regulating Myod1 mRNA levels via direct binding to the CE element of the promoter.This research was supported by National Institute of Health grants KAE (AR045617) and JJM (AR053641).