Homeobox protein Nkx-2.1
transcription cis-regulatory region binding
Functional study of the human thyroid peroxidase gene promoter.
Structure/function relationships in the human thyroid peroxidase gene promoter have been studied by deletion and mutation analyses and confronted with footprint patterns obtained with thyroid nuclear extracts and the purified thyroid transcription factor TTF-1. Crude nuclear extracts from dog thyroid primary cultures were shown to contain a binding activity recognizing the -119 to -105 segment of the promoter (coordinates relative to the transcriptional start site). Deletion, or site-directed mutagenesis of this segment dramatically reduced transcriptional activity in transient expression experiments on gene fusions of the thyroid peroxidase promoter and the growth hormone reporter. This binding activity was increased in nuclear extracts from thyrocytes cultured in the presence of the cAMP-agonist forskolin. A mutation that decreased the promoter function in forskolin-stimulated thyrocytes resulted in weakening of the corresponding footprint. The binding site displays no significant sequence similarities with known cAMP-responsive elements. Mutagenesis of another region of the promoter (-99 to -94) induced the binding of an additional factor, resulting in a dramatically enhanced promoter activity. We show that the thyroid-specific transcriptional factor TTF-1 is not directly involved in the above-mentioned interactions and provide evidence suggesting that, in spite of displaying a similar binding pattern to thyroperoxidase and thyroglobulin promoters in vitro, TTF-1 plays a less important role in the former. Altogether, our data delineate the minimal thyroid peroxidase gene promoter in the human and identify the binding sites of two trans-activating factors, one of them being potentially the mediator of a non-conventional cAMP control, independent of the cAMP-responsive element and factor AP-2.