Twist-mediated activation of the NK-4 homeobox gene in the visceral mesoderm of Drosophila requires two distinct clusters of E-box regulatory elements.
NK-4, also called msh2 and tinman, encodes a homeodomain transcription factor that is required for the development of the dorsal mesoderm and its derivatives in the Drosophila embryo. Genetic analyses indicate that NK-4 resides downstream of the mesodermal determinant twist, which encodes a basic helix-loop-helix-type transcription factor. However, the regulation of NK-4 by twist remains poorly understood. Using expression assays in cultured cells and transgenic flies, we show that two distinct clusters of E-box regulatory sequences, present upstream of the NK-4 gene, mediate NK-4 expression in the visceral mesoderm. These elements are conserved between the Drosophila melanogaster and Drosophila virilis NK-4 genes and serve as binding sites for Twist (E1 cluster) and NK-4 (E2 cluster) proteins. In cultured cells, Twist and NK-4 binding results in activation of NK-4 gene expression. In transgenic animals, the E1 and E2 clusters are functionally connected, and both elements are required for NK-4 activation in cells of the visceral mesoderm and also for NK-4 repression in cells of the somatic musculature. These results demonstrate that NK-4 is a direct transcriptional target for Twist and its own gene product in visceral mesodermal cells, supporting the idea that twist and NK-4 function in the subdivision of the mesoderm during Drosophila embryogenesis.
Lee
Y M
YM
Laboratory of Molecular Cardiology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Park
T
T
Schulz
R A
RA
Kim
Y
Y
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D004330:Drosophila / Q000196:embryology* / Q000235:genetics; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000196:embryology / Q000235:genetics; D018507:Gene Expression Regulation, Developmental; D005801:Genes, Homeobox; D017930:Genes, Reporter; D018257:Helix-Loop-Helix Motifs; D018398:Homeodomain Proteins / Q000235:genetics*; D006706:Homeostasis; D008648:Mesoderm / Q000378:metabolism*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D012097:Repressor Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D014158:Transcription, Genetic; D051879:Twist-Related Protein 1; D014781:Viscera / Q000196:embryology
D029721:Drosophila Proteins; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C497753:Twi protein, Drosophila; D051879:Twist-Related Protein 1; C102933:tin protein, Drosophila