DNase I Footprinting Analysis of Transcription Factors Recognizing Adrenergic Receptor Gene Promoter Sequences

Abstract

Adrenergic receptors (ARs) are G-protein-coupled transmembrane glyco-proteins that play a key role in mediating the sympathoadrenal response to stress. Pharmacological studies have suggested the existence of multiple adrenergic receptor subtypes and sub-subtypes, and to date, nine distinct AR genes have been identified by molecular cloning, including three α₁-AR subtypes (α_1A/D, α_1B, and α_1C), three α₂-AR (α_2A, α_2B, and α_2C) and three β-AR (β₁, β₂, and β₃) (1,2). The expression of these receptors is regulated in a complex tissue-specific manner. Such regulation by hormones or developmental factors has been shown to occur at the transcriptional level under many of these conditions. To understand the molecular mechanisms involved in regulating the transcription of the AR under a variety of physiological and pathological conditions, many AR genes have been isolated. After the promoter sequences of receptor genes have been defined through the use of chloroamphenicol acetyltransferase (CAT) reporter assays, a computer-based search of the Sitedata database of previously identified cis-elements can be used to check whether these promoter sequences contain known sequence-specific protein binding sites. To determine whether, in a given tissue, a protein does in fact bind to a consensus recognition sequence on the DNA and/or to identify novel cis-elements, DNase I footprinting techniques can be used. Once protein binding to DNA is verified by using this technique, a number of other methods, including DNA-gel mobility shift and supershift assays and mutational analyses can be used to characterize the trans-acting factors bound to any specific site.