MED15::ATF1-Rearranged Tumor: A Novel Cutaneous Tumor With Melanocytic Differentiation.

We recently described novel dermal tumors with melanocytic differentiation and morphologic and biological similarities to cutaneous clear cell sarcoma, including CRTC1::TRIM11 cutaneous tumor, and clear cell tumors with melanocytic differentiation and either ACTIN::MITF or MITF::CREM. Here, we describe a series of 3 patients presenting with tumors reminiscent of CRTC1::TRIM11 cutaneous tumor, found to demonstrate a novel MED15::ATF1 fusion. All 3 patients were children (5-16 years old). Primary excision of case 1 showed a circumscribed wedge-shaped silhouette with peripheral intercalation into collagen fibers and scattered lymphoid aggregates. All 3 tumors abutted the epidermis; one showed a junctional component. Tumors were highly cellular and comprised of monomorphic, oval-to-round epithelioid cells arranged in vague nests and short fascicles in variably fibrotic stroma. Mitotic rate was high (hotspot 6-12/mm2), without atypical mitoses. Necrosis was focally present in case 3. All cases showed strong, diffuse nuclear staining for SOX10 and MITF (2/2) but showed variable expression for S100 protein (1/3) and other melanocytic markers-Melan-A (focal in 2/3), HMB45 (focal in 1/3), and Pan-Melanoma (patchy in 1/1). Whole-exome RNA sequencing demonstrated a MED15::ATF1 fusion without any other notable alterations. Cases 1 and 2 were completely excised without recurrence (12 months). Case 3 developed a grossly apparent regional lymph node spread shortly after primary biopsy. The patient was treated with wide excision, radiation, cervical lymph node dissection (4/46 with >75% lymph node replacement), and neoadjuvant and adjuvant nivolumab (alive without disease at cycle 11). This series is presented to aid in future diagnosis of this novel dermal tumor with melanocytic differentiation and emphasize the potential for aggressive biologic behavior, which should be considered in patient management planning.

An Unexpected Encounter: Respiratory Syncytial Virus Nonstructural Protein 1 Interacts with Mediator Subunit MED25.

Human respiratory syncytial virus (RSV) is the leading cause of severe acute lower respiratory tract infections in infants worldwide. Nonstructural protein NS1 of RSV modulates the host innate immune response by acting as an antagonist of type I and type III interferon (IFN) production and signaling in multiple ways. Likely, NS1 performs this function by interacting with different host proteins. In order to obtain a comprehensive overview of the NS1 interaction partners, we performed three complementary protein-protein interaction screens, i.e., BioID, MAPPIT, and KISS. To closely mimic a natural infection, the BioID proximity screen was performed using a recombinant RSV in which the NS1 protein is fused to a biotin ligase. Remarkably, MED25, a subunit of the Mediator complex, was identified in all three performed screening methods as a potential NS1-interacting protein. We confirmed the interaction between MED25 and RSV NS1 by coimmunoprecipitation, not only upon overexpression of NS1 but also with endogenous NS1 during RSV infection. We also demonstrate that the replication of RSV can be enhanced in MED25 knockout A549 cells, suggesting a potential antiviral role of MED25 during RSV infection. Mediator subunits function as transcriptional coactivators and are involved in transcriptional regulation of their target genes. Therefore, the interaction between RSV NS1 and cellular MED25 might be beneficial for RSV during infection by affecting host transcription and the host immune response to infection. IMPORTANCE Innate immune responses, including the production of type I and III interferons, play a crucial role in the first line of defense against RSV infection. However, only a poor induction of type I IFNs is observed during RSV infection, suggesting that RSV has evolved mechanisms to prevent type I IFN expression by the infected host cell. A unique RSV protein, NS1, is largely responsible for this effect, probably through interaction with multiple host proteins. A better understanding of the interactions that occur between RSV NS1 and host proteins may help to identify targets for an effective antiviral therapy. We addressed this question by performing three complementary protein-protein interaction screens and identified MED25 as an RSV NS1-interacting protein. We propose a role in innate anti-RSV defense for this Mediator complex subunit.

Guizhi Fuling Capsule inhibits uterine fibroids growth by modulating Med12-mediated Wnt/ß-Catenin signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Guizhi Fuling Capsule (GFC) is a famous traditional Chinese medicine (TCM) formula recorded in Synopsis of the Golden Chamber, which has achieved obvious effects in the treatment of uterine fibroids (UFs). AIM OF STUDY: Mediator complex subunit 12 (Med12) mutations were closely related to UFs in 85% of fibroid cases. The Wnt/ß-Catenin signaling pathway plays an important role in the occurrence and development of UFs. This study aims to explore the pharmacological mechanism of GFC against UFs in which the Med12-mediated Wnt/ß-Catenin pathway is involved. MATERIALS AND METHODS: Med12 was silenced in uterine fibroid cells (UFCs) using a lentivirus-based Med12 gene-specific RNA interference (RNAi) strategy. Cell proliferation was performed by CCK-8 assay, cell apoptosis and cell cycle were measured by flow cytometry. The rat model of UFs was established by injecting estradiol benzoate and progesterone. Forty-eight rats were divided into six groups, the low-dose GFC (L-GFC) group, the medium-dose GFC (M-GFC) group and the high-dose GFC (H-GFC) group were intragastrically treated with GFC solution at 0.25 g/kg, 0.50 g/kg and 1.00 g/kg per day for 8 weeks, the positive control (PC) group was administrated with mifepristone (2.70 mg/kg/day), the normal control (NC) group and the model control (MC) group were given equal volume of normal saline once a day for 8 weeks. The histopathological changes of uterine tissues were evaluated by H&E staining. The expression of Med12 in uterine tissues were detected by immunohistochemistry. The protein and mRNA levels of associated genes were evaluated by western bolt and real time-PCR, respectively. Related indicators involved in Wnt/ß-Catenin pathway, such as Wnt1, ß-Catenin, Cyclin D1, TCF1/TCF7 and C-myc, were compared among different groups. RESULTS: The Wnt/ß-Catenin signaling pathway was inhibited after Med12 gene was knocked out in UFCs. GFC-containing serum could induce cell apoptosis, make the cell cycle stagnated in G0/G1 phase to inhibiting the proliferation and reduce the expression of Wnt1, ß-Catenin, Cyclin D1, TCF1/TCF7, and C-myc in control-shRNA cells, while had no significant effect on Med12-shRNA cells. Compared with the MC group, the weight, endometrial thickness, and pathological structure of the uterus in the GFC treated groups were significantly improved. The expression of Med12, Wnt1, ß-Catenin, Cyclin D1, TCF1/TCF7, and C-myc that related to Wnt/ß-Catenin pathway in the GFC treated groups were decreased with the increase of dosage administration. CONCLUSIONS: GFC inhibited UFs growth, which was directly associated with Med12 modulated Wnt/ß-Catenin signaling pathway. This study provided new perspective to understand the therapeutic mechanism of UFs.

White Tea Reduced Bone Loss by Suppressing the TRAP/CTX Pathway in Ovariectomy-Induced Osteoporosis Model Rats.

Osteoporosis is an important skeletal disease characterized by bone weakness and high risk of fracture in postmenopausal women. Tea consumption is known to play an important role in the prevention or alleviation of osteoporosis. However, the therapeutic effects of aqueous extracts of white tea (WT) have not been evaluated in osteoporosis rat models. The aim of this study was to investigate the potential anti-osteoporotic role of WT in ovariectomized (OVX) rats. WT was given orally at 0.5% w/v doses for 12 weeks in OVX rats. Biochemical parameters in blood samples, bone tartrate-resistant acid phosphatase (TRAP), C-terminal telopeptide of type 1 collagen (CTX) and estradiol levels were evaluated. Bone mineral density and bone mineral content values were measured in the left femur. In addition to histopathological examination, osteolcalcin, osteopontin and TUNEL levels were determined. OVX group data demonstrated that bone loss occurred by thinning of the metaphyseal growth plates of the femur. Similarly, the levels of TRAP and CTX, markers of osteoclastic activity, were found to be high concurrently with a decrease in femoral bone mineral density. In addition, increased osteolcalcin and osteopontin levels were present in the metaphyseal growth zones. On the other hand, while TRAP and CTX levels were suppressed in the OVX-WT group, bone mineral content increased. In ad-dition, TUNEL, osteocalcin and osteopontin positivity decreased in the right femoral metaphysis growth zones, proliferating zone and resting zone cells. These results showed that chronic WT consumption has a protective effect by reducing bone resorption in OVX-induced osteoporotic rats.

Interaction map of Arabidopsis Mediator complex expounding its topology.

Understanding of mechanistic details of Mediator functioning in plants is impeded as the knowledge of subunit organization and structure is lacking. In this study, an interaction map of Arabidopsis Mediator complex was analyzed to understand the arrangement of the subunits in the core part of the complex. Combining this interaction map with homology-based modeling, probable structural topology of core part of the Arabidopsis Mediator complex was deduced. Though the overall topology of the complex was similar to that of yeast, several differences were observed. Many interactions discovered in this study are not yet reported in other systems. AtMed14 and AtMed17 emerged as the key component providing important scaffold for the whole complex. AtMed6 and AtMed10 were found to be important for linking head with middle and middle with tail, respectively. Some Mediator subunits were found to form homodimers and some were found to possess transactivation property. Subcellular localization suggested that many of the Mediator subunits might have functions beyond the process of transcription. Overall, this study reveals role of individual subunits in the organization of the core complex, which can be an important resource for understanding the molecular mechanism of functioning of Mediator complex and its subunits in plants.

Developmental role of the tomato Mediator complex subunit MED18 in pollen ontogeny.

Pollen development is a crucial step in higher plants, which not only makes possible plant fertilization and seed formation, but also determines fruit quality and yield in crop species. Here, we reported a tomato T-DNA mutant, pollen deficient1 (pod1), characterized by an abnormal anther development and the lack of viable pollen formation, which led to the production of parthenocarpic fruits. Genomic analyses and the characterization of silencing lines proved that pod1 mutant phenotype relies on the tomato SlMED18 gene encoding the subunit 18 of Mediator multi-protein complex involved in RNA polymerase II transcription machinery. The loss of SlMED18 function delayed tapetum degeneration, which resulted in deficient microspore development and scarce production of viable pollen. A detailed histological characterization of anther development proved that changes during microgametogenesis and a significant delay in tapetum degeneration are associated with a high proportion of degenerated cells and, hence, should be responsible for the low production of functional pollen grains. Expression of pollen marker genes indicated that SlMED18 is essential for the proper transcription of a subset of genes specifically required to pollen formation and fruit development, revealing a key role of SlMED18 in male gametogenesis of tomato. Additionally, SlMED18 is able to rescue developmental abnormalities of the Arabidopsis med18 mutant, indicating that most biological functions have been conserved in both species.

Immunohistochemical profiling of receptor tyrosine kinases, MED12, and TGF-ßRII of surgically resected small cell lung cancer, and the potential of c-kit as a prognostic marker.

The limited number of available treatments for patients with small-cell lung cancer (SCLC) has prompted us to further investigate the biology of SCLC by molecular profiling. We collected formalin-fixed paraffin-embedded tumor samples from 127 patients with SCLC, who had undergone surgery at 16 institutions between January 2003 and January 2013, and analyzed the association between disease-specific survival and protein expression of c-kit, c-Met, epidermal growth factor receptor, human EGFR-related 2, vascular endothelial growth factor receptor II, anaplastic lymphoma kinase, mediator complex subunit 12 (MED12), and transforming growth factor beta receptor II (TGF-ßRII) by immunohistochemistry (IHC). Of the 125 evaluable samples, all tumors expressed MED12, and 123 samples (98.4%) expressed TGF-ßRII. MED12 was highly expressed in the nucleus in 92% of the positive samples while TGF-ßRII was highly expressed in the cytoplasm in 55% of the positive samples. High c-kit expression was an independent favorable prognostic marker confirmed by multivariate analysis (hazard ratio: 0.543, 95% confidence interval: 0.310-0.953, p = 0.033). Both the relapse free-survival and overall survival of patients who underwent adjuvant chemotherapy were statistically longer in those with high c-kit expression (n = 38) than those with intermediate, low, or no c-kit expression (n = 19) (not reached vs 11.6 months, p = 0.021; not reached vs 25.9 months, p = 0.028). IHC for c-kit may offer a prognostic marker for early-stage SCLC, and the results for MED12 and TGF-ßRII may suggest the biological characteristics of SCLC. Further investigation of the roles of their related molecules in early stage SCLC is required.

Effect of l-arginine, asymmetric dimethylarginine, and symmetric dimethylarginine on ischemic heart disease risk: A Mendelian randomization study.

BACKGROUND: l-arginine is a commonly consumed dietary conditional essential amino acid found in food items and supplements, which is closely related to asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA). l-arginine is thought to increase nitric oxide and be cardioprotective, whereas ADMA and SDMA may inhibit nitric oxide synthesis and increase cardiovascular disease risk. Unexpectedly, l-arginine increased mortality in a small trial. To clarify the effects of these potential targets of intervention, we assessed the risk of ischemic heart disease (IHD) by genetically determined l-arginine, ADMA, and SDMA. METHODS: Single nucleotide polymorphisms (SNPs) contributing to l-arginine, ADMA, and SDMA, at genome-wide significance, were applied to the CARDIoGRAMplusC4D 1000 Genomes-based genome-wide association study IHD case (n=60,801, ~70% myocardial infarction)-control (n=123,504) study. We obtained unconfounded estimates using instrumental variable analysis by combining the Wald estimators for each SNP, taking into account any correlation between SNPs using weighted generalized linear regression. RESULTS: Higher l-arginine was associated with higher risk of IHD (odds ratio [OR] 1.18 per SD increase, 95% CI 1.03-1.36) and of myocardial infarction (OR 1.29, 95% CI 1.10-1.51), based on 2 SNPs from MED23. Symmetric dimethylarginine had an OR of 1.07 per SD (95% CI 0.99-1.17) for IHD based on 5 SNPs from AGXT2. Asymmetric dimethylarginine had and OR of 1.08 per SD (95% CI 0.99-1.19) for IHD based on 4 SNPs from DDAH1. CONCLUSION: l-arginine could possibly cause IHD. Given that l-arginine occurs in many common dietary items, investigation of its health effect is required.

Proximity biotinylation and affinity purification are complementary approaches for the interactome mapping of chromatin-associated protein complexes.

Mapping protein-protein interactions for chromatin-associated proteins remains challenging. Here we explore the use of BioID, a proximity biotinylation approach in which a mutated biotin ligase (BirA*) is fused to a bait of interest, allowing for the local activation of biotin and subsequent biotinylation of proteins in the bait vicinity. BioID allowed for successful interactome mapping of core histones and members of the mediator complex. We explored the background signal produced by the BioID approach and found that using distinct types of controls increased the stringency of our statistical analysis with SAINTexpress. A direct comparison of BioID with our AP-MS protocol optimized for chromatin-associated protein complexes revealed that the approaches identified few shared interaction partners and enriched for distinct biological processes; yet, both approaches permitted the recovery of biologically meaningful interactions. While no clear bias could be observed for either technique toward protein complexes of particular functions, BioID allowed for the purification of proteins of lower cellular abundance. Finally, we were able to identify a strong association of MED4 with the centrosome by BioID and validated this finding by immunofluorescence. In summary, BioID complements AP-MS for the study of chromatin-associated protein complexes. BIOLOGICAL SIGNIFICANCE: This manuscript describes the application of BioID, a proximity biotinylation approach, to chromatin-associated proteins, namely core histones and members of the mediator complex. We observed that BioID was successful at identifying known interaction partners for the baits tested, but also allowed novel putative interaction partners to be identified. By performing a detailed comparison of BioID versus a standard method for interactome mapping (affinity purification coupled to mass spectrometry, AP-MS), we show that the approaches were complementary, allowing for purification of different interaction partners. These interaction partners were different in the biological processes they are associated with, but also in their abundance. BioID represents a significant technical development in the field of chromatin research by expanding the search space for interactome mapping beyond what is possible with AP-MS. This article is part of a Special Issue entitled: Protein dynamics in health and disease. Guest Editors: Pierre Thibault and Anne-Claude Gingras.

MED18 interaction with distinct transcription factors regulates multiple plant functions.

Mediator is an evolutionarily conserved transcriptional regulatory complex. Mechanisms of Mediator function are poorly understood. Here we show that Arabidopsis MED18 is a multifunctional protein regulating plant immunity, flowering time and responses to hormones through interactions with distinct transcription factors. MED18 interacts with YIN YANG1 to suppress disease susceptibility genes glutaredoxins GRX480, GRXS13 and thioredoxin TRX-h5. Consequently, yy1 and med18 mutants exhibit deregulated expression of these genes and enhanced susceptibility to fungal infection. In addition, MED18 interacts with ABA INSENSITIVE 4 and SUPPRESSOR OF FRIGIDA4 to regulate abscisic acid responses and flowering time, respectively. MED18 associates with the promoter, coding and terminator regions of target genes suggesting its function in transcription initiation, elongation and termination. Notably, RNA polymerase II occupancy and histone H3 lysine tri-methylation of target genes are affected in the med18 mutant, reinforcing MED18 function in different mechanisms of transcriptional control. Overall, MED18 conveys distinct cues to engender transcription underpinning plant responses.

Resveratrol modulates MED28 (Magicin/EG-1) expression and inhibits epidermal growth factor (EGF)-induced migration in MDA-MB-231 human breast cancer cells.

Resveratrol and pterostilbene exhibit diverse biological activities. MED28, a subunit of the mammalian Mediator complex for transcription, was also identified as magicin, an actin cytoskeleton Grb2-associated protein, and as endothelial-derived gene (EG-1). Several tumors exhibit aberrant MED28 expression, whereas the underlying mechanism is unclear. Triple-negative breast cancers, often expressing epidermal growth factor (EGF) receptor (EGFR), are associated with metastasis and poor survival. The objective of this study is to compare the effect of resveratrol and pterostilbene and to investigate the role of MED28 in EGFR-overexpressing MDA-MB-231 breast cancer cells. Pretreatment of resveratrol, but not pterostlbene, suppressed EGF-mediated migration and expression of MED28 and matrix metalloproteinase (MMP)-9 in MDA-MB-231 cells. Moreover, overexpression of MED28 increased migration, and the addition of EGF further enhanced migration. Our data indicate that resveratrol modulates the effect of MED28 on cellular migration, presumably through the EGFR/phosphatidylinositol 3-kinase (PI3K) signaling pathway, in breast cancer cells.

Ovocalyxin-32, a novel chicken eggshell matrix protein. isolation, amino acid sequencing, cloning, and immunocytochemical localization.

The eggshell is a highly ordered structure resulting from the deposition of calcium carbonate concomitantly with an organic matrix upon the eggshell membranes. Mineralization takes place in an acellular uterine fluid, which contains the ionic and matrix precursors of the eggshell. We have identified a novel 32-kDa protein, ovocalyxin-32, which is expressed at high levels in the uterine and isthmus regions of the oviduct, and concentrated in the eggshell. Sequencing of peptides derived from the purified protein allowed expressed sequence tag sequences to be identified that were assembled to yield a full-length composite sequence whose conceptual translation product contained the complete amino acid sequence of ovocalyxin-32. Data base searches revealed that ovocalyxin-32 has limited identity (32%) to two unrelated proteins: latexin, a carboxypeptidase inhibitor expressed in the rat cerebral cortex and mast cells, and a skin protein, which is encoded by a retinoic acid receptor-responsive gene, TIG1. High level expression of ovocalyxin-32 was limited to the isthmus and uterus tissue, where immunocytochemistry at the light and electron microscope levels demonstrated that ovocalyxin-32 is secreted by surface epithelial cells. In the eggshell, ovocalyxin-32 localizes to the outer palisade layer, the vertical crystal layer, and the cuticle of the eggshell, in agreement with its demonstration by Western blotting at high levels in the uterine fluid during the termination phase of eggshell formation. Ovocalyxin-32 is therefore identified as a novel protein synthesized in the distal oviduct where hen eggshell formation occurs.

Isolation and characterization of a novel gene from the DiGeorge chromosomal region that encodes for a mediator subunit.

Hemizygous deletions on chromosome 22q11.2 result in developmental disorders referred to as DiGeorge syndrome (DGS)/velocardiofacial syndrome (VCFS). We report the isolation of a novel gene, PCQAP (PC2 glutamine/Q-rich-associated protein), that maps to the DiGeorge typically deleted region and encodes a protein identified as a subunit of the large multiprotein complex PC2. PC2 belongs to the family of the human Mediator complexes, which exhibit coactivator function in RNA polymerase II transcription. Furthermore, we cloned the homologous mouse Pcqap cDNA. There is 83% amino acid identity between the human and the mouse predicted protein sequences, with 96% similarity at the amino- and carboxy-terminal ends. To assess the potential involvement of PCQAP in DGS/VCFS, its developmental expression pattern was analyzed. In situ hybridization of mouse embryos at different developmental stages revealed that Pcqap is ubiquitously expressed. However, higher expression was detected in the frontonasal region, pharyngeal arches, and limb buds. Moreover, analysis of subjects carrying a typical 22q11 deletion revealed that the human PCQAP gene was deleted in all patients. Many of the structures affected in DGS/VCFS evolve from Pcqap-expressing cells. Together with the observed haploinsufficiency of PCQAP in DGS/VCFS patients, this finding is consistent with a possible role for this novel Mediator subunit in the development of some of the structures affected in DGS/VCFS.

A novel glutamine-rich putative transcriptional adaptor protein (TIG-1), preferentially expressed in placental and bone-marrow tissues.

We used a subtractive hybridization protocol to identify novel expressed sequence tags (ESTs) corresponding to mRNAs whose expression was induced upon exposure of the human leukemia cell line K562 to the phorbol ester 12-O-tetradecanolyphorbol-13-acetate (TPA). The complete open reading frame of one of the novel ESTs, named TIG-1, was obtained by screening K562 cell and placental cDNA libraries. The deduced open reading frame of the TIG-1 cDNA encodes for a glutamine repeat-rich protein with a predicted molecular weight of 63kDa. The predicted open reading frame also contains a consensus bipartite nuclear localization signal, though no specific DNA-binding domain is found. The corresponding TIG-1 mRNA is ubiquitously expressed. Placental tissue expresses the TIG-1 mRNA 200 times more than the lowest expressing tissues such as kidney and lung. There is also preferential TIG-1 mRNA expression in cells of bone-marrow lineage.In-vitro transcription/translation of the TIG-1 cDNA yielded a polypeptide with an apparent molecular weight of 97kDa. Using polyclonal antibodies obtained from a rabbit immunized with the carboxy-terminal portion of bacterially expressed TIG-1 protein, a polypeptide with molecular weight of 97kDa was identified by Western blot analyses of protein lysates obtained from K562 cells. Cotransfection assays of K562 cells, using a GAL4-TIG-1 fusion gene and GAL4 operator-CAT, indicate that the TIG-1 protein may have transcriptional regulatory activity when tethered to DNA. We hypothesize that this novel glutamine-rich protein participates in a protein complex that regulates gene transcription. It has been demonstrated by Naar et al. (Naar, A.M., Beaurang, P.A., Zhou, S., Abraham, S., Solomon, W.B., Tjian, R., 1999, Composite co-activator ARC mediates chromatin-directed transcriptional activation. Nature 398, 828-830) that the amino acid sequences of peptide fragments obtained from a polypeptide found in a complex of proteins that alters chromatin structure (ARC) are identical to portions of the deduced open reading frame of TIG-1 mRNA.

Identification of mouse TRAP100: a transcriptional coregulatory factor for thyroid hormone and vitamin D receptors.

Nuclear hormone receptors (NRs) regulate transcription in part by recruiting distinct transcriptional coregulatory complexes to target gene promoters. The thyroid hormone receptor (TR) was recently purified from thyroid hormone-cultured HeLa cells in association with a complex of novel nuclear proteins termed TRAPs (thyroid hormone receptor-associated proteins) ranging in size from 20 to 240 kDa. The TRAP complex markedly enhances TR-mediated transcription in vitro, suggesting a coactivator role for one or more of the TRAP components. Here we present the mouse cDNA for the 100-kDa component of the TRAP complex (mTRAP100). The mTRAP100 protein contains seven LxxLL motifs thought to be potential binding surfaces for liganded NRs, yet surprisingly fails to interact with TR and other NRs in vitro. By contrast, mTRAP100 coprecipitates in vivo with another component of the TRAP complex (TRAP220), which directly contacts TR and the vitamin D receptor in a ligand-dependent manner. Our findings thus suggest that TRAP100 is targeted to NRs in association with TRAP complexes specifically containing TRAP220. Transient overexpression of mTRAP100 in mammalian cells further enhances ligand-dependent transcription by both TR and the vitamin D receptor, revealing a functional role for mTRAP100 in NR-mediated transactivation. The presence of an intrinsic mTRAP100 transactivation function is suggested by the ability of mTRAP100 to activate transcription constitutively when tethered to the GAL4 DNA-binding domain. Collectively, these findings suggest that TRAP100, in concert with other TRAPs, plays an important functional role in mediating transactivation by specific NRs.