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Based on competition analysis of gel shift assays utilizing nuclear extracts from the mouse adrenal Y1 cell line, the cAMP-responsive element of the human CYP21B gene (-129/-96 base pairs (bp] is found to contain two overlapping nuclear protein binding elements. One, -126/-113 bp, interacts specifically with an adrenal-specific protein factor (ASP) while the other, -119/-110 bp, contains a GC boxlike sequence and binds Sp1. The nucleotide replacements (GG----CC at -113 and -112 bp) introduced within the sequence -129/-96 bp greatly decreased the binding affinities of both Sp1 and ASP to the fragment and resulted in a loss of cAMP-enhanced transcription of a reporter gene in transient transfection experiments. When the 14-bp ASP-binding sequence was inserted in front of the reporter gene, induced transcription by cAMP was observed that could be increased by multimerization of this -126/-113-bp sequence. The -126/-113-bp fragment revealed specific binding only with nuclear extracts from adrenal Y1 cells among various types of cells tested, indicating that the protein factor ASP is specifically expressed in adrenal cells. The nucleotide replacement (G----C at -117 bp) of the -126 -113-bp sequence not only abolishes the binding to ASP but also the cAMP-dependent transcription, strongly supporting the hypothesis that ASP is a tissue-specific transacting protein that directly functions as a transcription factor essential to the cAMP-dependent transcription. Furthermore, the -129/-115-bp sequence of the bovine cytochrome P450C21 gene corresponding to the human -126/-113-bp sequence is demonstrated to be functionally conserved with respect to both cAMP-dependent enhancement of transcription and ASP binding.