INTRODUCTION
The mu opioid receptor (MOR) plays an important role in mediating the actions of morphine and morphine-like drugs. Based largely on pharmacological and clinical observations, MOR has traditionally been considered the main site of interaction of the major clinically used analgesics, particularly morphine (1). Three major types of opioid receptors, μ, δ and κ, have been cloned and shown to belong to the G-protein-coupled receptor superfamily (2). Regulation of the opioid receptor gene expression may be in response to fluctuating levels of various agents in certain brain regions. Thus, study of the mechanism underlying the transcriptional regulation of opioid receptor genes may facilitate elucidation of the spatial and temporal expressions and the modulation of expression in different physiological states.
The expression of mouse MOR gene is known to be regulated by various cis-acting elements and trans-acting factors, which are important for promoter activity (3–9). It has been reported that a major species of mRNA and polyadenylation signal of MOR has promoter-dependent functional activity (10). We have also reported that MOR transcription is suppressed by the neuron-restrictive silencer element (NRSE) in the mouse MOR promoter through binding of neuron-restriction silence factor (NRSF) (11).
In both non-neuronal and neuronal cells, NRSF silences the expression of its target genes by its two independently acting repressor domains (12,13). The N-terminal repressor domain of NRSF has been shown to recruit co-repressors, such as mSIN3 and histone deacetylases (HDACs), into the vicinity of the promoter. Histone deacetylation leads to a more compact chromatin that prevents accessibility of transcription factors. The C-terminal repressor domain (CTRD) of NRSF has been shown to interact with at least one factor, the transcriptional co-repressor 2 (CoREST) that may serve as a platform protein for the recruitment of molecular machinery that imposes silencing across a chromosomal interval (14,15). CoREST is able to interact with HDAC1/2, indicating that the CTRD also contributes to deacetylation of histones (16).
Some studies have revealed the existence of additional repression mechanisms mediated by NRSF. This has been postulated because HDAC inhibitors fail to derepress the CTRD repression of some NRSF target genes, such as GluR2 receptor and SCG10 (17,18). In addition, overexpression of the dominant-negative NRSF and knock-out of NRSF failed to release the repression of its target gene completely, even though NRSF was originally defined as a repressor for neuronal-specific gene in non-neuronal cells. Therefore, from recent studies, it has become clear that genetic context is extremely important in determining the function of NRSE/NRSF repression mechanisms.
In this study, we have identified a conserved GC box (Sp family binding site) sequence downstream of NRSE region in the mouse MOR gene. Our results have showed that Sp3 specifically binds to this mouse GC box and interacts with NRSF to synergistically repress the MOR expression.