MyoD binds cooperatively to two sites in a target enhancer sequence: occupancy of two sites is required for activation.
MyoD is a master regulatory gene for myogenesis. Its product, the MyoD protein, appears to act by binding to muscle-specific enhancer sequences. We show that MyoD binds cooperatively to two sites in the muscle-specific creatine kinase enhancer; this is dramatically reflected in dissociation-rate measurements. A deletion of the acidic N terminus (residues 3-56) results in a protein that binds normally to single sites but fails to bind cooperatively to two adjacent sites, suggesting a role of the N terminus in cooperative interactions. In transfection assays, a reporter gene flanked by a single MyoD binding site fails to be activated by cotransfected MyoD expression vectors. In contrast, a reporter with two or more MyoD binding sites is activated by wild-type MyoD but not by N-terminally deleted MyoD. A reporter gene with a single binding site, although not activated by MyoD, can nonetheless compete for expression with a reporter gene containing three sites. Thus, in vivo, a single site can bind MyoD, but occupancy of two or more sites is required for subsequent transcriptional activation.
Weintraub
H
H
Fred Hutchinson Cancer Research Center, Seattle, WA 98104.
Davis
R
R
Lockshon
D
D
Lassar
A
A
D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation / Q000235:genetics*; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D003402:Creatine Kinase / Q000235:genetics*; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D005809:Genes, Regulator; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000235:genetics; D007527:Isoenzymes; D008969:Molecular Sequence Data; D009132:Muscles / Q000166:cytology* / Q000201:enzymology; D015345:Oligonucleotide Probes; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis
D007527:Isoenzymes; D015345:Oligonucleotide Probes; D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase; D005982:Glutathione Transferase; D003402:Creatine Kinase