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Thyroid Hormone-Dependent Gene Expression in Differentiated Embryonic Stem Cells and Embryonal Carcinoma Cells: Identification of Novel Thyroid Hormone Target Genes by Deoxyribonucleic Acid Microarray Analysis

Molecular Endocrinology Laboratory, Departments of Medicine and Physiology, David Geffen School of Medicine at University of California, Los Angeles, and Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California 90073

Address all correspondence and requests for reprints to: Gregory A. Brent, Molecular Endocrinology Laboratory, VA Greater Los Angeles Healthcare System, Building 114, Room 230, 11301 Wilshire Boulevard, Los Angeles, California 90073. E-mail: gbrent{at}ucla.edu.

T₃ is required for normal early development, but relatively few T₃-responsive target genes have been identified. In general, in vitro stem cell differentiation techniques stimulate a wide range of developmental programs, including thyroid hormone receptor (TR) pathways. We developed several in vitro stem cell models to more specifically identify TR-mediated gene expression in early development. We found that embryonic carcinoma (EC) cells have reduced T₃ nuclear binding capacity and only modestly express the known T₃ target genes, neurogranin (RC3) and Ca²⁺/calmodulin-dependent protein kinase IV (CaMKIV), in response to T₃. Full T₃ induction in transient transfection of EC cells was restored with cotransfection of a TR expression vector. We, therefore, performed gene expression profiles in wild-type embryonic stem (ES) cells compared with expression in cells with deficient (EC) or mutant TR (TR P398H mutant ES cells), to identify T₃ target genes. T₃ stimulation of wild-type ES cells altered mRNA expression of 610 known genes (26% of those studied), although only approximately 60 genes (1%) met criteria for direct T₃ stimulation based on the magnitude of induction and requirement for the presence of TR. We selected five candidate T₃ target genes, neurexophilin 2, spermatid perinuclear RNA-binding protein (SPNR), kallikrein-binding protein (KBP), prostate-specific membrane antigen (PSMA), and synaptotagmin II, for more detailed study. T₃ responsiveness of these genes was evaluated in both in vitro endogenous gene expression and in vivo mouse model systems. These genes identified in a novel stem cell system, including those induced and repressed in response to T₃, may mediate thyroid hormone actions in early development.