Characterization of the nonmuscle myosin heavy chain IIB promoter: regulation by E2F.
To identify DNA sequences important for the transcriptional regulation of the nonmuscle myosin heavy chain IIB (NMMHC-IIB) gene we isolated and sequenced genomic clones that contain the promoter of the gene for both human and mouse. In addition to considerable homology in the first (untranslated) exon (91%) we found 80% sequence identity in the 700 base pairs immediately upstream of the major start of transcription (+1) as well as significant homologies as far as 1500 base pairs upstream. The promoter region was characterized using luciferase reporter constructs transiently transfected into NIH3T3 cells. Consensus binding sites for several known transcription factors are present that are completely conserved between the mouse and human genes, including CRE/ATF, Sp1, CAAT, and the cell-cycle transcription factor E2F. Gel shift assays indicated that E2F can bind to its putative binding site in vitro. To test whether this site is functional we cotransfected NMMHC-IIB promoter constructs driving luciferase with a vector expressing E2F-1. The E2F-1 vector stimulated luciferase activity from an intact promoter whereas mutation of the site eliminates binding and diminishes transactivation. These data provide strong evidence that E2F or an E2F-related transcription factor is involved in the regulation of nonmuscle myosin expression.
Weir
L
L
Department of Medicine (Cardiology), St. Elizabeth's Medical Center, Tufts University School of Medicine, Boston, MA 02135, USA.
Chen
D
D
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004247:DNA; D004268:DNA-Binding Proteins / Q000378:metabolism*; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009132:Muscles; D018995:Myosin Heavy Chains / Q000235:genetics*; D010442:Peptide Chain Initiation, Translational; D011401:Promoter Regions, Genetic; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation
C533431:Arid4a protein, mouse; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; C493754:E2f1 protein, mouse; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors; D004247:DNA; D018995:Myosin Heavy Chains