Two binding sites for thyroid transcription factor 1 (TTF-1) determine theactivity of the bovine thyroglobulin gene upstream enhancer element

A thyroid-specific enhancer element located upstream from the bovine thyrog lobulin gene had been shown to contain three contiguous regions that are pr otected by thyroid transcription factor 1 (TTF-I) in footprinting experimen ts in vitro. The functional relevance of the individual TTF-I binding sites was investigated in a transient assay in primary cultured thyrocytes. Usin g reporter constructs containing synthetic oligonucleotides overlapping the protected sequences we were able to show that only two out of the three TT F-1 binding sites exhibit transcription enhancing activity. Within the cont ext of the complete enhancer sequence, the central 'inactive' TTF-1 site co uld be deleted whithout any consequence on the activity of the enhancer in the assay, whereas the presence of both terminal 'active' TTF-I sites had p reviously been shown to be strictly required for enhancer function. Our res ults thus show that the activity of the bovine thyroglobulin upstream enhan cer relies on the presence of a pair of TTF-I binding sites separated by ab out 30 bp. These results also emphasize the need to assess experimentally t he functional relevance of TTF-I binding sites identified in footprinting e xperiments. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.