Distinct adjacent protein-binding domains in the glycoprotein hormone alpha gene interact independently with a cAMP-responsive enhancer.

The glycoprotein hormone alpha gene contains a duplicated cAMP-responsive element (CRE) that functions as a classical enhancer and interacts with a transcription factor termed CRE-binding protein. Adjacent to the CRE resides an upstream regulatory element (URE) that binds tissue-specific factors. The URE stimulates basal transcription but requires the CRE to impart its transcriptional effects. Protein binding to the URE and CRE domains is enhanced by linking the elements together, suggesting that interactions occur between the proteins that bind to these regulatory domains. Using gel mobility shift assays, we show that the URE forms distinct complexes with proteins extracted from JEG-3 cells. Two distinct protein-binding domains within the URE were delineated using mutational analyses and methylation interference assays. An upstream domain (-177 to -156 base pairs) interacts with a relatively abundant protein, whereas a downstream domain (-172 to -151 base pairs) binds a less abundant factor. Simultaneous occupancy of the two URE domains was not found, suggesting that binding of proteins to these regions may be mutually exclusive. In transient expression assays, the upstream and downstream domains of the URE were shown to independently enhance CRE-mediated transcription of the alpha gene. However, the downstream domain of the URE imparts greater transcriptional activity than does the upstream domain, and a DNA element that contains both URE domains is less active than is the downstream domain alone. These studies support a mechanism in which the transcriptional activity of a CRE can be differentially modulated by factors that bind to adjacent, but distinct upstream regulatory elements.