Transcription of the dominant-negative helix-loop-helix protein Id1 is regulated by a protein complex containing the immediate-early response gene Egr-1.
The expression of Id1, a helix-loop-helix protein which inhibits the activity of basic helix-loop-helix transcription factors, is down-regulated during cellular differentiation and cell cycle withdrawal both in tissue culture models and in mouse embryos. In order to study the mechanism of control of Idl expression, we have isolated a 210-bp enhancer element in the upstream region of the Id1 gene whose activity recapitulates Id1 expression in C2C12 muscle cells and C3H10T1/2 fibroblasts: i.e., this element is active in proliferating cells in the presence of serum and completely inactivated upon mitogen depletion, cell cycle withdrawal, and (in the case of C2C12) induced myoblast differentiation. Using linker-scanning mutations and site-directed mutagenesis in transient transfection experiments, we have identified two functional elements within the 210-bp enhancer which are required for proper serum responsiveness. One element (A) contains a consensus Egr-1 binding site and additional flanking sequences required for optimal activity, and the other element (B) fits no known consensus. Gel shift experiments demonstrate that the protein complex binding to the A site contains Egr-1 and other proteins. This complex as well as a protein complex that binds to the B site is lost within 24 h of serum depletion, correlating with the down-regulation of Id1 expression. On the basis of these findings, we propose that the regulation of the Id1 response to serum is mediated in part by the early response gene Egr-1 and as such provides a signaling link between the early-growth-response transcription factors and dominant-negative helix-loop-helix proteins.
Tournay
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Cell Biology program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Benezra
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D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D016384:Consensus Sequence; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D051766:Early Growth Response Protein 1; D004622:Embryo, Mammalian; D004742:Enhancer Elements, Genetic; D005347:Fibroblasts; D005786:Gene Expression Regulation; D017781:Genes, Immediate-Early; D018257:Helix-Loop-Helix Motifs; D017874:Immediate-Early Proteins; D051798:Inhibitor of Differentiation Protein 1; D051379:Mice; D008809:Mice, Inbred C3H; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins; D012689:Sequence Homology, Nucleic Acid; D014157:Transcription Factors / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D016335:Zinc Fingers
D017931:DNA Primers; D004268:DNA-Binding Proteins; D051766:Early Growth Response Protein 1; C486109:Egr1 protein, mouse; C497455:Idb1 protein, mouse; D017874:Immediate-Early Proteins; D051798:Inhibitor of Differentiation Protein 1; D012097:Repressor Proteins; D014157:Transcription Factors