Mouse μ Opioid Receptor Distal Promoter Transcriptional Regulation by SOX Proteins

Abstract

We have identified transcription factors that bind to specific sequences in 5 -distal promoter regulatory sequences of the mouse opioid receptor (mor) promoter using the yeast one-hybrid system. The sequence between ؊746 and ؊707 in mor distal promoter was used as the bait because it acts as a functional promoter element and binds several DNA-binding proteins. From an adult mouse brain cDNA library, five cDNA clones encoding three Sox gene family (Sry like high mobility group (HMG) box gene) transcriptional factors, mSOX18, mSOX21, and mSOX6, were isolated. Electrophoretic mobility shift assays confirmed the presence of a binding site for SOX proteins in the ؊731/؊725 region. Additionally, we have also established that the flanking regions outside the core Sox-binding site play an essential role in high affinity binding. DNase I footprint analysis indicates that proteins from mouse brain interact with the Sox-binding site within the mor distal promoter. Finally, we demonstrated that overexpression of mSOX18 and/or mSOX21 was able to up-regulate mouse mor distal promoter activity in mor-expressing neuronal cells (NMB). These data indicate that SOX proteins might contribute to the transcriptional activity of the mor gene and suggest that opioid receptor could mediate some of the developmental processes in which SOX proteins are included.Opioids have many pharmacological and physiological effects, including analgesia, sedation, euphoria, and respiratory depression and also induce tolerance and physical dependence when administered chronically. Pharmacological, physiological, receptor binding, and molecular cloning studies (1) have revealed that opioids interact with three major types of opioid receptors in the mammalian central nervous system and peripheral tissues, , ␦, and . All three types of receptors belong to the superfamily of G-protein-coupled receptors (2), and the opioid receptor (mor) 1 is known to play the essential role in morphine-induced analgesia, tolerance, and dependence as indicated from pharmacological studies. This has also been confirmed by additional in vivo pharmacological analyses of mice where mor was deleted by homologous recombination (3).Mor is expressed mainly in the central nervous system, where it exhibits distinct temporal and spatial patterns of distribution (1). Recently, in situ hybridization studies and other methods (e.g. RPA (RNase protection assay) and reverse transcriptase-PCR) indicate that expression of mor mRNA is regulated at the transcriptional level, especially during embryonic development (4 -7). The molecular mechanisms underlying this regulation are not completely understood, but analysis of the 5Ј-upstream sequences from the translation initiation sites of the mor gene has shown that expression of mor is driven by two promoters, a distal and a proximal promoter (8). Both promoters exhibit characteristics of housekeeping genes lacking a TATA box (9, 10), with the proximal promoter regulating mor transcription from four major initiation sites located in a region 291-...

A Proteomics Approach for Identification of Single Strand DNA-binding Proteins Involved in Transcriptional Regulation of Mouse μ Opioid Receptor Gene

“…Electrophoretic Mobility Shift Assay (EMSA)-The EMSA was performed as described previously (25). The sense strand of the probe (5Ј-CAATCCACTCCTTCTCTCTCCTCCCTCCCCTCTAGCCTCTG-3Ј) was end-labeled with [␥-32 P]dATP.…”

A Major Species of Mouse μ-opioid Receptor mRNA and Its Promoter-Dependent Functional Polyadenylation Signal

“…Extensive studies have been carried out to elucidate transcriptional regulation of MOR gene expression, primarily for the 5Ј upstream region. For example, our lab has identified multiple regulatory elements in the MOR promoter region, such as Sp1 (Ko et al, 1998), single-stranded DNA-binding site (Ko and Loh, 2001), Sox (Hwang et al, 2003), PU.1 , neuronrestrictive silencer factor , and Sp3 isoforms (Choi et al, 2005). Regulatory sequences in the 5Ј-UTR have also been defined by other labs, including STAT6 (Kraus et al, 2001), cAMP response element-binding protein (Lee and Lee, 2003), and neurorestrictive suppressor element (Andria and Simon, 2001).…”

Evidence of Endogenous Mu Opioid Receptor Regulation by Epigenetic Control of the Promoters

“…The promoter sequences of MORs from mouse, rat, and human are not exactly homologous, except for some regions known to be conserved (12,33,34,41,45,47,48). To study regulation of the mouse MOR gene, we had to find a mouse model cell line in which the MOR gene could be regulated while the endogenous MOR gene was not expressed constitutively.…”