YAO, Meng; ROH , Hyunsuk ; DENVER, Robert J.; Univ of Michigan, Ann Arbor; Univ of Michigan, Ann Arbor; Univ of Michigan, Ann Arbor: Structural and Functional Analysis of Corticotropin-Releasing Factor Genes in Xenopus laevis

Corticotropin releasing factor (CRF) plays a central role in the regulation of neuroendocrine, autonomic, immune, and behavioral responses to physical and emotional stress. To understand the evolution of the regulation of the CRF gene in vertebrates we analyzed the gene structure and up to 1.5 kb of upstream sequence of two CRF genes of Xenopus laevis. Using a comparative genomics approach, we searched for conserved regions of the CRF genes among vertebrates. Our analyses revealed that the structures and nucleotide sequences of the X. laevis CRF genes are highly conserved with mammalian CRF genes. Several potential glucocorticoid response elements (GREs) were identified by bioinformatics analysis, which could mediate transcriptional regulation by activated glucocorticoid receptor (GR). Using electrophoretic mobility shift assay we found that the X. laevis GR specifically bound to the putative GRE sequences. The X. laevis CRF genes, as the mammalian genes, possess consensus AP1 and cyclic AMP response element (CRE) sites to which Fos-Jun heterodimers or phosphorylated CREB, respectively, could bind to regulate CRF gene expression. We tested the functionality of the identified CRE and GRE sites using cell transfection. In transiently transfected PC-12 cells, the proximal promoters of the X. laevis CRF genes fused to firefly luciferase were activated by forskolin, which was partially blocked by treatment with dexamethasone. Point mutation of the putative CRE site in the core promoter significantly reduced its upregulation by forskolin. Our data show that the basic regulatory elements of the CRF gene responsible for stress-induced expression arose early in vertebrate evolution. (Supported by NSF grant IBN 0235401 to R.J.D.)