Smooth muscle alpha-actin CArG elements coordinate formation of a smooth muscle cell-selective, serum response factor-containing activation complex.
Previous studies have shown that multiple serum response factor (SRF)-binding CArG elements were required for smooth muscle cell (SMC)-specific regulation of smooth muscle (SM) alpha-actin expression. However, a critical question remains as to the mechanisms whereby a ubiquitously expressed transcription factor such as SRF might contribute to SMC-specific expression. The goal of the present study was to investigate the hypothesis that SMC-selective expression of SM alpha-actin is due at least in part to (1) unique CArG flanking sequences that distinguish the SM alpha-actin CArGs from other ubiquitously expressed CArG-dependent genes such as c-fos, (2) cooperative interactions between CArG elements, and (3) SRF-dependent binding of SMC-selective proteins to the CArG-containing regions of the promoter. Results demonstrated that specific sequences flanking CArG B were important for promoter activity in SMCs but not in bovine aortic endothelial cells. We also provided evidence indicating that the structural orientation between CArGs A and B was an important determinant of promoter function. Electrophoretic mobility shift assays and methylation interference footprinting demonstrated that a unique SRF-containing complex formed that was selective for SMCs and, furthermore, that this complex was probably stabilized by protein-protein interactions and not by specific interactions with CArG flanking sequences. Taken together, the results of these studies provide evidence that SM alpha-actin expression in SMCs is complex and may involve the formation of a unique multiprotein initiation complex that is coordinated by SRF complexes bound to multiple CArG elements.
Mack
C P
CP
Department of Molecular Physiology and Biological Physics, University of Virginia Medical School, Charlottesville, USA.
Thompson
M M
MM
Lawrenz-Smith
S
S
Owens
G K
GK
D000199:Actins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001011:Aorta / Q000166:cytology; D002417:Cattle; D002478:Cells, Cultured; D018983:DNA Footprinting; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D004730:Endothelium, Vascular / Q000737:chemistry / Q000166:cytology / Q000502:physiology*; D050981:Erythroid-Specific DNA-Binding Factors; D005786:Gene Expression Regulation / Q000502:physiology; D009131:Muscle, Smooth, Vascular / Q000737:chemistry / Q000166:cytology / Q000502:physiology*; D016296:Mutagenesis / Q000502:physiology; D009687:Nuclear Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic / Q000502:physiology*; D011485:Protein Binding / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000032:analysis; D051381:Rats; D026362:Serum Response Factor; D014157:Transcription Factors / Q000032:analysis; D014158:Transcription, Genetic / Q000502:physiology; D050807:ets-Domain Protein Elk-1; D050808:ets-Domain Protein Elk-4
D000199:Actins; D004268:DNA-Binding Proteins; C494114:Elk1 protein, rat; D050981:Erythroid-Specific DNA-Binding Factors; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D026362:Serum Response Factor; D014157:Transcription Factors; D050807:ets-Domain Protein Elk-1; D050808:ets-Domain Protein Elk-4