A GC Box in the Human Sodium Iodide Symporter Gene Promoter is Essential for Full Activity

Abstract

We previously reported that the human sodium iodide symporter (hNIS) 5′-flanking region between-603 and -415 is essential for full expression. In this study, we further localized sequences within this region required for the basal expression of the hNIS promoter and identified a functional GC box. Activity of the hNIS promoter was assessed by transient transfection of luciferase reporter gene constructs with progressive 5′ deletions into the human papillary thyroid cancer cell line BHP 2-7. Deletion from -603 to -535 enhanced promoter activity, further deletion to -469 decreased promoter activity, and deletion to -415 nearly abolished promoter activity. The DNA sequence within this critical 55 bp region from -469 to -415 contains a GC box. Introduction of mutations into the GC box of the deletion constructs -603, -535 and -469 decreased promoter activity in both thyroid cells (BHP 2-7 and rat thyroid cell FRTL-5) and nonthyroid cells (human prostate cancer cell LNCaP-2 and human breast cancer cell MCF-7). The magnitude of reduction for the -603 mutation construct was significantly greater than that for the -469 mutation construct in thyroid cells compared to nonthyroid cells. In vitro transcription using nuclear extracts isolated from HeLa cells was reduced from DNA templates with the GC box mutation and nearly abolished from templates with 5′ deletion to -415. Identification of proteins interacting with the GC box was performed by gel retardation assays with or without Sp1 specific antibodies. Sp1 and an "Sp1-like" protein bound to the wild-type GC box sequence but not to the GC box mutant. In summary, the GC box is a positive regulatory element in the hNIS basal promoter.