Somatic MED12 Mutations in Myometrial Cells.

Over 70% of leiomyoma (LM) harbor MED12 mutations, primarily in exon 2 at c.130-131 (GG). Myometrial cells are the cell origin of leiomyoma, but the MED12 mutation status in non-neoplastic myometrial cells is unknown. In this study, we investigated the mutation burden of MED12 in myometrium. As traditional Sanger or even NGS sequencing may not be able to detect MED12 mutations that are lower than 0.1% in the testing sample, we used duplex deep sequencing analysis (DDS) to overcome this limitation. Tumor-free myometria (confirmed by pathology evaluation) were dissected, and genomic DNA from MED12 exon 2 (test) and TP53 exon 5 (control) were captured by customer-designed probe sets, followed by DDS. Notably, DDS demonstrated that myometrial cells harbored a high frequency of mutations in MED12 exon 2 and predominantly in code c.130-131. In contrast, the baseline mutations in other coding sequences of MED12 exon 2 as well as in the TP53 mutation hotspot, c.477-488 were comparably low in myometrial cells. This is the first report demonstrating a non-random accumulation of MED12 mutations at c.130-131 sites in non-neoplastic myometrial cells which provide molecular evidence of early somatic mutation events in myometrial cells. This early mutation may contribute to the cell origin for uterine LM development in women of reproductive age.

MED12 and CDK8/19 Modulate Androgen Receptor Activity and Enzalutamide Response in Prostate Cancer.

Prostate cancer progression is driven by androgen receptor (AR) activity, which is a target for therapeutic approaches. Enzalutamide is an AR inhibitor that prolongs the survival of patients with advanced prostate cancer. However, resistance mechanisms arise and impair its efficacy. One of these mechanisms is the expression of AR-V7, a constitutively active AR splice variant. The Mediator complex is a multisubunit protein that modulates gene expression on a genome-wide scale. MED12 and cyclin-dependent kinase (CDK)8, or its paralog CDK19, are components of the kinase module that regulates the proliferation of prostate cancer cells. In this study, we investigated how MED12 and CDK8/19 influence cancer-driven processes in prostate cancer cell lines, focusing on AR activity and the enzalutamide response. We inhibited MED12 expression and CDK8/19 activity in LNCaP (AR+, enzalutamide-sensitive), 22Rv1 (AR-V7+, enzalutamide-resistant), and PC3 (AR-, enzalutamide-insensitive) cells. Both MED12 and CDK8/19 inhibition reduced cell proliferation in all cell lines, and MED12 inhibition reduced proliferation in the respective 3D spheroids. MED12 knockdown significantly inhibited c-Myc protein expression and signaling pathways. In 22Rv1 cells, it consistently inhibited the AR response, prostate-specific antigen (PSA) secretion, AR target genes, and AR-V7 expression. Combined with enzalutamide, MED12 inhibition additively decreased the AR activity in both LNCaP and 22Rv1 cells. CDK8/19 inhibition significantly decreased PSA secretion in LNCaP and 22Rv1 cells and, when combined with enzalutamide, additively reduced proliferation in 22Rv1 cells. Our study revealed that MED12 and CDK8/19 regulate AR activity and that their inhibition may modulate response to enzalutamide in prostate cancer.

Transcriptional noise, gene activation, and roles of SAGA and Mediator Tail measured using nucleotide recoding single-cell RNA-seq.

We describe a time-resolved nascent single-cell RNA sequencing (RNA-seq) approach that measures gene-specific transcriptional noise and the fraction of active genes in S. cerevisiae. Most genes are expressed with near-constitutive behavior, while a subset of genes show high mRNA variance suggestive of transcription bursting. Transcriptional noise is highest in the cofactor/coactivator-redundant (CR) gene class (dependent on both SAGA and TFIID) and strongest in TATA-containing CR genes. Using this approach, we also find that histone gene transcription switches from a low-level, low-noise constitutive mode during M and M/G1 to an activated state in S phase that shows both an increase in the fraction of active promoters and a switch to a noisy and bursty transcription mode. Rapid depletion of cofactors SAGA and MED Tail indicates that both factors play an important role in stimulating the fraction of active promoters at CR genes, with a more modest role in transcriptional noise.

The essential role of MED27 in stabilizing the mediator complex for cardiac development and function.

AIM: Transcriptional regulation of gene expression plays a crucial role in orchestrating complex morphogenetic and molecular events during heart development and function. Mediator complex is an essential multi-subunit protein complex that governs gene expression in eukaryotic cells. Although Mediator subunits (MEDs) work integrally in the complex, individual MED component displays specialized functions. MED27, categorized as an Upper Tail subunit, possesses an as-yet-uncharacterized function. In this study, we aimed to investigate the physiological role of MED27 in cardiomyocytes. MATERIALS AND METHODS: we generated a Med27 floxed mouse line, which was further used to generate constitutive (cKO) and inducible (icKO) cardiomyocyte-specific Med27 knockout mouse models. Morphological, histological analysis and cardiac physiological studies were performed in Med27 cKO and icKO mutants. Transcriptional profiles were determined by RNA sequencing (RNAseq) analysis. KEY FUNDINGS: Ablation of MED27 in developing mouse cardiomyocytes results in embryonic lethality, while its deletion in adult cardiomyocytes leads to heart failure and mortality. Similar to the ablation of another Upper Tail subunit, MED30 in cardiomyocytes, deletion of MED27 leads to decreased protein levels of most MEDs in cardiomyocytes. Interestingly, overexpression of MED30 fails to restore the protein levels of Mediator subunits in MED27-deficient cardiomyocytes, demonstrating that the role of MED27 in maintaining the integrity and stability of the Mediator complex is independent of MED30. SIGNIFICANCE: Our results revealed an essential role of MED27 in cardiac development and function by maintaining the stability of the Mediator core.

Quality of life after myomectomy according to the surgical approach and MED12 mutation status.

OBJECTIVE: Molecular status of uterine leiomyomas has been shown to affect both tumor characteristics and treatment response. Mutations in mediator complex subunit 12 (MED12), the most prevalent alterations in leiomyomas, are associated with tumor size and number of leiomyomas. Myomectomy can be performed by laparoscopy or by open abdominal surgery, depending on the size and number of leiomyomas removed. The aim of this study was to examine the association between MED12 mutation status and surgical approach of myomectomy. We also evaluated myomectomy patients' quality of life after laparoscopic or abdominal surgery and according to the MED12 mutation status. STUDY DESIGN: The prospective cohort study included 104 women who underwent laparoscopic or abdominal myomectomy at the Helsinki University Hospital during 2015-2019. Patients filled in the validated Uterine Fibroid Symptom and Quality of Life (UFS-QOL) questionnaire before the operation and 6 and 12 months after the operation. Medical records were reviewed to collect clinical data. Leiomyoma tissue samples were collected and screened for MED12 mutations. RESULTS: Patients undergoing abdominal myomectomy had larger and more numerous leiomyomas compared to patients with laparoscopic myomectomy (10 cm vs 7.4 cm, p < 0.001 and 3 vs 1 leiomyomas, p < 0.001, respectively). A mean change of over 20 points was seen in UFS-QOL scores at 6 months after both laparoscopic and abdominal myomectomy (p < 0.001). MED12 mutations were detected in 178/242 (74 %) of leiomyomas. Of the patients, 45/97 (46 %) had only MED12 positive leiomyomas, while 39/97 (40 %) had only MED12 wild type leiomyomas. The number of leiomyomas removed was higher among patients with MED12 positive leiomyomas than in patients with MED12 wild type tumors (p < 0.001). Laparoscopic approach was equally common in both groups (62 % and 64 %), and there was no statistically significant difference in the UFS-QOL scores. CONCLUSION: Both laparoscopic and abdominal myomectomy significantly improved the quality of life. While MED12 mutations were related with multiple leiomyomas and therefore potentially generated a greater leiomyoma burden, they were not associated with the surgical approach. Pre- and postoperative quality of life was comparable between patients regardless of MED12 status.

MED23 pathogenic variant: genomic-phenotypic analysis.

The mediator complex subunit 23 (MED23) gene encodes a protein that acts as a tail module mediator complex, a multi-subunit co-activator involved in several cellular activities. MED23 has been shown to have substantial roles in myogenesis and other molecular mechanisms. The functions of MED23 in the neurological system remain unclear and the clinical phenotype is not thoroughly described. Whole exome sequencing was used to identify a novel mutation in the MED23 gene. DNA capture probes using next-generation sequencing-based copy number variation analysis with Illumina array were performed. The clinical, demographic, neuroimaging, and electrophysiological data of the patients were collected, and similarly, the data of all reported cases in the literature were extracted to compare findings. Screening a total of 9,662 articles, we identified 22 main regulatory processes for the MED23 gene, including suppressive activity for carcinogenic processes. MED23 is also involved in the brain's neurogenesis and functions. The identified cases mainly presented with intellectual disability (87.5%) and developmental delay (50%). Seizures were present in only 18.75% of the patients. Slow backgrounds and spike and sharp-wave complexes were reported on the electroencephalogram (EEG) of a few patients and delayed myelination, thin corpus callosum, and pontine hypoplasia on magnetic resonance imaging (MRI). The MED23 gene regulates several processes in which its understanding promotes considerable therapeutic potential for patients. It is crucial to consider genetic and laboratory testing, particularly when encountering potential carriers. Intellectual disability and developmental delay are the most notable clinical signs with heterogeneous features on EEG and MRI.

A C->T Variation in 3'-Untranslated Region Elevates MED12 Protein Level in Breast Cancer That Relates to Better Prognosis.

Objective: Mediator complex subunit 12 (MED12) is among the most frequently mutated genes in various types of human cancers. However, there is still a lack of understanding regarding the role of MED12 in breast cancer patient. Therefore, the aim of this study is to explore the roles of MED12 in breast cancer. Materials and Methods: We utilized the UALCAN platform (http://ualcan.path.uab.edu/) for analyzing the transcriptional expression, protein expression, and protein phosphorylation data of MED12. Our study involved 35 breast cancer patients. From these samples, we extracted proteins and RNA. To obtain the sequence of MED12 3'-UTR, we performed reverse transcription-polymerase chain reaction and sequencing. We then used TargetScan to predict the miRNA targets of MED12 3'-UTR and confirmed the interactions between miRNAs and MED12 3'-UTR through dual luciferase assay. Results: The protein level of MED12 was upregulated in breast cancer, while the mRNA level did not show significant changes. Interestingly, higher levels of MED12 mRNA were associated with better prognosis, whereas patients with increased MED12 protein levels tended to have a poorer prognosis. Furthermore, through our analysis of the MED12 3'-UTR sequence, we identified a specific C->T variation that was unique to breast tumors. We also identified four miRNAs (miR-204, -211, -450 b, and -518a) that directly target MED12 3'-UTR. Most important, this C->T variation disrupts the interaction between MED12 3'-UTR and miR-450b, ultimately leading to the upregulation of MED12 in breast cancer. Conclusion: Our study revealed a significant finding regarding a mutation site in the MED12 3'-UTR that contributes to the upregulation of MED12 in breast cancer. This mutation disrupts the interactions between specific miRNAs and MED12 mRNA, leading to increased expression of MED12. These findings have important implications for breast cancer diagnosis, as this mutation site can serve as a potent biomarker.

An intrinsically disordered region in MED13 turns Mediator on/off on cue.

Complementary studies by Zhao et al.1 and Chen et al.2 reveal how an intrinsically disordered region in MED13 controls mutually exclusive binding of RNA Polymerase II and CDK8 kinase module to Mediator, switching Mediator and transcription activation on and off.

3D chromatin architecture, BRD4, and Mediator have distinct roles in regulating genome-wide transcriptional bursting and gene network.

Discontinuous transcription is evolutionarily conserved and a fundamental feature of gene regulation; yet, the exact mechanisms underlying transcriptional bursting are unresolved. Analyses of bursting transcriptome-wide have focused on the role of cis-regulatory elements, but other factors that regulate this process remain elusive. We applied mathematical modeling to single-cell RNA sequencing data to infer bursting dynamics transcriptome-wide under multiple conditions to identify possible molecular mechanisms. We found that Mediator complex subunit 26 (MED26) primarily regulates frequency, MYC regulates burst size, while cohesin and Bromodomain-containing protein 4 (BRD4) can modulate both. Despite comparable effects on RNA levels among these perturbations, acute depletion of MED26 had the most profound impact on the entire gene regulatory network, acting downstream of chromatin spatial architecture and without affecting TATA box-binding protein (TBP) recruitment. These results indicate that later steps in the initiation of transcriptional bursts are primary nodes for integrating gene networks in single cells.

Med3-mediated NADPH generation to help Saccharomyces cerevisiae tolerate hyperosmotic stress.

Hyperosmotic stress tolerance is crucial for Saccharomyces cerevisiae in producing value-added products from renewable feedstock. The limited understanding of its tolerance mechanism has impeded the application of these microbial cell factories. Previous studies have shown that Med3 plays a role in hyperosmotic stress in S. cerevisiae. However, the specific function of Med3 in hyperosmotic stress tolerance remains unclear. In this study, we showed that the deletion of the mediator Med3 impairs S. cerevisiae growth under hyperosmotic stress. Phenotypic analyses and yeast two-hybrid assays revealed that Med3 interacts with the transcription factor Stb5 to regulate the expression of the genes gnd1 and ald6, which are involved in NADPH production under hyperosmotic stress conditions. The deletion of med3 resulted in a decrease in intracellular NADPH content, leading to increased oxidative stress and elevated levels of intracellular reactive oxygen species under hyperosmotic stress, thereby impacting bud formation. These findings highlight the significant role of Med3 as a regulator in maintaining NADPH generation and redox homeostasis in S. cerevisiae during hyperosmotic stress.IMPORTANCEHyperosmotic stress tolerance in the host strain is a significant challenge for fermentation performance in industrial production. In this study, we showed that the S. cerevisiae mediator Med3 is essential for yeast growth under hyperosmotic conditions. Med3 interacts with the transcription factor Stb5 to regulate the expression of genes involved in the NADPH-generation system during hyperosmotic stress. Adequate NADPH ensures the timely removal of excess reactive oxygen species and supports bud formation under these conditions. This work highlights the crucial role of Med3 as a regulator in maintaining NADPH generation and redox homeostasis in S. cerevisiae during hyperosmotic stress.

Catatonia and Maintenance Electroconvulsive Therapy in a 15-Year-Old Patient With MED13L Haploinsufficiency Syndrome in the Context of Epilepsy Diathesis.

ABSTRACT: This is the first report of pediatric catatonia syndrome in MED13L haploinsufficiency syndrome. This report describes unique challenges in diagnosis and management of catatonia in rare genetic conditions. The case also illustrates the use of electroconvulsive therapy in patients with epilepsy, epileptic encephalopathy, or other epileptic diathesis and the clinical conundrum in determining the course of maintenance electroconvulsive therapy.

An IDR-dependent mechanism for nuclear receptor control of Mediator interaction with RNA polymerase II.

The essential Mediator (MED) coactivator complex plays a well-understood role in regulation of basal transcription in all eukaryotes, but the mechanism underlying its role in activator-dependent transcription remains unknown. We investigated modulation of metazoan MED interaction with RNA polymerase II (RNA Pol II) by antagonistic effects of the MED26 subunit and the CDK8 kinase module (CKM). Biochemical analysis of CKM-MED showed that the CKM blocks binding of the RNA Pol II carboxy-terminal domain (CTD), preventing RNA Pol II interaction. This restriction is eliminated by nuclear receptor (NR) binding to CKM-MED, which enables CTD binding in a MED26-dependent manner. Cryoelectron microscopy (cryo-EM) and crosslinking-mass spectrometry (XL-MS) revealed that the structural basis for modulation of CTD interaction with MED relates to a large intrinsically disordered region (IDR) in CKM subunit MED13 that blocks MED26 and CTD interaction with MED but is repositioned upon NR binding. Hence, NRs can control transcription initiation by priming CKM-MED for MED26-dependent RNA Pol II interaction.

Structures and compositional dynamics of Mediator in transcription regulation.

The eukaryotic Mediator, comprising a large Core (cMED) and a dissociable CDK8 kinase module (CKM), functions as a critical coregulator during RNA polymerase II (RNAPII) transcription. cMED recruits RNAPII and facilitates the assembly of the pre-initiation complex (PIC) at promoters. In contrast, CKM prevents RNAPII binding to cMED while simultaneously exerting positive or negative influence on gene transcription through its kinase function. Recent structural studies on cMED and CKM have revealed their intricate architectures and subunit interactions. Here, we explore these structures, providing a comprehensive insight into Mediator (cMED-CKM) architecture and its potential mechanism in regulating RNAPII transcription. Additionally, we discuss the remaining puzzles that require further investigation to fully understand how cMED coordinates with CKM to regulate transcription in various events.

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.

Mediator complex: an important regulator of root system architecture.

Mediator, a multiprotein complex, is an important component of the transcription machinery. In plants, the latest studies have established that it functions as a signal processor that conveys transcriptional signals from transcription factors to RNA polymerase II. Mediator has been found to be involved in different developmental and stress-adaptation conditions, ranging from embryo, root, and shoot development to flowering and senescence, and also in responses to different biotic and abiotic stresses. In the last decade, significant progress has been made in understanding the role of Mediator subunits in root development. They have been shown to transcriptionally regulate development of almost all the components of the root system architecture-primary root, lateral roots, and root hairs. They also have a role in nutrient acquisition by the root. In this review, we discuss all the known functions of Mediator subunits during root development. We also highlight the role of Mediator as a nodal point for processing different hormone signals that regulate root morphogenesis and growth.

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.

Malignant phyllodes tumor with EGFR variant III mutation: A rare case report with immunohistochemical and genomic studies.

A female in her 60's presented with a left-sided breast mass. A core needle biopsy specimen showed diffuse proliferation of a round cell tumor, which was positive for vimentin, NKX2.2, BCOR, and focal CD99 on immunohistochemistry (IHC). No fusion genes of the Ewing family sarcomas were detected. With a tentative diagnosis of primary breast sarcoma (PBS), total mastectomy was performed after chemotherapy. The resected tissues showed proliferation of round or spindle-shaped tumor cells with a high nuclear-to-cytoplasmic ratio, exhibiting solid and fascicular arrangements but no epithelial component or organoid pattern. While IHC indicated no particular histological diagnosis, genomic examination revealed gene alterations in MED12 p.G44D, MLL2 (KMT2D) p.T1496fs*27, and EGFR variant III (vIII). Moreover, a retrospective IHC study showed overexpression of EGFRvIII. A malignant phyllodes tumor (PT) with extensive sarcomatous overgrowth was indicated as an integrative diagnosis. This is a rare case of a malignant PT harboring EGFRvIII. The present case provides an importance of accurate diagnosis and genomic analysis of rare breast tumors, as malignant PT and PBS are different in its treatment strategy and prognosis.

Mediator complex in transcription regulation and DNA repair: Relevance for human diseases.

The Mediator complex is an essential coregulator of RNA polymerase II transcription. More recent developments suggest Mediator functions as a link between transcription regulation, genome organisation and DNA repair mechanisms including nucleotide excision repair, base excision repair, and homologous recombination. Dysfunctions of these processes are frequently associated with human pathologies, and growing evidence shows Mediator involvement in cancers, neurological, metabolic and infectious diseases. The detailed deciphering of molecular mechanisms of Mediator functions, using interdisciplinary approaches in different biological models and considering all functions of this complex, will contribute to our understanding of relevant human diseases.

Rare MED12L Variants Are Associated with Susceptibility to Guttate Psoriasis in the Han Chinese Population.

INTRODUCTION: According to the common disease/rare variant hypothesis, it is important to study the role of rare variants in complex diseases. The association of rare variants with psoriasis has been demonstrated, but the association between rare variants and specific clinical subtypes of psoriasis has not been investigated. METHODS: Gene-based and gene-level meta-analyses were performed on data extracted from our previous study data sets (2,483 patients with guttate psoriasis and 8,292 patients with non-guttate psoriasis) for genotyping. Then, haplotype analysis was performed for rare loss-of-function variants located in MED12L, and protein function prediction was performed for MED12L. Gene-based analysis at each stage had a moderate significance threshold (p < 0.05). A χ2 test was then conducted on the three potential genes, and the merged gene-based analysis was used to confirm the results. We also conducted association analysis and meta-analysis for functional variants located on the identified gene. RESULTS: Through these gene-level analyses, we determined that MED12L is a guttate psoriasis susceptibility gene (p = 9.99 × 10-5), and the single-nucleotide polymorphism with the strongest association was rs199780529 (p_combine = 1 × 10-3, p_meta = 2 × 10-3). CONCLUSIONS: In our study, a guttate psoriasis-specific subtype-associated susceptibility gene was confirmed in a Chinese Han population. These findings contribute to a better genetic understanding of different subtypes of psoriasis.