Two cysteine residues in the DNA-binding domain of CREB control binding toCRE and CREB-mediated gene expression

The cAMP-responsive element-binding protein (CREB) has been implicated in t he regulation of numerous physiological functions including those of severa l hypoxia-responding genes. All CREB transcription-regulated genes harbor t he eight base-pair cAMP-responsive element (CRE) or the seven base-pair AP- 1 sequence. Utilizing mutational analysis and biochemical assays, we found that reduction of two cysteine residues located in the DNA-binding basic do main of CREB, enhances the binding efficiency of CREB to DNA and regulates CRE-mediated gene expression. Substitution of these residues to serine rend ers insensitivity to reduction, hypoxia and to the sulfhydryl-specific modi fying agent, N-ethylmaleimide. These substitutions enhance the binding of C REB to its cognate DNA sites under oxidative conditions, and of the CREB-de pendent gene expression during non-noxia. These findings are supported by r esults of molecular modeling of the CREB-CRE interactions. We also found th at HTLV-1 Tax enhancement of CREB binding to the cellular and the viral DNA sites and activation of the CRE-dependent gene expression are independent of CREB activation exerted by redox conditions. The genetic biochemical and molecular modeling presented in this work indicate that the two cysteine r esidues in the bZIP domain of CREB regulate the binding efficiency of CREB to its cognate DNA sites and as a consequence the activation of CREB-mediat ed gene expression. (C) 2001 Academic Press.