A heterodimer of HEB and an E12-related protein interacts with the CD4 enhancer and regulates its activity in T-cell lines.
A T-lymphocyte-specific enhancer located 13 kb upstream of the murine CD4 gene was recently shown to be required for the developmentally regulated expression of CD4. We have previously identified three nuclear protein binding sites in this enhancer; one of these sites, CD4-3, is essential for expression and contains two E-box core motifs (CANNTG) adjacent to each other in the sequence TAACAGGTGTCAGCTGGT. In electrophoretic mobility shift assays using the CD4-3 oligonucleotide as a probe, three nuclear protein complexes, termed CD4-3A, -B, and -C, were detected with nuclear extracts from T-cell lines. CD4-3A, which involves nuclear protein binding to the 5' E-box, was detected only with nuclear extracts from lymphoid cells. Specific antisera were used to show that the CD4-3A complex contains a heterodimer or heterooligomer of basic helix-loop-helix transcriptional factors, E12 or a related factor and HEB, which is expressed predominantly in thymus. Consistent with this finding, in vitro-translated E12 and HEB proteins, as homodimers or heterodimers, bound preferentially to the 5' E-box. Point mutations in the 5' E-box, but not in the 3' E-box, abolished CD4 enhancer activity. Furthermore, overexpression of Id, a protein that forms inactive heterodimers with E12/E47, blocked CD4 enhancer activity in T cells. These results suggest that a heterodimer composed of HEB and E12 or a closely related protein plays a critical role in CD4 enhancer function by interacting with the 5' E-box motif of the CD4-3 site in vivo.
Sawada
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Department of Microbiology and Immunology, University of California, San Francisco 94143-0414.
Littman
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DR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins; D001665:Binding Sites; D001667:Binding, Competitive; D015704:CD4 Antigens / Q000235:genetics*; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D017354:Point Mutation; D013601:T-Lymphocytes / Q000378:metabolism*; D051820:TCF Transcription Factors; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors
D051792:Basic Helix-Loop-Helix Proteins; D015704:CD4 Antigens; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D051820:TCF Transcription Factors; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors; C489532:Tcf12 protein, mouse; C544913:Tcf7l1 protein, mouse