Novel regulatory sequence -82/-62 functions as a key element to drive the somite-specificity of zebrafish myf-5.
Myf-5, a transcription factor that controls muscle differentiation, is expressed in somites during early embryogenesis. However, gene regulation of myf-5 is poorly understood and detailed functional analysis of the regulatory cis-elements is needed. In zebrafish, the myf-5 upstream sequence from -82 to -62 (-82/-62) was fused with a basal promoter and transferred to fertilized zebrafish eggs. The -82/-62 cassette drove green fluorescent protein (GFP) reporter gene expression specifically in the somites. Moreover, GFP signals were detected exclusively in the somites of 28-hpf embryos derived from eggs injected with pCMV-5x(-82/-62), which contained five copies of the -82/-62 cassette inserted within cytomegalovirus promoter/enhancer. Thus, the -82/-62 cassette, conserved in mouse myf-5, functions to drive somite-specific expression and to repress nonspecific expression during the early development of zebrafish embryos. Mutated sequence analysis of -82/-62 cassette showed that the -70/-62 sequence was the key element for controlling myf-5 specificity. The putative CCAAT-like box, located at -66/-62, could not direct somite-specific expression. A DNA-protein complex was specifically formed between the -70/-62 probe and embryonic nuclear extracts. We conclude that the -70/-62 motif is essential for controlling somite-specific expression and the CCAAT-like box is essential for activating gene transcription.
Chen
Yau-Hung
YH
Institute of Molecular and Cell Biology, National Taiwan University, Taipei, Taiwan.
Lee
Hung-Chieh
HC
Liu
Chia-Feng
CF
Lin
Cheng-Yung
CY
Tsai
Huai-Jen
HJ
D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D003587:Cytomegalovirus / Q000235:genetics; D004268:DNA-Binding Proteins; D018507:Gene Expression Regulation, Developmental; D005809:Genes, Regulator; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D008954:Models, Biological; D009124:Muscle Proteins / Q000737:chemistry / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000254:growth & development; D016297:Mutagenesis, Site-Directed; D051787:Myogenic Regulatory Factor 5; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017384:Sequence Deletion; D019170:Somites / Q000378:metabolism*; D013997:Time Factors; D015534:Trans-Activators; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D015027:Zebrafish / Q000196:embryology*
D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; D009124:Muscle Proteins; D051787:Myogenic Regulatory Factor 5; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D049452:Green Fluorescent Proteins