Novel cis-element in intron 1 represses somite expression of zebrafish myf-5

Myf-5 is a basic helix-loop-helix (bHLH) transcription factor that controls muscle differentiation. During early embryogenesis, myf-5 expression is transient, somite- and stage-specific. However, the negative regulation of myf-5 is poorly understood. We constructed a plasmid [(-9977/-1)/E1/I1/E2/GFP] that contains the sequence -9977 to -1, exon 1 (E1), intron 1 (I1), and exon 2 (E2) of zebrafish (Danio rerio) myf-5 and a reporter GFP gene. This plasmid was microinjected into zebrafish zygotes. Surprisingly, the somite-specific expression rate of reporter GFP in the transgenic embryos was extremely low (2%, n=392), compared to that of (-9977/-1)/GFP (92%, n=210). Dramatic repression of myf-5 expression was also observed in embryos microinjected with plasmids in which the sequence -8600/-1, -2937/-1 or -290/-1 was linked to E1/I1/E2/GFP. Thus, intron 1 contains a silencer that specifically represses the activity of myf-5. Functional analysis of intron 1 showed a strong, negative, cis-regulatory element was located at +502/+835. Its function was orientation- and position-dependent. The repressive capability of this silencer was completely dependent on two core motifs, IE1 (+502/+527) and IE2 (+816/+835), and a 156-bp spanning sequence that lies between them. This is the first study to identify a novel, cis-acting silencer in intron 1 that is crucial to negatively regulating zebrafish myf-5 expression.