Upstream Enhancer Activity in the Human Surfactant Protein B Gene Is Mediated by Thyroid Transcription Factor 1

Abstract

Surfactant protein B (SP-B) is selectively expressed in bronchiolar and alveolar epithelial cells of the lung. We identified an upstream enhancer located in the 5′-flanking region of the human SP-B gene (−439 to −331 base pair, hSP-B(−439/−331)) by deletion analysis of SP-B-luciferase constructs assessed in transfection assays in vitro. The element cis-activated the expression of an SV40 promoter-luciferase reporter gene in a human pulmunary adenocarcinoma cell line (H441-4). Three distinct binding sites for the nuclear transcription protein, thyroid transcription factor 1 (TTF-1), were identified, and the purified TTF-1 homeodomain was bound to the region of hSP-B(−439/−331). Co-transfection of H441-4 cells with the expression vector pCMV-TTF-1 trans-activated the native human SP-B promoter and the SV40 promoter fused with the SP-B enhancer. Mutations of the TTF-1 binding sites in the upstream enhancer blocked TTF-1 binding and transactivation activity. In summary, TTF-1 interacts with distinct proximal (−80 to −110) and distal (−439 to −331) cis-acting elements that regulate lung epithelial cell-specific transcription of the human SP-B gene. Surfactant protein B (SP-B) is selectively expressed in bronchiolar and alveolar epithelial cells of the lung. We identified an upstream enhancer located in the 5′-flanking region of the human SP-B gene (−439 to −331 base pair, hSP-B(−439/−331)) by deletion analysis of SP-B-luciferase constructs assessed in transfection assays in vitro. The element cis-activated the expression of an SV40 promoter-luciferase reporter gene in a human pulmunary adenocarcinoma cell line (H441-4). Three distinct binding sites for the nuclear transcription protein, thyroid transcription factor 1 (TTF-1), were identified, and the purified TTF-1 homeodomain was bound to the region of hSP-B(−439/−331). Co-transfection of H441-4 cells with the expression vector pCMV-TTF-1 trans-activated the native human SP-B promoter and the SV40 promoter fused with the SP-B enhancer. Mutations of the TTF-1 binding sites in the upstream enhancer blocked TTF-1 binding and transactivation activity. In summary, TTF-1 interacts with distinct proximal (−80 to −110) and distal (−439 to −331) cis-acting elements that regulate lung epithelial cell-specific transcription of the human SP-B gene.