Two binding sites for thyroid transcription factor 1 (TTF-1) determine the activity of the bovine thyroglobulin gene upstream enhancer element.
A thyroid-specific enhancer element located upstream from the bovine thyroglobulin gene had been shown to contain three contiguous regions that are protected by thyroid transcription factor 1 (TTF-1) in footprinting experiments in vitro. The functional relevance of the individual TTF-1 binding sites was investigated in a transient assay in primary cultured thyrocytes. Using reporter constructs containing synthetic oligonucleotides overlapping the protected sequences we were able to show that only two out of the three TTF-1 binding sites exhibit transcription enhancing activity. Within the context of the complete enhancer sequence, the central 'inactive' TTF-1 site could be deleted whithout any consequence on the activity of the enhancer in the assay, whereas the presence of both terminal 'active' TTF-1 sites had previously been shown to be strictly required for enhancer function. Our results thus show that the activity of the bovine thyroglobulin upstream enhancer relies on the presence of a pair of TTF-1 binding sites separated by about 30 bp. These results also emphasize the need to assess experimentally the functional relevance of TTF-1 binding sites identified in footprinting experiments.
Christophe-Hobertus
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IRIBHN, Université Libre de Bruxelles, Faculté de Médecine, Belgium.
Christophe
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D
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002417:Cattle; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics; D018983:DNA Footprinting; D004742:Enhancer Elements, Genetic; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D017384:Sequence Deletion; D013954:Thyroglobulin / Q000235:genetics*; D013961:Thyroid Gland / Q000378:metabolism*; D000074482:Thyroid Nuclear Factor 1; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection
D009687:Nuclear Proteins; D000074482:Thyroid Nuclear Factor 1; D014157:Transcription Factors; D004247:DNA; D013954:Thyroglobulin