Objectives To identify basic helix-loop-helix transcription factors expressed in the primate placenta. The basic helix-loop-helix (bHLH) class of transcription factors plays a critical role in cell-type determination and cell-specific gene transcription in a variety of tissues, including the central nervous system, the hematopoetic system, the pancreas, and skeletal muscle. We have hypothesized that there are placenta-specific members of this family which are critical for determining trophoblast differentiation at the time of early placental formation, and also play a role in trophoblast differentiation throughout placental development. We used RT-PCR to determine whether rhesus placenta, human placenta, differentiated rhesus embryonic stem cells, and human JEG-3 choriocarcinoma cells express components of the bHLH regulatory system. All cells or tissues expressed the negative regulatory modulators Id-2 and Id-3, as well as the positive heterodimeric partner E12. The E2A gene products E12 and E47 form heterodimers with cell-specific bHLH transcription factors to activate cell-specific target genes by binding to a conserved element, the E box. We examined whether placental cell nuclear extracts contain E box-binding activity, using enhancer motifs from the muscle creatine kinase, insulin, and immunoglobulin kappa genes, and a potential E box from the 5'-flanking DNA of the human chorionic gonadotropin beta subunit gene. With all probes, specific DNA-protein interactions were detected, and the different probes gave quantitatively or quantitatively distinct patterns in gel-mobility shift assays. With JEG-3 choriocarcinoma cells, treatment with 8-bromo-cAMP, which up-regulates hormone gene expression and differentiated function, dramatically reduced the E box-binding activities. The specific role of the bHLH regulatory system in primate trophoblast differentiation remains to be investigated. Key words placenta, gene transcription, helix-loop-helix, chorionic gonadotropin
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