A novel cAMP-dependent regulatory region including a sequence like the cAMP-responsive element, far upstream of the...

Deletion mutants in the 5' upstream sequence of the human CYP21A2 gene demonstrated a novel regulatory DNA element responsible for cAMP-dependent expression of the gene in the transient expression system using Y-1 cells (mouse adrenocortical tumor cell line). This regulatory element (-2574 to -2489 bp) was also found to exhibit a strong enhancer activity through heterologous promoters in response to cAMP and to contain a sequence like the cAMP-responsive element (CRE) and a CAAT-like sequence. The CRE-like sequence has a five-base motif (5'-TGACG-3') of the palindromic CRE consensus (TGACGTCA). Competitive gel mobility shift assays using nuclear extracts of bovine adrenal cortex with sequences typical of the binding sites for the binding proteins of CRE and the CAAT-like sequence revealed that these binding proteins, or related factors, bound to their cognate DNA binding sites in the upstream enhancer region of the CYP21A2 gene. These two enhancer elements and their cognate binding factors cooperate with previously identified tissue-specific enhancers (adrenal-specific protein factor and Ad4-like sequences) and their binding factors to express a high level of cAMP-responsive expression of the CYP21A2 gene.