DUAL DNA-BINDING SPECIFICITY OF ADD1 SREBP1 CONTROLLED BY A SINGLE AMINO-ACID IN THE BASIC HELIX-LOOP-HELIX DOMAIN/

Adipocyte determination- and differentiation-dependent factor 1 (ADD1) , a member of the basic helix-loop-helix (bHLH) family of transcriptio n factors, has been associated with both adipocyte differentiation and cholesterol homeostasis (in which case it has been termed SREBP1). Us ing PCR-amplified binding analysis, we demonstrate that ADD1/SREBP1 ha s dual DNA sequence specificity, binding to both an E-box motif (ATCAC GTGA) and a non-E-box sequence previously shown to be important in cho lesterol metabolism, sterol regulatory element 1 (SRE-1; ATCACCCCAC). The ADD1/SREBP1 consensus E-box site is similar to a regulatory sequen ce designated the carbohydrate response element, defined by its abilit y to regulate transcription in response to carbohydrate in genes invol ved in fatty acid and triglyceride metabolism in liver and fat. When e xpressed in fibroblasts, ADD1/SREBP1 activates transcription through b oth the carbohydrate response E-box element and SRE-1. Substitution of an atypical tyrosine in the basic region of ADD1/SREBP1 to an arginin e found in most bHLH protein causes a restriction to only E-box bindin g. Conversely, substitution of a tyrosine for the equivalent arginine in another bHLH protein, upstream stimulatory factor, allows this fact or to acquire a dual binding specificity similar to that of ADD1/SREBP 1. Promoter activation by ADD1/SREBP1 through the carbohydrate respons e element E box is not sensitive to the tyrosine-to arginine mutation, while activation through SRE-1 is completely suppressed. These data i llustrate that ADD1/SREBP1 has dual DNA sequence specificity controlle d by a single amino acid residue; this dual specificity may provide a novel mechanism to coordinate different pathways of lipid metabolism.