Twist-mediated Activation of the NK-4 Homeobox Gene in the Visceral Mesoderm of Drosophila Requires Two Distinct Clusters of E-box Regulatory Elements*
- From the ‡Laboratory of Molecular Cardiology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892 and the¶Department of Biochemistry and Molecular Biology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030
Abstract
NK-4, also called msh2and 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 ofNK-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 twistand NK-4 function in the subdivision of the mesoderm duringDrosophila embryogenesis.
Footnotes
-
↵* This research was supported by the NHLBI Intramural Research Program (to Y. K.) and a grant from the Human Frontier Science Program (to R. A. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) AF00436.
-
↵§ Present address: Dept. of Molecular Biology, Kon-Kuk University, Chung-Ju 380, Korea.
-
↵‖ To whom correspondence should be addressed: LMC, NHLBI, NIH, Bldg. 10, Rm. 8N228, 10 Center Dr., MSC 1762, Bethesda, MD 20892-1762. Tel.: 301-496-3672; Fax: 301-402-1542; E-mail:yongsok{at}helix.nih.gov.
-
↵1 The abbreviations used are: bHLH, basic helix-loop-helix; CAT, chloramphenicol acetyltransferase; kb, kilobase pair(s); PCR, polymerase chain reaction; bp, base pair(s); CMV, cytomegalovirus; GST, glutathione S-transferase.
-
↵2 Y. M. Lee, T. Park, and Y. Kim, unpublished results.
-
↵3 K. Chung and Y. Kim, unpublished results.
-
↵4 Y. M. Lee and Y. Kim, manuscript in preparation.
-
- Received February 19, 1997.
- Revision received May 5, 1997.
