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J Biol Chem, Vol. 273, Issue 33, 21115-21124, August 14, 1998

Transcriptional Activation of the Type II Transforming Growth Factor- Receptor Gene upon Differentiation of Embryonal Carcinoma Cells

David Kelly^§, Seong-Jin Kim, and Angie Rizzino^§

From the  Eppley Institute for Research in Cancer and Allied Diseases and ^§ Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska 68198 and the  NCI, National Institutes of Health, Bethesda, Maryland 20892

Previously, it has been shown that differentiation of embryonal carcinoma (EC) cells turns on the expression of functional transforming growth factor type- receptors. Here, we show that the type II receptor (TR-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 TR-II. In addition, the expression of TR-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 TR-II gene. Moreover, we identify a cAMP response element/activating transcription factor site that acts as a positive cis-regulatory element in the TR-II promoter, and we demonstrate that the transcription factor ATF-1 binds to this site and strongly stimulates the expression of the TR-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 TR-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 TR-II promoter expression. Together, these studies suggest that the transcription factors ATF-1 and NF-Y play important roles in the regulation of the TR-II gene.

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