Both muscle-specific and ubiquitous nuclear factors are required for muscle-specific expression of the myosin heavy-chain beta gene in cultured cells.
Expression of the myosin heavy-chain beta gene is controlled by multiple cis-acting regulatory elements in the 5' flanking region; two of these, referred to as A (-276 to -263) and B (-207 to -180), are essential for conferring muscle-specific activation on homologous and heterologous promoters. Here we report on the identification of nuclear protein factors that specifically bind to these two elements. By using the A element as a probe, as well as nuclear extracts from muscle cells, we found two protein-DNA complexes that displayed distinct bands in a gel mobility shift assay but had identical methylation interference patterns. One complex was present mainly in nuclear extracts from undifferentiated muscle and nonmuscle cells, whereas the other was observed mainly in nuclear extracts from differentiated muscle cells. Thus, the muscle-specific complex formation with the A element appears to be involved in determining tissue-specific expression. Furthermore, competition analysis demonstrated that the A-element-binding factors also bind to the muscle-CAT motif in the cardiac troponin T gene. By using the B element as a probe, we saw similar patterns of gel-shifted bands and methylation interference in nonmuscle and muscle nuclear extracts. In addition, both elements A and B were found to be necessary for tissue-specific expression, suggesting that the muscle-specific activation of the myosin heavy-chain beta gene may require interaction between a muscle-specific and a ubiquitous protein-DNA complex.
Shimizu
N
N
Department of Medicine, University of Chicago, Illinois 60637.
Dizon
E
E
Zak
R
R
D000818:Animals; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000235:genetics / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D008745:Methylation; D008969:Molecular Sequence Data; D009132:Muscles / Q000166:cytology / Q000378:metabolism*; D009218:Myosins / Q000235:genetics*; D009687:Nuclear Proteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013937:Thymidine Kinase / Q000235:genetics; D014336:Troponin / Q000235:genetics; D020107:Troponin T; D014407:Tumor Cells, Cultured
D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D014336:Troponin; D020107:Troponin T; D015500:Chloramphenicol O-Acetyltransferase; D013937:Thymidine Kinase; D009218:Myosins