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The homeodomain protein Distal-less 3 (Dlx3) is essential for normal placental development in mice. Dlx3-null mice die by embryonic day 10.0 due to placental failure. The aim of our studies was to examine the transcriptional regulation and expression of Dlx3 in choriocarcinoma cell lines and primary trophoblasts from human placenta. A Dlx3 promoter fragment coupled to a luciferase reporter gene was sufficient to increase luciferase activity more than 11-fold over a luciferase control vector in choriocarcinoma cells, but not in a heterologous gonadotrope cell line. A 5' deletion series of the Dlx3 promoter revealed that a 13-nucleotide CCAAT box-containing element was required for basal expression in choriocarcinoma cell lines. Mutation of the CCAAT box within the context of the full-length promoter resulted in reduced basal activation of the Dlx3 reporter gene, suggesting that the CCAAT box was required for full basal expression. Western blot analysis revealed that Dlx3, CCAAT/enhancer-binding protein alpha (C/EBP alpha), and C/EBP beta were present in choriocarcinoma cells and isolated trophoblasts from term human placentas. Electrophoretic mobility shift assays revealed the formation of a specific complex between choriocarcinoma cell nuclear extracts and the Dlx3 CCAAT box sequence. Competition and antibody electrophoretic mobility shift assays revealed that CCAAT/enhancer-binding protein beta (C/EBP beta) binds the Dlx3 CCAAT box sequence. Overexpression of C/EBP beta was sufficient to increase basal expression of a Dlx3 reporter gene in a dose-dependent manner. These studies provide the first insight into the mechanism(s) of Dlx3 gene expression in placental cells and suggest a role for C/EBP beta in the basal regulation of the Dlx3 gene.

A Role for CCAAT/Enhancer-Binding Protein β in the Basal Regulation of the Distal-Less 3 Gene Promoter in Placental Cells