The smooth muscle gamma-actin gene promoter is a molecular target for the mouse bagpipe homologue, mNkx3-1, and serum response factor.
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.
Carson
J A
JA
Department of Cellular and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
Fillmore
R A
RA
Schwartz
R J
RJ
Zimmer
W E
WE
D000199:Actins / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001717:Birds; D002454:Cell Differentiation; D002460:Cell Line; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D029721:Drosophila Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D000882:Haplorhini; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008648:Mesoderm / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D026362:Serum Response Factor; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
D000199:Actins; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D018398:Homeodomain Proteins; C491178:NKX3-1 protein, human; C102837:Nkx3-1 protein, mouse; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D026362:Serum Response Factor; D014157:Transcription Factors; C104163:bagpipe protein, Drosophila; D005982:Glutathione Transferase