Novel MED15::ATF1 fusion in a pediatric melanoma with spitzoid features and aggressive presentation.

Childhood melanoma is a rare and biologically heterogeneous pediatric malignancy. The differential diagnosis of pediatric melanoma is usually broad, including a wide variety of spindle cell or epithelioid neoplasms. Different molecular alterations affecting the MAPK and PI3K/AKT/mTOR pathways, tumor suppressor genes, and telomerase reactivation have been implicated in melanoma tumorigenesis and progression. Here, we report a novel MED15::ATF1 fusion in a pediatric melanoma with spitzoid features and an aggressive clinical course.

MED15::TFE3 fusion renal cell carcinoma with extensive cystic change: A clinicopathologic and molecular genetic study of 2 cases, with an emphasis on differential diagnosis.

OBJECTIVES: TFE3-rearranged renal cell carcinomas (RCCs) harbor gene fusions between TFE3 and 1 of many partner genes. MED15::TFE3 fusion RCC is rare, often cystic, and easily misdiagnosed. METHODS: This study aimed to characterize 2 cases of MED15::TFE3 fusion RCC with extensive cystic change using fluorescence in situ hybridization and targeted RNA sequencing. RESULTS: Both patients were young adult women aged 29 and 35 years. Radiologically, both presented with a cystic Bosniak category II renal lesion. The cysts measured 9.3 cm and 4.8 cm in greatest dimension. Both patients underwent cyst enucleation, and neither had tumor recurrence or metastasis at 26 and 6 months of follow-up, respectively. Microscopically, both tumors were entirely cystic, with thick, fibrous cystic walls lined by small clusters of cells with clear to eosinophilic cytoplasm and uniform, round nuclei with inconspicuous nucleoli. There were also small aggregations of similar clear cells within the cystic walls. Foci of basement membrane-like material depositions were noted in 1 case; calcifications were observed in both cases. Both cases demonstrated nuclear positivity for PAX8 and TFE3 and cytoplasmic staining for Melan-A; HMB45, CAIX, and CK7 were negative. Fluorescence in situ hybridization revealed that both tumors were positive for TFE3 rearrangements. RNA sequencing identified MED15::TFE3 gene fusions in both cases. CONCLUSIONS: The main differential diagnosis of MED15::TFE3 fusion RCC includes multilocular cystic renal neoplasm of low malignant potential and atypical renal cysts. Molecular confirmation of TFE3 fusion is essential for establishing the correct diagnosis.

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.

Identification of novel inhibitors against Med15a KIX domain of Candida glabrata.

Candida glabrata, the second most common cause of invasive fungal infections, exhibits multi-drug resistance to commonly used antifungal drugs. To counter this resistance, there is a critical need for novel antifungals. This study identifies small molecule inhibitors that target a three-helix bundle KIX domain in the Med15a Mediator subunit of Candida glabrata (CgMed15a KIX). This domain plays a crucial role by interacting with the Pleiotropic Drug Resistance transcription factor Pdr1, a key regulator of the multidrug resistance pathway in Candida glabrata. We performed high throughput computational screening of large chemical datasets against the binding sites of the CgMed15a KIX domain to identify novel inhibitors. We selected six potential candidates with high affinity and confirmed their binding with the CgMed15a KIX domain. A phytochemical compound, Chebulinic acid binds to the CgMed15a KIX domain with a KD value of 0.339 µM and shows significant inhibitory effects on the growth of Candida glabrata. Molecular dynamics simulation studies further revealed the structural stability of the CgMed15a KIX-Chebulinic acid complex. Thus, in conclusion, this study highlights Chebulinic acid as a novel potential antifungal compound against Candida glabrata.

A Mediator subunit imparts robustness to a polyphenism decision.

Polyphenism is a type of developmental plasticity that translates continuous environmental variability into discontinuous phenotypes. Such discontinuity likely requires a switch between alternative gene-regulatory networks, a principle that has been borne out by mechanisms found to promote morph-specific gene expression. However, whether robustness is required to execute a polyphenism decision has awaited testing at the molecular level. Here, we used a nematode model for polyphenism, Pristionchus pacificus, to identify the molecular regulatory factors that ensure the development of alternative forms. This species has a dimorphism in its adult feeding structures, specifically teeth, which are a morphological novelty that allows predation on other nematodes. Through a forward genetic screen, we determined that a duplicate homolog of the Mediator subunit MDT-15/MED15, P. pacificus MDT-15.1, is necessary for the polyphenism and the robustness of the resulting phenotypes. This transcriptional coregulator, which has a conserved role in metabolic responses to nutritional stress, coordinates these processes with its effects on this diet-induced polyphenism. Moreover, this MED15 homolog genetically interacts with two nuclear receptors, NHR-1 and NHR-40, to achieve dimorphism: Single and double mutants for these three factors result in morphologies that together produce a continuum of forms between the extremes of the polyphenism. In summary, we have identified a molecular regulator that confers discontinuity to a morphological polyphenism, while also identifying a role for MED15 as a plasticity effector.

Mediator subunit MDT-15 promotes expression of propionic acid breakdown genes to prevent embryonic lethality in Caenorhabditis elegans.

The micronutrient vitamin B12 is an essential cofactor for two enzymes: methionine synthase, which plays a key role in the one-carbon cycle; and methylmalonyl-CoA mutase, an enzyme in a pathway that breaks down branched-chain amino acids and odd-chain fatty acids. A second, vitamin B12-independent pathway that degrades propionic acid was recently described in Caenorhabditis elegans, the propionate shunt pathway. Activation of five shunt pathway genes in response to low vitamin B12 availability or high propionic acid levels is accomplished by a transcriptional regulatory mechanism involving two nuclear hormone receptors, NHR-10 and NHR-68. Here, we report that the C. elegans Mediator subunit mdt-15 is also essential for the activation of the propionate shunt pathway genes, likely by acting as a transcriptional coregulator for NHR-10. C. elegans mdt-15 mutants fed with a low vitamin B12 diet have transcriptomes resembling those of wild-type worms fed with a high vitamin B12 diet, with low expression of the shunt genes. Phenotypically, the embryonic lethality of mdt-15 mutants is specifically rescued by diets high in vitamin B12, but not by dietary polyunsaturated fatty acids, which rescue many other phenotypes of the mdt-15 mutants. Finally, NHR-10 binds to MDT-15 in yeast two-hybrid assays, and the transcriptomes of nhr-10 mutants share overlap with those of mdt-15 mutants. Our data show that MDT-15 is a key coregulator for an NHR regulating propionic acid detoxification, adding to roles played by NHR:MDT-15 partnerships in metabolic regulation and pinpointing vitamin B12 availability as a requirement for mdt-15 dependent embryonic development.

Mediator Subunit Med15 Regulates Cell Morphology and Mating in Candida lusitaniae.

Candida lusitaniae is an emerging opportunistic pathogenic yeast capable of shifting from yeast to pseudohyphae form, and it is one of the few Candida species with the ability to reproduce sexually. In this study, we showed that a dpp3Δ mutant, inactivated for a putative pyrophosphatase, is impaired in cell separation, pseudohyphal growth and mating. The defective phenotypes were not restored after the reconstruction of a wild-type DPP3 locus, reinforcing the hypothesis of the presence of an additional mutation that we suspected in our previous study. Genetic crosses and genome sequencing identified an additional mutation in MED15, encoding a subunit of the mediator complex that functions as a general transcriptional co-activator in Eukaryotes. We confirmed that inactivation of MED15 was responsible for the defective phenotypes by rescuing the dpp3Δ mutant with a wild-type copy of MED15 and constructing a med15Δ knockout mutant that mimics the phenotypes of dpp3Δ in vitro. Proteomic analyses revealed the biological processes under the control of Med15 and involved in hyphal growth, cell separation and mating. This is the first description of the functions of MED15 in the regulation of hyphal growth, cell separation and mating, and the pathways involved in C. lusitaniae.

Cystic MED15::TFE3 translocation renal cell carcinoma: histologic mimicker of multilocular cystic renal neoplasm of low malignant potential with review of the literature.

Presented are four cystic renal masses which harbored a MED15::TFE3 gene fusion detected by RNAseq, mimicking multilocular cystic neoplasm of low malignant potential. Clinicopathologic and outcomes data were collected for all cases. Radiologically, three cases were diagnosed as complex cystic masses and one case as a renal cyst, three years prior to surgery. The tumors ranged in size from 1.8 to 14.5 cm. Grossly, all masses were extensively cystic. Microscopically, cells with a clear or minimally granular cytoplasm and nuclei with inconspicuous nucleoli lined the cysts' septa. Focally, small mass-forming aggregates of malignant cells were present between septae and were associated with psammomatous calcifications. In case one, apparent prior cyst wall rupture was associated with reactive changes and cystic spaces filled with fibrin clots. Two of the tumors were staged as T1a, one as T1b, and the other as T2b. By immunohistochemistry, the tumors were positive for TFE3, MelanA, and P504S, with apical CD10 while CAIX and CK7 were negative. RNA sequencing was performed on all cases revealing a MED15::TFE3 gene fusion. The patients were alive and without evidence of disease 11-49 months (mean 29.5) after partial nephrectomy. To date, 12 of the 15 MED15::TFE3 fusion renal cell carcinomas published in the literature are cystic, with three being extensively cystic. Thus, if a multilocular cystic renal neoplasm is encountered in a kidney specimen, translocation renal cell carcinoma should be included in the differential diagnosis as cystic MED15::TFE3 tRCCs carry an uncertain prognosis making recognition for future characterization necessary.

MED15::TFE3 Renal Cell Carcinomas: Report of Two New Cases and Review of the Literature Confirming Nearly Universal Multilocular Cystic Morphology.

We report two novel cases of Xp11 translocation renal cell carcinomas with the MED15::TFE3 gene fusion in adult females aged 40 and 74 years. Both cases were extensively cystic and contained only minimal clear cells lining cysts and within septal walls, raising the differential diagnosis of multilocular cystic renal neoplasm of low malignant potential. By immunohistochemistry, both neoplasms labeled for PAX8, TFE3, cathepsin K and Melan A but not for HMB45. On review of the published literature and the two cases reported herein, over 90% of MED15::TFE3 renal cell carcinomas (RCCs) have been described as cystic. The correlation of the MED15::TFE3 fusion with extensively cystic morphology represents the strongest association of TFE3 fusion partner with clinicopathological features among TFE3-rearranged RCC reported to date.

Cryptic inhibitory regions nearby activation domains.

Previously, the Nine amino acid TransActivation Domain (9aaTAD) was identified in the Gal4 region 862-870 (DDVYNYLFD). Here, we identified 9aaTADs in the distal Gal4 orthologs by our prediction algorithm and found their conservation in the family. The 9aaTAD function as strong activators was demonstrated. We identified adjacent Gal4 region 871-811 (DEDTPPNPKKE) as a natural 9aaTAD inhibitory domain located at the extreme Gal4 terminus. Moreover, we identified conserved Gal4 region 172-185 (FDWSEEDDMSDGLP), which was capable to reverse the 9aaTAD inhibition. In conclusion, our results uncover the existence of the cryptic inhibitory domains, which need to be carefully implemented in all functional studies with transcription factors to avoid incorrect conclusions.

Possible Role for Allelic Variation in Yeast MED15 in Ecological Adaptation.

The propensity for Saccharomyces cerevisiae yeast to ferment sugars into ethanol and CO2 has long been useful in the production of a wide range of food and drink. In the production of alcoholic beverages, the yeast strain selected for fermentation is crucial because not all strains are equally proficient in tolerating fermentation stresses. One potential mechanism by which domesticated yeast may have adapted to fermentation stresses is through changes in the expression of stress response genes. MED15 is a general transcriptional regulator and RNA Pol II Mediator complex subunit which modulates the expression of many metabolic and stress response genes. In this study, we explore the role of MED15 in alcoholic fermentation. In addition, we ask whether MED15 alleles from wine, sake or palm wine yeast improve fermentation activity and grape juice fermentation stress responses. And last, we investigate to what extent any differences in activity are due to allelic differences in the lengths of three polyglutamine tracts in MED15. We find that strains lacking MED15 are deficient in fermentation and fermentation stress responses and that MED15 alleles from alcoholic beverage yeast strains can improve both the fermentation capacity and the response to ethanol stresses when transplanted into a standard laboratory strain. Finally, we find that polyglutamine tract length in the Med15 protein is one determinant in the efficiency of the alcoholic fermentation process. These data lead to a working model in which polyglutamine tract length and other types of variability within transcriptional hubs like the Mediator subunit, Med15, may contribute to a reservoir of transcriptional profiles that may provide a fitness benefit in the face of environmental fluctuations.

Formation of nuclear condensates by the Mediator complex subunit Med15 in mammalian cells.

BACKGROUND: The Mediator complex is an evolutionarily conserved multi-subunit protein complex that plays major roles in transcriptional activation and is essential for cell growth, proliferation, and differentiation. Recent studies revealed that some Mediator subunits formed nuclear condensates that may facilitate enhancer-promoter interactions and gene activation. The assembly, regulation, and functions of these nuclear condensates remain to be further understood. RESULTS: We found that Med15, a subunit in the tail module of the Mediator complex, formed nuclear condensates through a novel mechanism. Nuclear foci of Med15 were detected by both immunostaining of endogenous proteins and live cell imaging. Like Med1 foci and many other biomolecular condensates, Med15 foci were sensitive to 1, 6-Hexanediol and showed rapid recovery during fluorescence recovery after photobleaching. Interestingly, overexpressing DYRK3, a dual-specificity kinase that controls the phase transition of membraneless organelles, appeared to disrupt Med1 foci and Med15 foci. We identified two regions that are required to form Med15 nuclear condensates: the glutamine-rich intrinsically disordered region (IDR) and a short downstream hydrophobic motif. The optodroplet assay revealed that both the IDR and the C-terminal region of Med15 contributed to intracellular phase separation. CONCLUSIONS: We identified that the Mediator complex subunit Med15 formed nuclear condensates and characterized their features in living cells. Our work suggests that Med15 plays a role in the assembly of transcription coactivator condensates in the nucleus and identifies Med15 regions that contribute to phase separation.

Novel Immune Modulators Enhance Caenorhabditis elegans Resistance to Multiple Pathogens.

Traditionally, treatments for bacterial infection have focused on killing the microbe or preventing its growth. As antimicrobial resistance becomes more ubiquitous, the feasibility of this approach is beginning to wane and attention has begun to shift toward disrupting the host-pathogen interaction by improving the host defense. Using a high-throughput, fragment-based screen to identify compounds that alleviate Pseudomonas aeruginosa-mediated killing of Caenorhabditis elegans, we identified over 20 compounds that stimulated host defense gene expression. Five of these molecules were selected for further characterization. Four of five compounds showed little toxicity against mammalian cells or worms, consistent with their identification in a phenotypic, high-content screen. Each of the compounds activated several host defense pathways, but the pathways were generally dispensable for compound-mediated rescue in liquid killing, suggesting redundancy or that the activation of unknown pathway(s) may be driving compound effects. A genetic mechanism was identified for LK56, which required the Mediator subunit MDT-15/MED15 and NHR-49/HNF4 for its function. Interestingly, LK32, LK34, LK38, and LK56 also rescued C. elegans from P. aeruginosa in an agar-based assay, which uses different virulence factors and defense mechanisms. Rescue in an agar-based assay for LK38 entirely depended upon the PMK-1/p38 MAPK pathway. Three compounds-LK32, LK34, and LK56-also conferred resistance to Enterococcus faecalis, and the two lattermost, LK34 and LK56, also reduced pathogenesis from Staphylococcus aureus This study supports a growing role for MDT-15 and NHR-49 in immune response and identifies five molecules that have significant potential for use as tools in the investigation of innate immunity.IMPORTANCE Trends moving in opposite directions (increasing antimicrobial resistance and declining novel antimicrobial development) have precipitated a looming crisis: the nearly complete inability to safely and effectively treat bacterial infections. To avert this, new approaches are needed. One idea is to stimulate host defense pathways to improve the clearance of bacterial infection. Here, we describe five small molecules that promote resistance to infectious bacteria by activating C. elegans' innate immune pathways. Several are effective against both Gram-positive and Gram-negative pathogens. One of the compounds was mapped to the action of MDT-15/MED15 and NHR-49/HNF4, a pair of transcriptional regulators more generally associated with fatty acid metabolism, potentially highlighting a new link between these biological functions. These studies pave the way for future characterization of the anti-infective activity of the molecules in higher organisms and highlight the compounds' potential utility for further investigation of immune modulation as a novel therapeutic approach.

Precise Expression of Afmed15 Is Crucial for Asexual Development, Virulence, and Survival of Aspergillus fumigatus.

The rise of drug resistance in fungal pathogens is becoming a serious problem owing to the limited number of antifungal drugs available. Identifying and targeting factors essential for virulence or development unique to fungal pathogens is one approach to develop novel treatments for fungal infections. In this study, we present the identification and functional characterization of a novel developmental regulator in Aspergillus fumigatus, AfMed15, which contained a conserved Med15_fungal domain, as determined by screening of a mutant library that contained more than 2,000 hygromycin-resistant A. fumigatus transformants. Downregulating the expression of Afmed15 abolished the conidiation and decreased the fungal virulence in an insect model. Strikingly, the overexpression of Afmed15 caused fungal death accompanied by intensive autophagy. RNA sequencing of an Afmed15 overexpression strain revealed that altered gene expression patterns were associated with carbon metabolism, energy metabolism, and translation. Interestingly, the addition of metal ions could partially rescue fungal death caused by the overexpression of Afmed15, indicating that disordered ion homeostasis is a potential reason for the fungal death caused by the overexpression of Afmed15 Considering that the precise expression of Afmed15 is crucial for fungal development, virulence, and survival and that no ortholog was found in humans, Afmed15 is an ideal target for antifungal-drug development.IMPORTANCE The identification and characterization of regulators essential for virulence or development constitute one approach for antifungal drug development. In this study, we screened and functionally characterized Afmed15, a novel developmental regulator in A. fumigatus We demonstrate that the precise transcriptional expression of Afmed15 is crucial for fungal asexual development, virulence, and survival. Downregulating the expression of Afmed15 abolished the conidiation and decreased the fungal virulence in an insect model. In contrast, the overexpression of Afmed15 caused fungal death accompanied by intensive autophagy. Our study provides a foundation for further studies to identify compounds perturbing the expression of Afmed15 that may be used for the prevention of invasive A. fumigatus infections.

The Polymorphic PolyQ Tail Protein of the Mediator Complex, Med15, Regulates the Variable Response to Diverse Stresses.

The Mediator is composed of multiple subunits conserved from yeast to humans and plays a central role in transcription. The tail components are not required for basal transcription but are required for responses to different stresses. While some stresses are familiar, such as heat, desiccation, and starvation, others are exotic, yet yeast can elicit a successful stress response. 4-Methylcyclohexane methanol (MCHM) is a hydrotrope that induces growth arrest in yeast. We found that a naturally occurring variation in the Med15 allele, a component of the Mediator tail, altered the stress response to many chemicals in addition to MCHM. Med15 contains two polyglutamine repeats (polyQ) of variable lengths that change the gene expression of diverse pathways. The Med15 protein existed in multiple isoforms and its stability was dependent on Ydj1, a protein chaperone. The protein level of Med15 with longer polyQ tracts was lower and turned over faster than the allele with shorter polyQ repeats. MCHM sensitivity via variation of Med15 was regulated by Snf1 in a Myc-tag-dependent manner. Tagging Med15 with Myc altered its function in response to stress. Genetic variation in transcriptional regulators magnified genetic differences in response to environmental changes. These polymorphic control genes were master variators.

The evolution of the 9aaTAD domain in Sp2 proteins: inactivation with valines and intron reservoirs.

The universal nine-amino-acid transactivation domains (9aaTADs) have been identified in numerous transcription activators. Here, we identified the conserved 9aaTAD motif in all nine members of the specificity protein (SP) family. Previously, the Sp1 transcription factor has been defined as a glutamine-rich activator. We showed by amino acid substitutions that the glutamine residues are completely dispensable for 9aaTAD function and are not conserved in the SP family. We described the origin and evolutionary history of 9aaTADs. The 9aaTADs of the ancestral Sp2 gene became inactivated in early chordates. We next discovered that an accumulation of valines in 9aaTADs inactivated their transactivation function and enabled their strict conservation during evolution. Subsequently, in chordates, Sp2 has duplicated and created new paralogs, Sp1, Sp3, and Sp4 (the SP1-4 clade). During chordate evolution, the dormancy of the Sp2 activation domain lasted over 100 million years. The dormant but still intact ancestral Sp2 activation domains allowed diversification of the SP1-4 clade into activators and repressors. By valine substitution in the 9aaTADs, Sp1 and Sp3 regained their original activator function found in ancestral lower metazoan sea sponges. Therefore, the vertebrate SP1-4 clade could include both repressors and activators. Furthermore, we identified secondary 9aaTADs in Sp2 introns present from fish to primates, including humans. In the gibbon genome, introns containing 9aaTADs were used as exons, which turned the Sp2 gene into an activator. Similarly, we identified introns containing 9aaTADs used conditionally as exons in the (SP family-unrelated) transcription factor SREBP1, suggesting that the intron-9aaTAD reservoir is a general phenomenon.

The Mediator subunit OsMED15a is a transcriptional co-regulator of seed size/weight-modulating genes in rice.

Although several transcription factors (TFs) that regulate seed size/weight in plants are known, the molecular landscape regulating this important trait is unclear. Here, we report that a Mediator subunit, OsMED15a, links rice grain size/weight-regulating TFs to their target genes. Expression analysis and high-resolution quantitative trait loci (QTL) mapping suggested that OsMED15a is involved in rice seed development. OsMED15a has an N-terminal, three-helical KIX domain. Two of these helices, α1 and α3, and three amino acids, 76LRC78, within OsMED15a helix α3 were important for its interaction with several proteins, including interactions with the transactivation domains of two NAC-type TFs, OsNAC024 and OsNAC025. Moreover, OsMED15a, OsNAC024, and OsNAC025 all exhibited increased expression during seed development, and we identified several grain size/weight-associated SNPs in these genes in 509 low- and high-grain-weight rice genotypes. RNAi-mediated repression of OsMED15a expression down-regulated the expression of the grain size/weight regulating genes GW2, GW5 and DR11 and reduced grain length, weight, and yield. Of note, both OsNAC024 and OsNAC025 bound to the promoters of these three genes. We conclude that the transactivation domains of OsNAC024 and OsNAC025 target the KIX domain of OsMED15a in the regulation of grain size/weight-associated genes such as GW2, GW5, and D11. We propose that the integrated molecular-genetics approach used here could help identify networks of functional alleles of other regulator and co-regulator genes and thereby inform efforts for marker-assisted introgression of useful alleles in rice crop improvement.

Nuclear hormone receptors: Ancient 9aaTAD and evolutionally gained NCoA activation pathways.

In higher metazoans, the nuclear hormone receptors activate transcription trough their specific adaptors, nuclear hormone receptor adaptors NCoA, which are absent in lower metazoans. The Nine amino acid TransActivation Domain, 9aaTAD, was reported for a large number of the transcription activators that recruit general mediators of transcription. In this study, we demonstrated that the 9aaTAD from NHR-49 receptor of nematode C.elegans activates transcription as a small peptide. We showed that the ancient 9aaTAD domains are conserved in the nuclear hormone receptors including human HNF4, RARa, VDR and PPARg. Also their small 9aaTAD peptides effectively activated transcription in absence of the NCoA adaptors. We also showed that adjacent H11 domains in ancient and modern hormone receptors have an inhibitory effect on their 9aaTAD function.

The knockdown of the Mediator complex subunit MED15 restrains urothelial bladder cancer cells' malignancy.

The Mediator complex, a multi-subunit protein complex, plays an integral role in regulating transcription. Genetic alterations of the mediator subunit 15 (MED15) in separate tumor entities have been described previously. However, till now, not much is known about the role of MED15 in urothelial bladder cancer (BCa). Using cBioPortal, database analysis was executed for the mRNA expression and survival analysis of MED15 in BCa. Immunohistochemistry (IHC) analysis against MED15 was performed on tissue microarrays with 18 benign, 126 BCa, and 38 metastases samples. The intensity evaluation was performed using a staining intensity score from 0 to 3 and associated with clinicopathological data. The BCa cell lines T24 and TCCSUP were used for the functional investigation. After the MED15 knockdown by small interfering (si)RNA, cell proliferation, migration and invasion were investigated. On the mRNA level, only a low number of alterations (2%) was found for MED15 in BCa. Due to the small count of events, there were no significant differences or tendencies in survival. For IHC, MED15 was found to have a higher expression in non-muscle invasive BCa compared with benign and muscle invasive BCa. For survival analysis, no significant differences between samples with or without overexpression of MED15 were found. In the functional analysis, proliferation, migration, and invasion were significantly reduced in BCa-cells following the transient siRNA-mediated MED15 knockdown. In summary, MED15 appears to play a role in the tumor parameters proliferation, migration, and invasion in BCa, but further investigations are necessary.