Transcriptional Regulation of Mouse δ-Opioid Receptor Gene

Deoxycytidine kinase (EC 2.7.1.74, dCK) is central to drug activity of anticancer and antiviral agents such as cytosine arabinoside (araC) and gemcitabine. HepG2 hepatocellular carcinoma cells were used to study the transcriptional regulation of dCK. 5-Deletion and sitedirected mutagenesis of the dCK upstream region (positions ؊464 to ؊27) confirmed the importance of two GC-boxes (positions ؊317 to ؊309 and ؊213 to ؊206) and two E-boxes (positions ؊302 to ؊297 and ؊278 to ؊273). In vitro electromobility shift assays with HepG2 nuclear extracts and in vivo chromatin immunoprecipitation assays with HepG2 chromatin extracts confirmed the presence of bound Sp1/Sp3 and USF1/2. Co-transfections in HepG2 cells showed that USF1 and USF2a stimulated and Sp1 repressed promoter activity from a dCK-luciferase reporter gene construct. In Sp-and USF-null Drosophila Mel-2 cells, both Sp1 and USF1 stimulated dCK promoter activity in a dose-dependent manner, however, both Sp3 and USF2a were effectively inert. Combined Sp1 and USF1 showed additive transactivation at lower concentrations of Sp1. Sp1 was inhibitory at higher levels. Stimulation by combined USF1/USF2a with Sp1 was similar to that for USF1 alone with Sp1, whereas transactivation by Sp1 and USF2a without USF1 was synergistic. Physical interactions between USF and Sp proteins were confirmed by immunoprecipitations with Sp-and USF-specific antibodies. Domain mapping of USF1 and USF2a localized the functional interactions between USF and Sp proteins to the DNA binding domain of USF. Identifying the physical and functional interactions between Sp and USF proteins may lead to a better understanding of the basis for differential expression of the dCK gene in tumor cells and may foster strategies for up-regulating dCK gene expression and improving chemotherapy with araC and gemcitabine.

Section: Discussionmentioning

confidence: 99%

Physical and Functional Interactions between USF and Sp1 Proteins Regulate Human Deoxycytidine Kinase Promoter Activity

Jensen

Matherly

et al. 2003

“…Most notably, the CGRP 18-bp enhancer is controlled by USF and the cell-specific FoxA2 protein in thyroid C-cell lines (12). Similar partnerships with other factors have been reported for USF (41,47), including in the nervous system (48,49). Although we cannot rule out an unknown accessory factor or co-activator, the FoxA2 protein is not a candidate because mutation of the FoxA2 site did not decrease reporter activity in cultured neurons (27), and neither FoxA2 RNA nor protein could be detected in rat trigeminal ganglia by RT-PCR or Western blots (data not shown).…”

Section: Discussionmentioning

confidence: 58%

Control of the Calcitonin Gene-related Peptide Enhancer by Upstream Stimulatory Factor in Trigeminal Ganglion Neurons

Park

Russo

2008

The neuropeptide calcitonin gene-related peptide (CGRP) is a key player in migraine. However, the transcription factors controlling CGRP expression in the migraine-relevant trigeminal ganglion neurons are unknown. Previous in vitro studies demonstrated that upstream stimulatory factor (USF) 1 and USF2 bind to the CGRP neuroendocrine-specific 18-bp enhancer, yet discrepant overexpression results in cell lines, and the ubiquitous nature of the USF cast doubts about its role. To test the functional role of USF, we first demonstrated that small interfering RNAs directed against USF1 and USF2 reduced endogenous CGRP RNA and preferentially targeted the USF binding site at the 18-bp enhancer in the neuronal-like CA77 cell line. In cultured rat trigeminal ganglion neurons, knockdown of either USF1 or USF2 reduced CGRP promoter activity. Conversely, overexpression of USF1 or USF2 increased promoter activity. The activation was even greater upon cotransfection with an upstream activator of mitogen-activated protein kinases and was synergistic in a heterologous cell line. To begin to address the paradox of how ubiquitous USF proteins might direct neuronal-specific activity, we examined USF expression and used a series of adenoviral reporters in the cultured ganglia. Unexpectedly, there was more intense USF immunostaining in neurons than nonneuronal cells. Importantly, the 18-bp USF enhancer driving a minimal promoter was sufficient for neuronal specificity, although it was not the only site that directed neuronal expression. These results demonstrate that USF1 and USF2 are important contributors to neuronal-specific and mitogen-activated protein kinase regulation of the CGRP gene in trigeminal ganglion neurons.

“…In view of this, the fact that the mDOR gene is expressed in a tissue-and cell type-specific manner is rather surprising, because the housekeeping genes are usually ubiquitously expressed. Previous studies demonstrated that the mDOR promoter region from Ϫ262 to Ϫ141 containing Sp1/ Sp3, upstream stimulatory factor, and Ets-1-binding sites is critical for its basal promoter activity (5,6). Given that these transcriptional factors are also ubiquitously expressed, the mechanisms involved in the regulation of cell type-specific expression of mDOR are not clear.…”

Section: Discussionmentioning

confidence: 97%

“…Several transcription factors have been shown to bind to the mDOR promoter region and play a critical role in the regulation of mDOR promoter activity ( Fig. 2A) (5,6). Among these transcription factors, Sp1/Sp3 accounts for about 70% of the promoter activity in a transient transfection assay (5).…”

Section: Involvement Of Sp3 In the Mecp1-like Complex Formed By Me-26mentioning

confidence: 99%

“…Thus understanding the molecular mechanisms underlying the cell type-specific expression of DOR will provide insights into the regulation of the pharmacological benefits of ␦-opioids by manipulation of the DOR expression levels in certain cell types. We reported previously (4) the isolation of mouse genomic clones of DOR (mDOR), and we identified transcription upstream stimulatory factors Sp1/Sp3 and Ets-1 that functionally interact with the mDOR promoter and activate it in a mouse neuronal cell line NS20Y (5,6). As yet, no data are available on the mechanisms involved in the cell type-specific expression of mDOR.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Transcriptional Regulation of Mouse δ-Opioid Receptor Gene by CpG Methylation

Wang

Wei

Loh

2003

Self Cite

Opioid receptors are expressed in a cell type-specific manner. Here we show that the mouse ␦-opioid receptor (mDOR) gene is regulated by promoter region CpG methylation. The mDOR promoter containing a putative CpG island is highly methylated in Neuro2A cells, correlating with the repression of this gene in these cells. This is in contrast with the unmethylated state of the mDOR promoter in NS20Y cells, which express a high level of mDOR. Repression of mDOR transcription in Neuro2A cells could be partially relieved by chemically induced demethylation with 5-aza-2-deoxycytidine. In addition, in vitro methylation of the luciferase reporter gene driven by the mDOR promoter resulted in an inhibition of transcription in NS20Y cells. Methyl-CpG-binding protein complex 1 (MeCP1) has been implicated in methylation-mediated transcriptional repression of several genes. Electrophoretic mobility shift assays showed that fully methylated, but not unmethylated, mDOR promoter fragment formed a MeCP1-like protein complex that contained methyl-CpG-binding domain protein 2 (MBD2) and Sp3. Furthermore, the expression level of Sp3 was decreased when Neuro2A cells were demethylated with 5-aza-2-deoxycytidine, and increasing Sp3 levels in Schneider's Drosophila line 2 cells led to the repression of mDOR promoter activity when the promoter was methylated. These results demonstrate that Sp3 and MBD2 are involved in the transcriptional repression of mDOR in Neuro2A cells through binding to the methylated CpG sites in the promoter region and may play a role in the cell type-specific expression of mDOR.Opioids display strong analgesic effects as well as addictive properties through the activation of three major types of Gprotein-coupled receptors referred to as -, ␦-, and -opioid receptors (1). Whereas all three opioid receptors mediate opioid-induced analgesia, each receptor type exerts a distinct pharmacological profile as well as a unique cell type-specific expression pattern (2, 3). The expression of the ␦-opioid receptor (DOR) 1 is mainly confined to certain regions of the adult central nervous system, although it is found in the peripheral nervous system and some immune cells. It has been reported that the expression levels of DOR determine the activities of the DOR agonist, and the localization of DOR generally matches the pharmacological action sites of ␦-opioids. Thus understanding the molecular mechanisms underlying the cell type-specific expression of DOR will provide insights into the regulation of the pharmacological benefits of ␦-opioids by manipulation of the DOR expression levels in certain cell types. We reported previously (4) the isolation of mouse genomic clones of DOR (mDOR), and we identified transcription upstream stimulatory factors Sp1/Sp3 and Ets-1 that functionally interact with the mDOR promoter and activate it in a mouse neuronal cell line NS20Y (5, 6). As yet, no data are available on the mechanisms involved in the cell type-specific expression of mDOR.Methylation of the cytosine residue in the 5Ј-CpG-3Ј seq...