Alpha-myosin heavy chain gene regulation: delineation and characterization of the cardiac muscle-specific enhancer and muscle-specific promoter.
The alpha-myosin heavy chain (alpha-MHC) gene encodes a cardiac muscle-specific protein involved in active force generation. The mechanism responsible for restricting expression of this gene to the heart should provide clues for the identification of transcriptional regulatory events involved in the induction and maintenance of the cardiac cell lineage. In this report we dissect the alpha-MHC regulatory region to identify the components necessary for directing high levels of cardiac muscle-restricted expression. Deletion, site-specific mutant and heterologous promoter constructs were assayed for expression after injection into adult rat heart and skeletal muscle or transfection into non-muscle cells. These studies indicated that sequences from -344 to -156 directed high levels of cardiac-muscle specific expression from a heterologous promoter that was independent of position and orientation. This region includes a previously uncharacterized CArG box, alpha-MHC sequences from -86 to +16 promoted activity from two heterologous enhancers in a muscle-specific fashion. Mutational analysis of an E-box and a CArG box within the promoter revealed that they act as negative and positive regulatory elements, respectively. Based on competitive binding and supershift electrophoretic mobility shift assays, serum response factor was shown interact with the CArG boxes found in the promoter and enhancer. Similar experiments demonstrated that the E-box bound to a factor immunologically related to upstream stimulatory factor. Together, these results identify two distinct regions with different regulatory function that are critical for the tissue restricted expression of the alpha-MHC gene.
Molkentin
J D
JD
Department of Physiology, Medical College of Wisconsin, Milwaukee 53226, USA.
Jobe
S M
SM
Markham
B E
BE
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004252:DNA Mutational Analysis; D017931:DNA Primers; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000378:metabolism*; D018995:Myosin Heavy Chains / Q000096:biosynthesis* / Q000235:genetics; D009928:Organ Specificity; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D014162:Transfection
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D018995:Myosin Heavy Chains