Regulated activity of the distal promoter-like element of the human corticotropin-releasing hormone gene and secondary structural features of its corresponding transcripts

Abstract

Corticotropin-releasing hormone (CRH) plays a major role in the coordination of the stress response. Its gene is expressed in multiple brain regions, the peripheral sympathetic system and the placenta, as well as in peripheral inflammatory sites where CRH acts as a pro-inflammatory cytokine. The human (h) CRH gene, in addition to its primary promoter (TATA box I), has a second distal promoter-like structure (TATA box II) and a functional cyclic adenosine monophosphate-responsive element, all of which are preserved in the rat and ovine genes. To examine the functionality of TATA II, we positioned a 881-bp-long segment of the 5′ flanking region of the hCRH gene containing TATA II, but lacking TATA I, upstream from a chloramphenicol acetyltransferase (CAT) reporter gene cloned in a pUC vector. We transfected COS-7 cells with this construct and examined responsiveness of CAT activity to potential stimulants and inhibitors. Phorbol ester (TPA) and forskolin had mild but clear stimulatory effects on CAT expression (~ 1.5- and ~ 1.3-fold, respectively), with a combined effect of ~ 1.9-fold. Dexamethasone (DEX) inhibited TPA-stimulated CAT activity by ~ 2.6-fold. In contrast, in the presence of a co-transfected glucocorticoid receptor cDNA expression plasmid, DEX augmented TPA-stimulated CAT expression by ~ 3.1-fold. The predicted secondary structures of the primary transcripts employing the distal and proximal promoters had significant differences, which could affect their stability and translatability. These observations suggest that TATA II functions weakly in the heterologous system examined, independently of TATA I, and directs the synthesis of the second largest pool of CRH transcripts with a longer 5′ non-coding region than the transcripts driven by TATA I. The former possess structural features that may differentiate them from the latter in terms of stability and translatability.