Roles of the conserved CCAAT and GC boxes of the human and mouse type II transforming growth factor-beta receptor genes

Abstract

Embryonal carcinoma (EC) cells are used widely to study the molecular mechanisms that regulate the transcription of genes during mammalian embryogenesis. The type II transforming growth factor-beta receptor (TbetaR-II) gene is expressed at very low levels by mouse EC cells prior to differentiation. Differentiation of EC cells results in increases of both the steady-state levels of TbetaR-II mRNA and the activity of the TbetaR-II promoter. Several cis-regulatory elements have been shown previously to regulate the TbetaR-II gene. This study focuses on the role of a CCAAT box and three GC boxes in the regulation of the human and mouse TbetaR-II promoters in EC-differentiated cells. We demonstrate that the CCAAT box and two flanking GC boxes, Sp A and Sp B, function as positive regulatory elements in the human TbetaR-II promoter, and that the transcription factor complex NF-Y positively regulates the human TbetaR-II promoter through the CCAAT box motif. We also show that the CCAAT box and the downstream GC box Sp B, which are conserved between the human and mouse promoters, behave as positive regulatory elements in the mouse TbetaR-II promoter. In addition, we demonstrate that the transcription factor Sp1 can bind to the Sp B GC box in vitro. Finally, we show that a GC box located 25 bp upstream of the major transcription start site of the TbetaR-II gene plays a minimal role in the function of the TbetaR-II promoter in EC-differentiated cells. Together, our studies highlight important differences and similarities in the cis-regulatory elements that regulate the human and mouse TbetaR-II promoters.