Interaction between MED12 and ΔNp63 activates basal identity in pancreatic ductal adenocarcinoma.

The presence of basal lineage characteristics signifies hyperaggressive human adenocarcinomas of the breast, bladder and pancreas. However, the biochemical mechanisms that maintain this aberrant cell state are poorly understood. Here we performed marker-based genetic screens in search of factors needed to maintain basal identity in pancreatic ductal adenocarcinoma (PDAC). This approach revealed MED12 as a powerful regulator of the basal cell state in this disease. Using biochemical reconstitution and epigenomics, we show that MED12 carries out this function by bridging the transcription factor ΔNp63, a known master regulator of the basal lineage, with the Mediator complex to activate lineage-specific enhancer elements. Consistent with this finding, the growth of basal-like PDAC is hypersensitive to MED12 loss when compared to PDAC cells lacking basal characteristics. Taken together, our genetic screens have revealed a biochemical interaction that sustains basal identity in human cancer, which could serve as a target for tumor lineage-directed therapeutics.

OsMED14_2, a tail module subunit of Mediator complex, controls rice development and involves jasmonic acid.

The Mediator complex is essential for eukaryotic transcription, yet its role and the function of its individual subunits in plants, especially in rice, remain poorly understood. Here, we investigate the function of OsMED14_2, a subunit of the Mediator tail module, in rice development. Overexpression and knockout of OsMED14_2 resulted in notable changes in panicle morphology and grain size. Microscopic analysis revealed impact of overexpression on pollen maturation, reflected by reduced viability, irregular shapes, and aberrant intine development. OsMED14_2 was found to interact with proteins involved in pollen development, namely, OsMADS62, OsMADS63 and OsMADS68, and its overexpression negatively affected the expression of OsMADS68 and the expression of other genes involved in intine development, including OsCAP1, OsGCD1, OsRIP1, and OsCPK29. Additionally, we found that OsMED14_2 overexpression influences jasmonic acid (JA) homeostasis, affecting bioactive JA levels, and expression of OsJAZ genes. Our data suggest OsMED14_2 may act as a regulator of JA-responsive genes through its interactions with OsHDAC6 and OsJAZ repressors. These findings contribute to better understanding of the Mediator complex's role in plant traits regulation.

The Mediator Med23 controls a transcriptional switch for muscle stem cell proliferation and differentiation in muscle regeneration.

Muscle stem cells (MuSCs) contribute to a robust muscle regeneration process after injury, which is highly orchestrated by the sequential expression of multiple key transcription factors. However, it remains unclear how key transcription factors and cofactors such as the Mediator complex cooperate to regulate myogenesis. Here, we show that the Mediator Med23 is critically important for MuSC-mediated muscle regeneration. Med23 is increasingly expressed in activated/proliferating MuSCs on isolated myofibers or in response to muscle injury. Med23 deficiency reduced MuSC proliferation and enhanced its precocious differentiation, ultimately compromising muscle regeneration. Integrative analysis revealed that Med23 oppositely impacts Ternary complex factor (TCF)-targeted MuSC proliferation genes and myocardin-related transcription factor (MRTF)-targeted myogenic differentiation genes. Consistently, Med23 deficiency decreases the ETS-like transcription factor 1 (Elk1)/serum response factor (SRF) binding at proliferation gene promoters but promotes MRTF-A/SRF binding at myogenic gene promoters. Overall, our study reveals the important transcriptional control mechanism of Med23 in balancing MuSC proliferation and differentiation in muscle regeneration.

Expanding phenotype of MED13-associated syndrome presenting novel de novo missense variant in a patient with multiple congenital anomalies.

BACKGROUND: Whole exome sequencing allows rapid identification of causative single nucleotide variants and short insertions/deletions in children with congenital anomalies and/or intellectual disability, which aids in accurate diagnosis, prognosis, appropriate therapeutic interventions, and family counselling. Recently, de novo variants in the MED13 gene were described in patients with an intellectual developmental disorder that included global developmental delay, mild congenital heart anomalies, and hearing and vision problems in some patients. RESULTS: Here we describe an infant who carried a de novo p.Pro835Ser missense variant in the MED13 gene, according to whole exome trio sequencing. He presented with congenital heart anomalies, dysmorphic features, hydrocephalic changes, hypoplastic corpus callosum, bilateral optic nerve atrophy, optic chiasm atrophy, brain stem atrophy, and overall a more severe condition compared to previously described patients. CONCLUSIONS: Therefore, we propose to expand the MED13-associated phenotype to include severe complications that could end up with multiple organ failure and neonatal death.

DELLA proteins recruit the Mediator complex subunit MED15 to coactivate transcription in land plants.

DELLA proteins are negative regulators of the gibberellin response pathway in angiosperms, acting as central hubs that interact with hundreds of transcription factors (TFs) and regulators to modulate their activities. While the mechanism of TF sequestration by DELLAs to prevent DNA binding to downstream targets has been extensively documented, the mechanism that allows them to act as coactivators remains to be understood. Here, we demonstrate that DELLAs directly recruit the Mediator complex to specific loci in Arabidopsis, facilitating transcription. This recruitment involves DELLA amino-terminal domain and the conserved MED15 KIX domain. Accordingly, partial loss of MED15 function mainly disrupted processes known to rely on DELLA coactivation capacity, including cytokinin-dependent regulation of meristem function and skotomorphogenic response, gibberellin metabolism feedback, and flavonol production. We have also found that the single DELLA protein in the liverwort Marchantia polymorpha is capable of recruiting MpMED15 subunits, contributing to transcriptional coactivation. The conservation of Mediator-dependent transcriptional coactivation by DELLA between Arabidopsis and Marchantia implies that this mechanism is intrinsic to the emergence of DELLA in the last common ancestor of land plants.

A novel MED12 pathogenic variant in a female fetus with facial cleft and cardiac defects identified in the first trimester.

Trio exome sequencing was performed on a female fetus with an increased nuchal translucency, along with nasal bone hypoplasia, suspected cleft palate and abnormal outflow tract of the heart. A de novo heterozygous variant c.5500_5507del, p.(Tyr1834Argfs × 58) in the MED12 gene was detected. Loss-of-function variants in MED12 in females are associated with Hardikar syndrome (HS). A follow-up ultrasound at 15+5 weeks of gestation identified multiple fetal anomalies including bilateral cleft lip and palate, diaphragmatic hernia, atrioventricular septal defect, persistent truncus arteriosus, and bilateral renal pelvis dilation. Fetal autopsy confirmed the prenatal sonographic findings, and the MED12 variant was discussed by our multidisciplinary team to be the cause of fetal anomalies. Our case is the first prenatal one in which HS was diagnosed due to first trimester structural malformations. This case report presents another example of early identification of a major anomaly which allows earlier genetic diagnosis and more time for clinical management.

Nuclear receptor signaling via NHR-49/MDT-15 regulates stress resilience and proteostasis in response to reproductive and metabolic cues.

The ability to sense and respond to proteotoxic insults declines with age, leaving cells vulnerable to chronic and acute stressors. Reproductive cues modulate this decline in cellular proteostasis to influence organismal stress resilience in Caenorhabditis elegans We previously uncovered a pathway that links the integrity of developing embryos to somatic health in reproductive adults. Here, we show that the nuclear receptor NHR-49, an ortholog of mammalian peroxisome proliferator-activated receptor α (PPARα), regulates stress resilience and proteostasis downstream from embryo integrity and other pathways that influence lipid homeostasis and upstream of HSF-1. Disruption of the vitelline layer of the embryo envelope, which activates a proteostasis-enhancing intertissue pathway in somatic cells, triggers changes in lipid catabolism gene expression that are accompanied by an increase in fat stores. NHR-49, together with its coactivator, MDT-15, contributes to this remodeling of lipid metabolism and is also important for the elevated stress resilience mediated by inhibition of the embryonic vitelline layer. Our findings indicate that NHR-49 also contributes to stress resilience in other pathways known to change lipid homeostasis, including reduced insulin-like signaling and fasting, and that increased NHR-49 activity is sufficient to improve proteostasis and stress resilience in an HSF-1-dependent manner. Together, our results establish NHR-49 as a key regulator that links lipid homeostasis and cellular resilience to proteotoxic stress.

CDK7 kinase activity promotes RNA polymerase II promoter escape by facilitating initiation factor release.

Cyclin-dependent kinase 7 (CDK7), part of the general transcription factor TFIIH, promotes gene transcription by phosphorylating the C-terminal domain of RNA polymerase II (RNA Pol II). Here, we combine rapid CDK7 kinase inhibition with multi-omics analysis to unravel the direct functions of CDK7 in human cells. CDK7 inhibition causes RNA Pol II retention at promoters, leading to decreased RNA Pol II initiation and immediate global downregulation of transcript synthesis. Elongation, termination, and recruitment of co-transcriptional factors are not directly affected. Although RNA Pol II, initiation factors, and Mediator accumulate at promoters, RNA Pol II complexes can also proceed into gene bodies without promoter-proximal pausing while retaining initiation factors and Mediator. Further downstream, RNA Pol II phosphorylation increases and initiation factors and Mediator are released, allowing recruitment of elongation factors and an increase in RNA Pol II elongation velocity. Collectively, CDK7 kinase activity promotes the release of initiation factors and Mediator from RNA Pol II, facilitating RNA Pol II escape from the promoter.

Mediator kinase module proteins, genetic alterations and expression of super-enhancer regulated genes in colorectal cancer.

BACKGROUND: Genetic alterations are well characterized as contributors to the pathogenesis of cancers. Epigenetic abnormalities can lead to perturbations of the expression of genes in cancer cells without structural defects. Deregulation of proteins of the transcription machinery may result in perturbations of target genes. Mediator, a multiprotein component of the transcription machinery facilitates the function of RNA polymerase II, which transcribes most human genes. A part of the mediator with kinase activity, called the Mediator kinase module shows genetic alterations in a sub-set of colorectal cancers. METHODS: Data from publicly available genomic series of colorectal cancer patients were examined to determine alterations of Mediator kinase module component genes, including MED12, MED12L, MED13, MED13L, CDK8, CDK19, and CCNC. The prevalence of alterations in genomically defined colorectal cancer sub-sets was also interrogated. The effect of Mediator kinase module member gene expression on colorectal cancer relapse-free survival was investigated. RESULTS: Mutations in genes of the Mediator kinase module were present in a small percentage of colorectal cancers, ranging between 2 to 10% for MED12 and MED13 and alternative units MED12L and MED13L and below 2% for kinases CDK8 and CDK19 and cyclin C. Amplifications of the CDK8 gene were observed in 3% to 5% of colorectal cancers. The highest prevalence of mutations was observed in MSI cancers and the equivalent CMS1 group, with other genomic groups showing much lower frequency. An association of higher expression of MED12 with inferior relapse-free survival was observed. In contrast, higher expression of cyclin C was associated with improved survival. Colorectal cancer cell lines with CDK8 amplifications displayed sensitivity to several small molecule inhibitors of the KRAS/PI3K pathway but not to BET inhibitors. CONCLUSION: The Mediator kinase module is deregulated in a sub-set of colorectal cancers with differences observed in genomically defined groups. These variations may result in differences in sensitivity to targeted therapies and may have to be taken into consideration as such therapies are developed.

Growth-regulated co-occupancy of Mediator and Lsm3 at intronic ribosomal protein genes.

Mediator is a well-known transcriptional co-regulator and serves as an adaptor between gene-specific regulatory proteins and RNA polymerase II. Studies on the chromatin-bound form of Mediator revealed interactions with additional protein complexes involved in various transcription-related processes, such as the Lsm2-8 complex that is part of the spliceosomal U6 small nuclear ribonucleoprotein complex. Here, we employ Chromatin Immunoprecipitation sequencing (ChIP-seq) of chromatin associated with the Lsm3 protein and the Med1 or Med15 Mediator subunits. We identify 86 genes co-occupied by both Lsm3 and Mediator, of which 73 were intron-containing ribosomal protein genes. In logarithmically growing cells, Mediator primarily binds to their promoter regions but also shows a second, less pronounced occupancy at their 3'-exons. During the late exponential phase, we observe a near-complete transition of Mediator from these promoters to a position in their 3'-ends, overlapping the Lsm3 binding sites ∼250 bp downstream of their last intron-exon boundaries. Using an unbiased RNA sequencing approach, we show that transition of Mediator from promoters to the last exon of these genes correlates to reduction of both their messenger RNA levels and splicing ratios, indicating that the Mediator and Lsm complexes cooperate to control growth-regulated expression of intron-containing ribosomal protein genes at the levels of transcription and splicing.

Identification of a molecular network regulated by multiple ASD high risk genes.

Genetic sequencing has identified high-confidence ASD risk genes with loss-of-function mutations. How the haploinsufficiency of distinct ASD risk genes causes ASD remains to be elucidated. In this study, we examined the role of four top-ranking ASD risk genes, ADNP, KDM6B, CHD2, and MED13, in gene expression regulation. ChIP-seq analysis reveals that gene targets with the binding of these ASD risk genes at promoters are enriched in RNA processing and DNA repair. Many of these targets are found in ASD gene database (SFARI), and are involved in transcription regulation and chromatin remodeling. Common gene targets of these ASD risk genes include a network of high confidence ASD genes associated with gene expression regulation, such as CTNNB1 and SMARCA4. We further directly examined the transcriptional impact of the deficiency of these ASD risk genes. Our mRNA profiling with qPCR assays in cells with the knockdown of Adnp, Kdm6b, Chd2 or Med13 has revealed an intricate pattern of their cross-regulation, as well as their influence on the expression of other ASD genes. In addition, some synaptic genes, such as Snap25 and Nrxn1, are strongly regulated by deficiency of the four ASD risk genes, which could be through the direct binding at promoters or indirectly through the targets like Ctnnb1 or Smarca4. The identification of convergent and divergent gene targets that are regulated by multiple ASD risk genes will help to understand the molecular mechanisms underlying common and unique phenotypes associated with haploinsufficiency of ASD-associated genes.

Assessment of machine-learning predictions for the Mediator complex subunit MED25 ACID domain interactions with transactivation domains.

The human Mediator complex subunit MED25 binds transactivation domains (TADs) present in various cellular and viral proteins using two binding interfaces, named H1 and H2, which are found on opposite sides of its ACID domain. Here, we use and compare deep learning methods to characterize human MED25-TAD interfaces and assess the predicted models to published experimental data. For the H1 interface, AlphaFold produces predictions with high-reliability scores that agree well with experimental data, while the H2 interface predictions appear inconsistent, preventing reliable binding modes. Despite these limitations, we experimentally assess the validity of MED25 interface predictions with the viral transcriptional activators Lana-1 and IE62. AlphaFold predictions also suggest the existence of a unique hydrophobic pocket for the Arabidopsis MED25 ACID domain.

Familial Dilated Cardiomyopathy: A Novel MED9 Short Isoform Identification.

Familial dilated cardiomyopathy (DCM) is among the leading indications for heart transplantation. DCM alters the transcriptomic profile. The alteration or activation/silencing of physiologically operating transcripts may explain the onset and progression of this pathological state. The mediator complex (MED) plays a fundamental role in the transcription process. The aim of this study is to investigate the MED subunits, which are altered in DCM, to identify target crossroads genes. RNA sequencing allowed us to identify specific MED subunits that are altered during familial DCM, transforming into human myocardial samples. N = 13 MED subunits were upregulated and n = 7 downregulated. MED9 alone was significantly reduced in patients compared to healthy subjects (HS) (FC = -1.257; p < 0.05). Interestingly, we found a short MED9 isoform (MED9s) (ENSG00000141026.6), which was upregulated when compared to the full-transcript isoform (MED9f). Motif identification analysis yielded several significant matches (p < 0.05), such as GATA4, which is downregulated in CHD. Moreover, although the protein-protein interaction network showed FOG2/ZFPM2, FOS and ID2 proteins to be the key interacting partners of GATA4, only FOG2/ZFPM2 overexpression showed an interaction score of "high confidence" ≥ 0.84. A significant change in the MED was observed during HF. For the first time, the MED9 subunit was significantly reduced between familial DCM and HS (p < 0.05), showing an increased MED9s isoform in DCM patients with respect to its full-length transcript. MED9 and GATA4 shared the same sequence motif and were involved in a network with FOG2/ZFPM2, FOS, and ID2, proteins already implicated in cardiac development.

Cochleovestibular Phenotype in a Rare Genetic MED13L Mutation.

The gene MED13 participates in transcription. The MED13L gene is a paralog of MED13 that is involved in developmental gene expression. Mutations in the gene have been shown to result in a heterogenous phenotype affecting several physiological systems. Hearing loss has been reported very rarely, and vestibular weakness has never been reported in the condition. In this report, we present a mutation of MED13L in c.1162A > T (p.Arg388Ter), where we detail and describe a cochleovestibular phenotype with objective vestibulometry for the first time. The child showed bilateral sloping sensorineural hearing loss, a bilateral vestibular weakness, and an inner ear vestibular structural abnormality on imaging. Early intervention with hearing aids and vestibular rehabilitation led to a favorable outcome in terms of speech, communication, and balance. We emphasize the importance of comprehensive audiovestibular assessment in children diagnosed with MED13L mutations for effective management of these children.

Establishment of a human induced pluripotent stem cell line, KMUGMCi010-A, from a patient with X-linked Ohdo syndrome bearing missense mutation in the MED12 gene.

X-linkded Ohdo syndrome is characterized mainly by intellectual disability, delays in reaching development, feeding difficulties, thyroid dysfunction, and dysmorphic appearance with blepharophimosis, immobile mask-like face and bulbous nose. The X-linked Ohdo syndrome is caused by loss of function mutation in MED12 gene on X chromosome. The peripheral blood mononuclear cells from a patient carrying missense mutation of the MED12 gene were reprogrammed using the CytoTune-iPS2.0 Sendai Reprogramming Kit. The missense mutation in MED12 gene causes the abnormal protein variant. The established human induced pluripotent cell line will enable proper in vitro disease modelling of X-linked Ohdo syndrome.

A de novo frameshift variant in MED13 gene in a patient with autism spectrum disorder and magnetic resonance imaging abnormalities mimicking tuberous sclerosis.

The mediator complex subunit 13 (MED13) gene is implicated in neurodevelopmental disorders including autism spectrum disorder (ASD), intellectual disability, and speech delay with varying severity and course. Additional, extra central nervous system, features include eye or vision problems, hypotonia, congenital heart abnormalities, and dysmorphisms. We describe a 7-year- and 4-month-old girl evaluated for ASD whose brain magnetic resonance imaging was suggestive of multiple cortical tubers. The exome sequencing (ES - trio analysis) uncovered a unique, de novo, frameshift variant in the MED13 gene (c.4880del, D1627Vfs*17), with a truncating effect on the protein. This case report thus expands the phenotypic spectrum of MED13-related disorders to include brain abnormalities.

Hypoxic Bone Mesenchymal Stem Cell-Derived Exosomes Direct Schwann Cells Proliferation, Migration, and Paracrine to Accelerate Facial Nerve Regeneration via circRNA_Nkd2/miR-214-3p/MED19 Axis.

Background: Facial nerves have the potential for regeneration following injury, but this process is often challenging and slow. Schwann cells (SCs) are pivotal in this process. Bone mesenchymal stem cells (BMSC)-derived exosomes promote tissue repair through paracrine action, with hypoxic preconditioning enhancing their effects. The main purpose of this study was to determine whether hypoxia-preconditioned BMSC-derived exosomes (Hypo-Exos) exhibit a greater therapeutic effect on facial nerve repair/regeneration and reveal the mechanism. Methods: CCK-8, EdU, Transwell, and ELISA assays were used to evaluate the functions of Hypo-Exos in SCs. Histological analysis and Vibrissae Movements (VMs) recovery were used to evaluate the therapeutic effects of Hypo-Exos in rat model. circRNA array was used to identify the significantly differentially expressed exosomal circRNAs between normoxia-preconditioned BMSC-derived exosomes (Nor-Exos) and Hypo-Exos. miRDB, TargetScan, double luciferase assay, qRT-PCR and WB were used to predict and identify potential exosomal cirRNA_Nkd2-complementary miRNAs and its target gene. The function of exosomal circRNA_Nkd2 in facial nerve repair/regeneration was evaluated by cell and animal experiments. Results: This study confirmed that Hypo-Exos more effectively promote SCs proliferation, migration, and paracrine function, accelerating facial nerve repair following facial nerve injury (FNI) compared with Nor-Exos. Furthermore, circRNA analysis identified significant enrichment of circRNA_Nkd2 in Hypo-Exos compared with Nor-Exos. Exosomal circRNA_Nkd2 positively regulates mediator complex subunit 19 (MED19) expression by sponging rno-miR-214-3p. Conclusion: Our results demonstrated a mechanism by which Hypo-Exos enhanced SCs proliferation, migration, and paracrine function and facial nerve repair and regeneration following FNI through the circRNA_Nkd2/miR-214-3p/Med19 axis. Hypoxic preconditioning is an effective and promising method for optimizing the therapeutic action of BMSC-derived exosomes in FNI.

NEAT1 repression by MED12 creates chemosensitivity in p53 wild-type breast cancer cells.

Breast cancer is often treated with chemotherapy. However, the development of chemoresistance results in treatment failure. Long non-coding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) has been shown to contribute to chemoresistance in breast cancer cells. In studying the transcriptional regulation of NEAT1 using multi-omics approaches, we showed that NEAT1 is up-regulated by 5-fluorouracil in breast cancer cells with wild-type cellular tumor antigen p53 but not in mutant-p53-expressing breast cancer cells. The regulation of NEAT1 involves mediator complex subunit 12 (MED12)-mediated repression of histone acetylation marks at the promoter region of NEAT1. Knockdown of MED12 but not coactivator-associated arginine methyltransferase 1 (CARM1) induced histone acetylation at the NEAT1 promoter, leading to elevated NEAT1 mRNAs, resulting in a chemoresistant phenotype. The MED12-dependent regulation of NEAT1 differs between wild-type and mutant p53-expressing cells. MED12 depletion led to increased expression of NEAT1 in a wild-type p53 cell line, but decreased expression in a mutant p53 cell line. Chemoresistance caused by MED12 depletion can be partially rescued by NEAT1 knockdown in p53 wild-type cells. Collectively, our study reveals a novel mechanism of chemoresistance dependent on MED12 transcriptional regulation of NEAT1 in p53 wild-type breast cancer cells.

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.