Abstract
Previously, it has been shown that differentiation of embryonal carcinoma (EC) cells turns on the expression of functional transforming growth factor type-beta receptors. Here, we show that the type II receptor (TbetaR-II) gene is activated at the transcriptional level when EC cells differentiate. We show that the differentiated cells, but not the parental EC cells, express transcripts for TbetaR-II. In addition, the expression of TbetaR-II promoter/reporter gene constructs are elevated dramatically when EC cells differentiate and we identify at least two positive and two negative regulatory regions in the 5' flanking region of the TbetaR-II gene. Moreover, we identify a cAMP response element/activating transcription factor site that acts as a positive cis-regulatory element in the TbetaR-II promoter, and we demonstrate that the transcription factor ATF-1 binds to this site and strongly stimulates the expression of the TbetaR-II promoter/reporter gene constructs when ATF-1 is overexpressed in EC-derived differentiated cells. Equally important, we identify a negative regulatory element in a 53-base pair region that had previously been shown to inhibit strongly the expression of TbetaR-II promoter/reporter gene constructs. Specifically, we demonstrate that this region, which contains an inverted CCAAT box motif, binds the transcription factor complex NF-Y (also referred to as CBF) in vitro. Furthermore, expression of a dominant-negative NF-YA mutant protein, which prevents DNA binding by NF-Y, enhances TbetaR-II promoter expression. Together, these studies suggest that the transcription factors ATF-1 and NF-Y play important roles in the regulation of the TbetaR-II gene.
Highlights
It has been shown that differentiation of embryonal carcinoma (EC) cells turns on the expression of functional transforming growth factor type- receptors
Transcriptional Regulation of the TR-II Gene—Due to the large increase in the steady-state levels of TR-II mRNA when F9 EC cells are induced to differentiate, we examined the transcriptional activity of the TR-II gene promoter in F9 EC cells and their differentiated counterparts
We examined the expression of the TR-II gene in F9 EC and F9-differentiated cells, as TR-II expression is normally associated with ligand binding and growth responsiveness of cells to TGF- [21,22,23, 58]
Summary
It has been shown that differentiation of embryonal carcinoma (EC) cells turns on the expression of functional transforming growth factor type- receptors. Plex family of genetically distinct polypeptides that are secreted by virtually all cells and which exert potent effects on cell proliferation, differentiation, extracellular matrix production, and immunoregulation [1,2,3,4] Based on these activities and the defined spatial and temporal pattern of expression of the three mammalian isoforms of TGF- (TGF-1, TGF-2, and TGF-3) during mouse embryogenesis, it has been argued that the TGF-s play important roles in the generation and modification of extracellular signals that direct critical processes during mammalian development [5,6,7,8,9,10,11,12]. They can be induced to differentiate into many of the cell types formed during mammalian embryogenesis [37], making them well suited for the investigation of the signal
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