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Characterization of the Promoter Region of the Rat CCAAT/Enhancer-Binding Protein Gene and Regulation by Thyroid Hormone in Rat Immortalized Brown Adipocytes¹

Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Cientificas-Universidad Autónoma de Madrid (A.M.-H., A.P.-C.), 28029 Madrid; and Departamento de Bioquímica y Biología Molecular, Facultad Medicina, Universidad Complutense de Madrid (A.S.), Madrid 28029, Spain

Address all correspondence and requests for reprints to: Dr. A. Pérez-Castillo, Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Cientificas-Universidad Autónoma de Madrid, 28029 Madrid, Spain. E-mail: aperez{at}iib.uam.es

CCAAT/enhancer binding proteins (C/EBP) are a family of transcription factors with a highly conserved basic/leucine zipper (bZIP) domain that has been implicated in the transcriptional control of genes involved in cell growth and differentiation. We have previously demonstrated that the expression of C/EBP and C/EBPß genes is regulated by thyroid hormone in rat liver during development. The aim of the present study was to explore the molecular mechanisms underlying the control of C/EBP gene expression by thyroid hormone. To achieve this goal, we isolated and characterized a genomic clone containing 1171 bp of the 5'-flanking region of the rat C/EBP gene. This fragment was an active promoter in MB492 cells, an immortalized brown adipocyte cell line that expresses the endogenous C/EBP gene in a T₃-dependent manner. Sequence analysis suggested the presence of three thyroid hormone response elements, TRE-1 (-602/-589), TRE2 (-411/-396), and TRE3 (-376/-350). The results of deletion, mutagenesis, and gel mobility shift analysis disclosed that only TRE-1, an ER2-type response element, represented a functional T₃ response element. Our results demonstrate that T₃ is a factor that positively regulates C/EBP gene expression in a direct fashion.

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