ABSTRACT
Ras signaling elicits diverse outputs, yet how Ras specificity is generated remains incompletely understood. We demonstrate that Wingless (Wg) and Decapentaplegic (Dpp) confer competence for receptor tyrosine kinase-mediated induction of a subset of Drosophila muscle and cardiac progenitors by acting both upstream of and in parallel to Ras. In addition to regulating the expression of proximal Ras pathway components, Wg and Dpp coordinate the direct effects of three signal-activated (dTCF, Mad, and Pointed-functioning in the Wg, Dpp, and Ras/MAPK pathways, respectively) and two tissue-restricted (Twist and Tinman) transcription factors on a progenitor identity gene enhancer. The integration of Pointed with the combinatorial effects of dTCF, Mad, Twist, and Tinman determines inductive Ras signaling specificity in muscle and heart development.
Subject(s)
Bacterial Proteins , Body Patterning/genetics , Cell Lineage/genetics , Drosophila Proteins , Signal Transduction/genetics , Transcription Factors/genetics , ras Proteins/genetics , ras Proteins/metabolism , Animals , Binding Sites/genetics , DNA-Binding Proteins , Drosophila/embryology , Drosophila/genetics , Drosophila/metabolism , Enhancer Elements, Genetic/genetics , Eye Proteins/genetics , Eye Proteins/metabolism , Gene Expression Regulation, Developmental/genetics , Heart/embryology , Homeodomain Proteins/genetics , Insect Proteins/genetics , Insect Proteins/metabolism , Mesoderm/metabolism , Muscle, Skeletal/cytology , Muscle, Skeletal/embryology , Muscle, Skeletal/metabolism , Myocardium/cytology , Myocardium/metabolism , Nerve Tissue Proteins , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/genetics , Receptor Protein-Tyrosine Kinases/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , Stem Cells/cytology , Stem Cells/metabolism , Wnt1 ProteinABSTRACT
The even-skipped (eve) gene of Drosophila melanogaster is a crucial member of the pair-rule class of segmentation genes. We report here the characterization of a 16-kb region sufficient for all known aspects of eve expression and the rescue of an eve null mutation. We began by examining 45 kb surrounding the eve coding sequence for DNaseI hypersensitive sites and other transcription units. We find that the previously identified eve regulatory elements, those for early stripes 2, 3, and 7 and the late element, do not generate prominent hypersensitive sites. However, strong, constitutive DNaseI hypersensitive sites flank a 16-kb region, within which one developmentally regulated site is found at the eve promoter region. P-element transformation of this 16-kb domain into eve mutants rescues them to adult viability. This 16-kb domain contains regulatory elements for all known features of eve expression: the seven major blastoderm stripes, minor stripe expression during germ band extension, and later expression in the lateral mesodermal muscle precursor cells, in the central nervous system, adjacent to the invaginating proctodeum, and in a ring around the anal pad. We have begun a preliminary dissection of the 16-kb domain into its constituent regulatory elements. Other major findings include the following: (1) There is a second element for late stripe expression adjacent to the traditional late element. (2) A stripe element 3' of the gene interacts with the late element to give rise to the minor stripes seen in the even-numbered parasegments. (3) Expression in the proctodeum and anal pad is driven by sequences both 5' and 3' of the gene. (4) Expression in different sites in the central nervous system is driven by separable elements widely dispersed throughout 8 kb 3' of the gene.
Subject(s)
Bacterial Proteins , Body Patterning/genetics , Chromatin/genetics , Drosophila Proteins , Drosophila melanogaster/genetics , Genes, Insect , Homeodomain Proteins/genetics , Transcription Factors , Animals , Chromosome Mapping , Cloning, Molecular , Deoxyribonuclease I/metabolism , Drosophila melanogaster/embryology , Gene Expression Regulation, Developmental/genetics , Genes, Reporter , In Situ Hybridization , Mutation , RNA, Messenger/genetics , TransgenesABSTRACT
In this study we investigate the gene regulatory functions of Drosophila Fish-hook (Fish), a high mobility group (HMG) Sox protein that is essential for embryonic segmentation. We show that the Fish HMG domain binds to the vertebrate Sox protein consensus DNA binding sites, AACAAT and AACAAAG, and that this binding induces an 85 degrees DNA bend. In addition, we use a heterologous yeast system to show that the NH2-terminal portion of Fish protein can function as a transcriptional activator. Fish directly regulates the expression of the pair rule gene, even-skipped (eve), by binding to multiple sites located in downstream regulatory regions that direct formation of eve stripes 1, 4, 5, and 6. Fish may function along with the Drosophila POU domain proteins Pdm-1 and Pdm-2 to regulate eve transcription, as genetic interactions were detected between fish and pdm mutants. Finally, we determined that Fish protein is expressed in a dynamic pattern throughout embryogenesis, and is present in nuclear and cytoplasmic compartments.
Subject(s)
DNA-Binding Proteins/physiology , Drosophila Proteins , Drosophila/genetics , Gene Expression Regulation, Developmental/physiology , High Mobility Group Proteins/physiology , Transcription Factors/physiology , Animals , Base Sequence , DNA Primers , Drosophila/embryology , SOX Transcription Factors , Transcriptional Activation/physiologyABSTRACT
BACKGROUND: The Drosophila gene runt plays multiple roles during embryogenesis, including one as a pair-rule class segmentation gene. The runt protein (Runt) contains an evolutionarily conserved domain (the Runt domain) that is found in several mammalian proteins including the human protein AML1, which is involved in many chromosome translocations associated with leukaemia. Specific DNA binding activity of a mammalian Runt domain is enhanced by a partner protein called PEBP2beta/CBFbeta. DNA binding activity of Drosophila Runt is also stimulated by this protein, suggesting the existence of a similar Runt partner protein in Drosophila. RESULTS: We report here the cloning of two closely linked Drosophila genes, runt domain partner (rp) beta1 and beta2, that encode homologues of mouse PEBP2beta/CBFbeta. They are highly homologous to each other and to the mammalian counterpart. Either of the rpb proteins is capable of forming a complex with Runt and stimulating its DNA binding activity, but their temporal and spatial distributions are quite dissimilar, suggesting that functional specificity of Runt may be conferred by the interacting partner. Runt represses transcription dominantly when coexpressed with either partner in cultured cells, a function consistent with a direct role for Runt in regulating expression of the even-skipped gene in Drosophila embryos. CONCLUSIONS: Drosophila Runt can interact with either of two Runt domain partners, and the resulting complex functions as an active repressor of transcription.
Subject(s)
DNA-Binding Proteins/metabolism , DNA-Binding Proteins/physiology , Drosophila Proteins , Drosophila/genetics , Transcription Factors/physiology , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Cells, Cultured , Cloning, Molecular , Consensus Sequence , DNA/metabolism , DNA-Binding Proteins/genetics , Drosophila/metabolism , Gene Expression Regulation , In Situ Hybridization , Mice , Molecular Sequence Data , Nuclear Proteins , Repressor Proteins , Sequence Homology, Amino Acid , Time Factors , Transcription Factors/geneticsABSTRACT
The even-skipped (eve) gene of Drosophila melanogaster has been intensively studied as a model for spatial and temporal control of gene expression, using in vitro and transgenic techniques. Here, the study of eve is extended, using evolutionary conservation of DNA sequences. Conservation of much of the protein, and of known regulatory elements, supports models for eve function and regulation that have previously been advanced, and extensive conservation found in noncoding sequences predicts that functional elements exist that have yet to be defined. In contrast, a part of the protein implicated in transcriptional repression has diverged extensively while preserving overall amino acid composition, highlighting potentially essential features of this domain. Also, the basal promoter has diverged extensively, indicating evolutionary flexibility of promoter function.
