No abstract

The mouse homologue of the human receptor-interacting protein 140 (RIP140) was isolated from a mouse embryonic cDNA library in yeast two-hybrid screening experiments by using the ligand binding domain (LBD) of nuclear orphan receptor TR2 as the bait. The receptor-interacting domains of mouse RIP140 were mapped to the regions containing the LXXLL motif (where L is leucine and X is any amino acid), and the RIP140-interacting domain of TR2 was mapped to its C-terminal 10-to 20-amino-acid sequence, a putative activation function 2 (AF-2) region. In a GAL4 reporter system and a reporter driven by the proximal region of the TR2 promoter, RIP140 functioned as a corepressor for both a GAL4 DNA binding domain (BD)-TR2 fusion and the wild-type receptor. When tethered to the BD of GAL4, RIP140 exerted a trans-repressive effect on the GAL4 reporter. In addition, RIP140 suppressed the retinoic acid (RA) receptor-mediated RA induction in a dosedependent manner. Finally, it was demonstrated that in the presence of RIP140, a cytosolic, green fluorescent protein-tagged TR2 LBD translocated into the nucleus, and TR2 and RIP140 were coimmunoprecipitated from the cell extract, indicating that the interaction between RIP140 and the LBD of TR2 occurred in vivo. The potential biological role of RIP140 in TR2-modulated transcriptional activity is discussed.Nuclear receptors regulate target gene expression by binding to their cognate DNA response elements and recruiting associate proteins to the transcription machinery (19, 45). Recently, different coactivators and corepressors for several nuclear receptors have been identified (16). For instance, the nuclear receptor corepressor (N-CoR) and the silencing mediator for retinoid and thyroid hormone receptors (SMRT) have been shown to function as corepressors for retinoic acid (RA) receptor ␣ (RAR␣), thyroid hormone receptors (T 3 Rs), and orphan receptor COUP-TFI (4, 15, 36). The newly identified nuclear protein Nab1 is a corepressor for the orphan receptor NGFI-A family (38), and SUN-CoR is able to potentiate transcriptional repression by T 3 Rs and RevErb (44). In the coactivator category, the transcriptional mediator/intermediary factor 2 and steroid receptor coactivator 1 (SRC-1) are known to mediate transcriptional activation of RAR, estrogen receptor (ER), retinoid X receptor (RXR), and T 3 Rs (8,14,33,39,42). With respect to the working mechanism of corepressors and coactivators, it has been demonstrated that their actions involve the alteration of chromatin structure, such as the acetylation status of histone proteins (13,34,37).The orphan receptor TR2 belongs to the superfamily of nuclear receptors (21,31). This receptor gene is expressed most abundantly in the testes of adult animals, particularly the developing germ cell populations (22,24,25). During embryonic stages, TR2 expression is highest between embryonic day 8 (E8) and E12 (22). It is also known that this gene encodes two isoform receptors, one retaining the entire ligand binding domain (LBD) and the other truncated at ...

The pharmacological effect of morphine as a painkiller is mediated mainly via the mu opioid receptor (MOR) and is dependent on the number of MORs in the cell surface membrane. While several studies have reported that the MOR gene is regulated by various cis-and trans-acting factors, many questions remain unanswered regarding in vivo regulation. The present study shows that epigenetic silencing and activation of the MOR gene are achieved through coordinated regulation at both the histone and DNA levels. In P19 mouse embryonal carcinoma cells, expression of the MOR was greatly increased after neuronal differentiation. MOR expression could also be induced by a demethylating agent (5-aza-2-deoxycytidine) or histone deacetylase inhibitors in the P19 cells, suggesting involvement of DNA methylation and histone deacetylation for MOR gene silencing. Analysis of CpG DNA methylation revealed that the proximal promoter region was unmethylated in differentiated cells compared to its hypermethylation in undifferentiated cells. In contrast, the methylation of other regions was not changed in either cell type. Similar methylation patterns were observed in the mouse brain. In vitro methylation of the MOR promoters suppressed promoter activity in the reporter assay. Upon differentiation, the in vivo interaction of MeCP2 was reduced in the MOR promoter region, coincident with histone modifications that are relevant to active transcription. When MeCP2 was disrupted using MeCP2 small interfering RNA, the endogenous MOR gene was increased. These data suggest that DNA methylation is closely linked to the MeCP2-mediated chromatin structure of the MOR gene. Here, we propose that an epigenetic mechanism consisting of DNA methylation and chromatin modification underlies the cell stage-specific mechanism of MOR gene expression.Opioids exert their pharmacological and physiological effects through binding to their endogenous receptors. Three types of opioid receptors, mu (), delta (␦), and kappa (), all belonging to the G-protein-coupled receptor superfamily, have been cloned. Upon agonist binding, these receptors couple to G proteins and affect several signal transduction pathways thought to mediate a broad range of functions and pharmacological effects of endogenous and exogenous opioids (51). Previous studies suggested that the opioid receptor (MOR) plays a key role in mediating the major clinical effects of analgesics, such as morphine, as well as the development of tolerance and physical dependence upon prolonged administration (39). MOR is mainly expressed in the central nervous system, with densities varying greatly in different regions, which can display different functional roles (55). During mouse embryonic development, the MOR message was specifically observed as early as embryonic day 8.5 (E8.5) using the reverse transcription (RT)-PCR method (44). In contrast, MOR transcripts were detected only beginning at E12 using the radioligand binding method (70) and at E10.5 by in situ hybridization (85). Transcript levels gradually incr...

Endotoxin tolerance (ET) triggered by prior exposure to Toll-like receptor (TLR) ligands provides a mechanism to dampen inflammatory cytokines. Receptor-interacting protein 140 (RIP140) interacts with NF-κB to regulate the expression of proinflammatory cytokine genes. We identify lipopolysaccharide (LPS) stimulation of Syk-mediated tyrosine phosphorylation on RIP140 and RelA interaction with RIP140. These events increase recruitment of SOCS1-Rbx1 (SCF) E3 ligase to tyrosine-phosphorylated RIP140, thereby degrading RIP140 to inactivate inflammatory cytokine genes. Macrophages expressing a non-degradable RIP140 were resistant to the establishment of ET for specific genes. The results reveal RelA as an adaptor for SCF ubiquitin ligase to fine-tune NF-κB target genes by targeting co-activator RIP140, and an unexpected role for RIP140 protein degradation in resolving inflammation and ET.

Receptor-interacting protein 140 (RIP140) encodes a histone deacetylase (HDAC) inhibitor-sensitive repressive activity. Direct interaction of RIP140 with HDAC1 and HDAC3 occurs in vitro and in vivo as demonstrated in co-immunoprecipitation and glutathione S-transferase pull-down experiments. The HDAC-interacting domain of RIP140 is mapped to its N-terminal domain, between amino acids 78 and 303 based upon glutathione S-transferase pull-down experiments. In chromatin immunoprecipitation assays, it is demonstrated that histone deacetylation occurs at the chromatin region of the Gal4 binding sites as a result of Gal4 DNA binding domain-tethered RIP expression. The immunocomplexes of RIP140 from cells transfected with RIP140 and HDAC are able to deacetylate histone proteins in vitro. This study presents the first evidence for RIP140 as a negative coregulator for nuclear receptor actions by directly recruiting histone deacetylases and categorizes RIP140 as a novel negative coregulator that is able to directly interact with HDACs.Nuclear receptors play important roles in gene regulation. In most cases, they regulate target gene expression by binding to their cognate DNA response elements and recruiting associate proteins to the transcription machinery (1, 2). A large number of associate proteins of nuclear receptors have been found and demonstrated as co-activators or co-repressors (3). One mechanism of co-repressor and co-activator action has been shown to involve chromatin modification such as acetylation of chromosomal histone proteins (4 -6). For instance, corepressors such as N-CoR/SMRT interact with apo-receptors in their hinge region and recruit histone deacetylases (HDACs) 1 indirectly through the Sin3 complexes (4, 7). However, recent studies have shown that co-repressors bind to the same cavity formed by helices 3-5 and 12 in the ligand binding domain of nuclear receptors as do co-activators (8 -10). Upon ligand binding, the ligand binding domain undergoes a conformational change, releases corepressors, and recruits coactivators by re-positioning helix 12 (AF-2 domain). Several coactivators, mainly the p160 family, have been identified, including SRC-1/NCoA-1, TIF2/GRIP1/NCoA-2, and p/CIP/RAC3/ACTR/AIB1 (11-16). The SRC-1, ACTR, and CBP/p300 have been shown to encode intrinsic histone acetyl transferase activities (5, 6, 17).Two major classes of HDACs have been cloned in higher eukaryotes. Class 1 HDACs (HDAC 1-3) are homologous to the yeast , and class 2 HDACs (HDAC 4 -7) are homologues of the yeast Hda1 (21). It has been demonstrated that disruption of either Rpd3 or Hda1 in yeast resulted in global hyperacetylation of histones (19). The acetylation of chromosomal histones is correlated strongly with active gene transcription; conversely, hypoacetylation of a gene regulatory region is usually indicative of gene silencing (22-25). Whereas acetylation of histones is not sufficient to dictate the efficiency of transcription, an overwhelmingly large volume of convincing evidence has supported the notion that t...