Two upstream enhancers collaborate to regulate the spatial patterning and timing of <i>MyoD</i> transcription during mouse development

<jats:title>Abstract</jats:title><jats:p>MyoD is a member of the basic‐helix‐loop‐helix (bHLH) transcription factor family, which regulates muscle determination and differentiation in vertebrates. While it is now well established that the <jats:italic>MyoD</jats:italic> gene is regulated by Sonic hedgehog, Wnts, and other signals, it is not known how <jats:italic>MyoD</jats:italic> transcription is initiated and maintained in response to these signals. We have investigated the <jats:italic>cis</jats:italic> control of <jats:italic>MyoD</jats:italic> expression to identify and characterize the DNA targets that mediate <jats:italic>MyoD</jats:italic> transcription in embryos. By monitoring <jats:italic>lacZ</jats:italic> reporter gene expression in transgenic mice, we show that regulatory information contained in 24 kb of human <jats:italic>MyoD</jats:italic> 5′ flanking sequence is sufficient to accurately control <jats:italic>MyoD</jats:italic> expression in embryos. Previous studies have identified two muscle‐specific regulatory regions upstream of <jats:italic>MyoD</jats:italic>, a 4‐kb region centered at −20 kb (designated fragment 3) that contains a highly conserved 258‐bp core enhancer sequence, and a more proximal enhancer at −5 kb, termed the distal regulatory region (DRR), that heretofore has been identified only in mice. Here, we identify DRR‐related sequences in humans and show that DRR function is conserved in humans and mice. In addition, transcriptional activity of <jats:italic>MyoD</jats:italic> 5′ flanking sequences in somites and limb buds is largely a composite of the individual specificities of the two enhancers. Deletion of fragment 3 resulted in dramatic but temporary expression defects in the hypaxial myotome and limb buds, suggesting that this regulatory region is essential for proper temporal and spatial patterning of <jats:italic>MyoD</jats:italic> expression. These data indicate that regulatory sequences in fragment 3 are important targets of embryonic signaling required for the initiation of <jats:italic>MyoD</jats:italic> expression. © 2001 Wiley‐Liss, Inc.</jats:p>