Genome-wide screens identify specific drivers of mutant hTERT promoters

Cancer-specific hTERT promoter mutations reported in 19% of cancers result in enhanced telomerase activity. Understanding the distinctions between transcriptional regulation of wild-type (WT) and mutant (Mut) hTERT promoters may open up avenues for development of inhibitors which specially block hTERT expression in cancer cells. To comprehensively identify physiological regulators of WT- or Mut-hTERT promoters, we generated several isogenic reporter cells driven by endogenous hTERT loci. Genome-wide CRISPR-Cas9 and small interfering RNA screens using these isogenic reporter lines identified specific regulators of Mut-hTERT promoters. We validate and characterize one of these hits, namely, MED12, a kinase subunit of mediator complex. We demonstrate that MED12 specifically drives expression of hTERT from the Mut-hTERT promoter by mediating long-range chromatin interaction between the proximal Mut-hTERT promoter and T-INT1 distal regulatory region 260 kb upstream. Several hits identified in our screens could serve as potential therapeutic targets, inhibition of which may specifically block Mut-hTERT promoter driven telomerase reactivation in cancers.

MED10 Drives the Oncogenicity and Refractory Phenotype of Bladder Urothelial Carcinoma Through the Upregulation of hsa-miR-590

BackgroundDysfunctional transcription machinery with associated dysregulated transcription characterizes many malignancies. Components of the mediator complex, a principal modulator of transcription, are increasingly implicated in cancer. The mediator complex subunit 10 (MED10), a vital kinase module of the mediator, plays a critical role in bladder physiology and pathology. However, its role in the oncogenicity, metastasis, and disease recurrence in bladder cancer (BLCA) remains unclear.ObjectiveThus, we investigated the role of dysregulated or aberrantly expressed MED10 in the enhanced onco-aggression, disease progression, and recurrence of bladder urothelial carcinoma (UC), as well as the underlying molecular mechanism.MethodsUsing an array of multi-omics big data analyses of clinicopathological data, in vitro expression profiling and functional assays, and immunocytochemical staining, we assessed the probable roles of MED10 in the progression and prognosis of BLCA/UC.ResultsOur bioinformatics-aided gene expression profiling showed that MED10 is aberrantly expressed in patients with BLCA, is associated with high-grade disease, is positively correlated with tumor stage, and confers significant survival disadvantage. Reanalyzing the TCGA BLCA cohort (n = 454), we showed that aberrantly expressed MED10 expression is associated with metastatic and recurrent disease, disease progression, immune suppression, and therapy failure. Interestingly, we demonstrated that MED10 interacts with and is co-expressed with the microRNA, hsa-miR-590, and that CRISPR-mediated knockout of MED10 elicits the downregulation of miR-590 preferentially in metastatic UC cells, compared to their primary tumor peers. More so, silencing MED10 in SW1738 and JMSU1 UC cell lines significantly attenuates their cell proliferation, migration, invasion, clonogenicity, and tumorsphere formation (primary and secondary), with the associated downregulation of BCL-xL, MKI67, VIM, SNAI1, OCT4, and LIN28A but upregulated BAX protein expression. In addition, we showed that high MED10 expression is a non-inferior biomarker of urothelial recurrence compared with markers of cancer stemness; however, MED10 is a better biomarker of local recurrence than any of the stemness markers.ConclusionThese data provide preclinical evidence that dysregulated MED10/MIR590 signaling drives onco-aggression, disease progression, and recurrence of bladder UC and that this oncogenic signal is therapeutically actionable for repressing the metastatic/recurrent phenotypes, enhancing therapy response, and shutting down stemness-driven disease progression and relapse in patients with BLCA/UC.

Identification of Therapeutic Targets and Prognostic Biomarkers among Genes from the Mediator Complex Family in the Hepatocellular Carcinoma Tumour-Immune Microenvironment

Background. Hepatocellular carcinoma (HCC) is predominant among all types of primary liver cancers characterised by high morbidity and mortality. Genes in the mediator complex (MED) family are engaged in the tumour-immune microenvironment and function as regulatory hubs mediating carcinogenesis and progression across diverse cancer types. Whereas research studies have been conducted to examine the mechanisms in several cancers, studies that systematically focused on the therapeutic and prognostic values of MED in patients with HCC are limited. Methods. The online databases ONCOMINE, GEPIA, UALCAN, GeneMANIA, cBioPortal, OmicStudio, STING, Metascape, and TIMER were used in this study. Results. The transcriptional levels of all members of the MED family in HCC presented an aberrant high expression pattern. Significant correlations were found between the MED1, MED6, MED8, MED10, MED12, MED15, MED17, MED19, MED20, MED21, MED22, MED23, MED24, MED25, MED26, and MED27 expression levels and the pathological stage in the patients with HCC. The patients with high expression levels of MED6, MED8, MED10, MED17, MED19, MED20, MED21, MED22, MED24, and MED25 were significantly associated with poor prognosis. Functional enrichment analysis revealed that the members of the MED family were mainly enriched in the nucleobase-containing compound catabolic process, regulation of chromosome organisation, and transcriptional regulation by TP53. Significant correlations were found between the MED6, MED8, MED10, MED17, MED19, MED20, MED21, MED22, MED24, and MED25 expression levels and all types of immune cells (B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells). B cells and MED8 were independent predictors of overall survival. We found significant correlations between the somatic copy number alterations of the MED6, MED8, MED10, MED20, MED21, MED22, MED24, and MED25 molecules and the abundance of immune infiltrates. Conclusions. Our study delineated a thorough landscape to investigate the therapeutic and prognostic potentials of the MED family for HCC cases, which yielded promising results for the development of immunotherapeutic drugs and construction of a prognostic stratification model.

Mediator Complex Subunit 19 Promotes the Development of Hepatocellular Carcinoma by Regulating the AKT/mTOR Signaling Pathway

BackgroundHepatocellular carcinoma (HCC) is one of the most common malignant tumors, the pathogenesis of which remains unclear. Mediator complex subunit 19 (MED19), a subunit of the Mediator complex, is a multi-protein co-activator necessary for DNA transcription factors to induce RNA polymerase II transcription. In the current study, we aimed to study the role of MED19 in HCC and elucidate its mechanism.MethodsMED19 expression in HCC tissues was determined. The relationship between MED19 and the clinical prognosis was explored. The influence of MED19 on HCC cell viability, migration, invasion, and apoptosis was studied. The expression of AKT/mTOR pathway genes and proteins was detected by qRT-PCR and western blot. The correlation between MED19 and immune infiltration was investigated.ResultsMED19 was upregulated in HCC tissues compared with tumor-adjacent tissues, and was associated with a poor prognosis. Furthermore, high MED19 expression was correlated with race, gender, etc. Knockdown of MED19 inhibited cell proliferation, migration, invasion, and promoted apoptosis. Knockdown of MED19 decreased p-AKT and p-mTOR protein expression. Additionally, the downstream effectors of the AKT/mTOR pathway, p70S6K1 and 4EBP1, were affected by MED19. Notably, MED19 expression was positively correlated with the infiltration levels of B cells, CD4+ T cells, CD8+ T cells, macrophages, etc.ConclusionMED19 is significantly upregulated in HCC tissues and cells. MED19 may promote the progression of HCC in vitro and may be related to immune infiltration. Together, our data show that MED19 could be considered as a new possible biomarker as well as a novel therapeutic target for HCC.

MED1 Deficiency in Macrophages Accelerates Intimal Hyperplasia via ROS Generation and Inflammation

Mediator complex subunit 1 (MED1) is a component of the mediator complex and functions as a coactivator involved in the regulated transcription of nearly all RNA polymerase II-dependent genes. Previously, we showed that MED1 in macrophages has a protective effect on atherosclerosis; however, the effect of MED1 on intimal hyperplasia and mechanisms regulating proinflammatory cytokine production after macrophage MED1 deletion are still unknown. In this study, we report that MED1 macrophage-specific knockout (MED1ΔMac) mice showed aggravated neointimal hyperplasia, vascular smooth muscle cells (VSMCs), and macrophage accumulation in injured arteries. Moreover, MED1ΔMac mice showed increased proinflammatory cytokine production after an injury to the artery. After lipopolysaccharide (LPS) treatment, MED1ΔMac macrophages showed increased generation of reactive oxygen species (ROS) and reduced expression of peroxisome proliferative activated receptor gamma coactivator-1α (PGC1α) and antioxidant enzymes, including catalase and glutathione reductase. The overexpression of PGC1α attenuated the effects of MED1 deficiency in macrophages. In vitro, conditioned media from MED1ΔMac macrophages induced more proliferation and migration of VSMCs. To explore the potential mechanisms by which MED1 affects inflammation, macrophages were treated with BAY11-7082 before LPS treatment, and the results showed that MED1ΔMac macrophages exhibited increased expression of phosphorylated-p65 and phosphorylated signal transducer and activator of transcription 1 (p-STAT1) compared with the control macrophages, suggesting the enhanced activation of NF-κB and STAT1. In summary, these data showed that MED1 deficiency enhanced inflammation and the proliferation and migration of VSMCs in injured vascular tissue, which may result from the activation of NF-κB and STAT1 due to the accumulation of ROS.

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.

MED13L-related intellectual disability due to paternal germinal mosaicism

The MED13L-related intellectual disability or MRFACD syndrome (Mental retardation and distinctive facial features with or without cardiac defects; MIM # 616789) is one of the most common form of syndromic intellectual disability with about a hundred cases reported so far. Affected individuals share overlapping features comprising intellectual disability, hypotonia, motor delay, remarkable speech delay, and a recognizable facial gestalt. De novo disruption of the MED13L gene by deletions, duplications or sequence variants has been identified deleterious. Siblings affected by intragenic deletion transmitted from a mosaic parent have been reported once in the literature. We now present the first case of paternal germinal mosaicism for a missense MED13L variant causing MRFACD syndrome in one of the father's children and be the likely cause of intellectual disability and facial dysmorphism in the other. As part of the Mediator complex, the MED proteins have an essential role in regulating transcription. 32 subunits of the Mediator complex genes have been linked to congenital malformations that are now acknowledged as transcriptomopathies. The MRFACD syndrome has been suggested to represent a recognizable phenotype.

High Expression of Mediator Complex Subunit 8 Acts as a Prognostic Biomarker in Hepatocellular Carcinoma

Background : Mediator complex subunit 8 ( MED8 ) encodes a subunit of the mediator complex ( MED ), which is critical for transcription. MED8 is highly expressed in some tumours and is associated with a poor prognosis. However, correlations between MED8 and clinical features of hepatocellular carcinoma (HCC) have not been reported. Results: A univariate analysis showed that high MED8 expression predicts poor overall survival (HR: 2.495; 95% confidence interval (CI) 1.740, 3.578; P < 0.001). A multivariate regression analysis showed that high MED8 (HR: 3.032 (1.817, 5.060); P < 0.001) expression and M stage (HR=4.075 (1.179-14.091) for M1 vs. M0, P=0.026) are independent prognostic indicator of poor overall survival in patients with HCC. The areas under the curve (AUC) for receiver operating characteristic (ROC) curves were used to describe the prognostic value of MED8 (AUC: 0.905 (0.849, 0.941)). Gene Set Enrichment Analysis (GSEA) and Immune infiltration Analysis were applied to reveal significant enrichment differences among TCGA data. A functional analysis showed that the cell cycle checkpoints, mitotic G2-G2–M phases, transcriptional regulation by TP53, and regulation of TP53 activity were significantly enriched in DEGs associated with high MED8 expression. Th2 cells were positively correlated with MED8 expression. Conclusions: MED8 predicts poor prognosis in HCC, potentially via the regulation of the cell cycle regulation and Th2 cells. Key words: Mediator complex subunit 8, Mediator , hepatocellular carcinoma, prognosis, diagnostic biomarker

Mediator complex proximal Tail subunit MED30 is critical for Mediator core stability and cardiomyocyte transcriptional network

Dysregulation of cardiac transcription programs has been identified in patients and families with heart failure, as well as those with morphological and functional forms of congenital heart defects. Mediator is a multi-subunit complex that plays a central role in transcription initiation by integrating regulatory signals from gene-specific transcriptional activators to RNA polymerase II (Pol II). Recently, Mediator subunit 30 (MED30), a metazoan specific Mediator subunit, has been associated with Langer-Giedion syndrome (LGS) Type II and Cornelia de Lange syndrome-4 (CDLS4), characterized by several abnormalities including congenital heart defects. A point mutation in MED30 has been identified in mouse and is associated with mitochondrial cardiomyopathy. Very recent structural analyses of Mediator revealed that MED30 localizes to the proximal Tail, anchoring Head and Tail modules, thus potentially influencing stability of the Mediator core. However, in vivo cellular and physiological roles of MED30 in maintaining Mediator core integrity remain to be tested. Here, we report that deletion of MED30 in embryonic or adult cardiomyocytes caused rapid development of cardiac defects and lethality. Importantly, cardiomyocyte specific ablation of MED30 destabilized Mediator core subunits, while the kinase module was preserved, demonstrating an essential role of MED30 in stability of the overall Mediator complex. RNAseq analyses of constitutive cardiomyocyte specific Med30 knockout (cKO) embryonic hearts and inducible cardiomyocyte specific Med30 knockout (icKO) adult cardiomyocytes further revealed critical transcription networks in cardiomyocytes controlled by Mediator. Taken together, our results demonstrated that MED30 is essential for Mediator stability and transcriptional networks in both developing and adult cardiomyocytes. Our results affirm the key role of proximal Tail modular subunits in maintaining core Mediator stability in vivo.