PMID:10993896

Citation	Carson, JA, Fillmore, RA, Schwartz, RJ and Zimmer, WE (2000) The smooth muscle gamma-actin gene promoter is a molecular target for the mouse bagpipe homologue, mNkx3-1, and serum response factor. J. Biol. Chem. 275:39061-72
Abstract	An evolutionarily conserved vertebrate homologue of the Drosophila NK-3 homeodomain gene bagpipe, Nkx3-1, is expressed in vascular and visceral mesoderm-derived muscle tissues and may influence smooth muscle cell differentiation. Nkx3-1 was evaluated for mediating smooth muscle gamma-actin (SMGA) gene activity, a specific marker of smooth muscle differentiation. Expression of mNkx3-1 in heterologous CV-1 fibroblasts was unable to elicit SMGA promoter activity but required the coexpression of serum response factor (SRF) to activate robust SMGA transcription. A novel complex element containing a juxtaposed Nkx-binding site (NKE) and an SRF-binding element (SRE) in the proximal promoter region was found to be necessary for the Nkx3-1/SRF coactivation of SMGA transcription. Furthermore, Nkx3-1 and SRF associate through protein-protein interactions and the homeodomain region of Nkx3-1 facilitated SRF binding to the complex NKE.SRE. Mutagenesis of Nkx3-1 revealed an inhibitory domain within its C-terminal segment. In addition, mNkx3-1/SRF cooperative activity required an intact Nkx3-1 homeodomain along with the MADS box of SRF, which contains DNA binding and dimerization structural domains, and the contiguous C-terminal SRF activation domain. Thus, SMGA is a novel target for Nkx3-1, and the activity of Nkx3-1 on the SMGA promoter is dependent upon SRF.
Links	PubMed Online version:10.1074/jbc.M006532200
Keywords	Actins/genetics; Animals; Base Sequence; Binding Sites; Birds; Cell Differentiation; Cell Line; Conserved Sequence; DNA-Binding Proteins/genetics; DNA-Binding Proteins/metabolism; Drosophila Proteins; Electrophoresis, Polyacrylamide Gel; Fibroblasts/metabolism; Glutathione Transferase/metabolism; Haplorhini; Homeodomain Proteins/genetics; Homeodomain Proteins/metabolism; Humans; Mesoderm/metabolism; Mice; Molecular Sequence Data; Muscle, Smooth/metabolism; Mutagenesis, Site-Directed; Nuclear Proteins/genetics; Nuclear Proteins/metabolism; Plasmids/metabolism; Precipitin Tests; Promoter Regions, Genetic; Protein Binding; Protein Structure, Tertiary; Recombinant Fusion Proteins/metabolism; Sequence Homology, Nucleic Acid; Serum Response Factor; Transcription Factors/genetics; Transcription Factors/metabolism; Transcription, Genetic; Transcriptional Activation; Transfection
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