Glial-Specific Deletion of Med12 Results in Rapid Hearing Loss via Degradation of the Stria Vascularis.

Mediator protein complex subunit 12 (Med12) is a core component of the basal transcriptional apparatus and plays a critical role in the development of many tissues. Mutations in Med12 are associated with X-linked intellectual disability syndromes and hearing loss; however, its role in nervous system function remains undefined. Here, we show that temporal conditional deletion of Med12 in astrocytes in the adult CNS results in region-specific alterations in astrocyte morphology. Surprisingly, behavioral studies revealed rapid hearing loss after adult deletion of Med12 that was confirmed by a complete abrogation of auditory brainstem responses. Cellular analysis of the cochlea revealed degeneration of the stria vascularis, in conjunction with disorganization of basal cells adjacent to the spiral ligament and downregulation of key cell adhesion proteins. Physiologic analysis revealed early changes in endocochlear potential, consistent with strial-specific defects. Together, our studies reveal that Med12 regulates auditory function in the adult by preserving the structural integrity of the stria vascularis.SIGNIFICANCE STATEMENT Mutations in Mediator protein complex subunit 12 (Med12) are associated with X-linked intellectual disability syndromes and hearing loss. Using temporal-conditional genetic approaches in CNS glia, we found that loss of Med12 results in severe hearing loss in adult animals through rapid degeneration of the stria vascularis. Our study describes the first animal model that recapitulates hearing loss identified in Med12-related disorders and provides a new system in which to examine the underlying cellular and molecular mechanisms of Med12 function in the adult nervous system.

Dynamic lineage priming is driven via direct enhancer regulation by ERK.

Central to understanding cellular behaviour in multi-cellular organisms is the question of how a cell exits one transcriptional state to adopt and eventually become committed to another. Fibroblast growth factor-extracellular signal-regulated kinase (FGF -ERK) signalling drives differentiation of mouse embryonic stem cells (ES cells) and pre-implantation embryos towards primitive endoderm, and inhibiting ERK supports ES cell self-renewal1. Paracrine FGF-ERK signalling induces heterogeneity, whereby cells reversibly progress from pluripotency towards primitive endoderm while retaining their capacity to re-enter self-renewal2. Here we find that ERK reversibly regulates transcription in ES cells by directly affecting enhancer activity without requiring a change in transcription factor binding. ERK triggers the reversible association and disassociation of RNA polymerase II and associated co-factors from genes and enhancers with the mediator component MED24 having an essential role in ERK-dependent transcriptional regulation. Though the binding of mediator components responds directly to signalling, the persistent binding of pluripotency factors to both induced and repressed genes marks them for activation and/or reactivation in response to fluctuations in ERK activity. Among the repressed genes are several core components of the pluripotency network that act to drive their own expression and maintain the ES cell state; if their binding is lost, the ability to reactivate transcription is compromised. Thus, as long as transcription factor occupancy is maintained, so is plasticity, enabling cells to distinguish between transient and sustained signals. If ERK signalling persists, pluripotency transcription factor levels are reduced by protein turnover and irreversible gene silencing and commitment can occur.

Mediator complex subunit Med12 regulates cardiac jelly development and AV valve formation in zebrafish.

The molecular mechanism essential for the formation of heart valves involves complex interactions of signaling molecules and transcription factors. The Mediator Complex (MC) functions as multi-subunit machinery to orchestrate gene transcription, especially for tissue-specific fine-tuning of transcriptional processes during development, also in the heart. Here, we analyzed the role of the MC subunit Med12 during atrioventricular canal (AVC) development and endocardial cushion formation, using the Med12-deficient zebrafish mutant trapped (tpd). Whereas primary heart formation was only slightly affected in tpd, we identified defects in AVC development and cardiac jelly formation. We found that although misexpression of bmp4 and versican in tpd hearts can be restored by overexpression of a modified version of the Sox9b transcription factor (harboring VP16 transactivation domain) that functions independent of its co-activator Med12, endocardial cushion development in tpd was not reconstituted. Interestingly, expression of tbx2b and its target hyaluronan synthase 2 (has2) - the synthase of hyaluronan (HA) in the heart - was absent in both uninjected and Sox9b-VP16 overexpressing tpd hearts. HA is a major ECM component of the cardiac jelly and required for endocardial cushion formation. Furthermore, we found secreted phosphoprotein 1 (spp1), an endocardial marker of activated AV endocardial cells, completely absent in tpd hearts, suggesting that crucial steps of the transformation of AV endocardial cells into endocardial cushions is blocked. We demonstrate that Med12 controls cardiac jelly formation Sox9-independently by regulating tbx2b and has2 expression and therefore the production of the glycosaminoglycan HA at the AVC to guarantee proper endocardial cushion development.

Silencing of MED27 inhibits adrenal cortical carcinogenesis by targeting the Wnt/ß-catenin signaling pathway and the epithelial-mesenchymal transition process.

This study aimed to explore the effect of MED27 on the expression of epithelial-mesenchymal transition (EMT)-related proteins and ß-catenin in adrenal cortical carcinoma (ACC). The functional mechanism of MED27 on ACC processes was also explored. The expression of MED27 was assessed by quantitative real-time polymerase chain reaction (qRT-PCR). siRNA was utilized to knockdown the expression of MED27. CCK8 assays were performed to evaluate SW-13 cell proliferation. Transwell assays were performed to assess the invasion ability, and wound healing assays were utilized to detect migration. A tumor xenograft mouse model was established to investigate the impact of silencing MED27 on tumor growth and metastasis. MED27 was highly expressed in ACC tissues and cells. Down-regulation of MED27 induced ACC cell apoptosis, and significantly attenuated ACC cell proliferation, invasion and metastasis in vivo and in vitro. MED27 knockdown regulated the expression of EMT-related proteins and Wnt/ß-catenin signaling pathway-related proteins. Our study investigated the function and mechanism of MED27 and validated that MED27 plays a negative role in ACC occurrence and progression and could be utilized as a new therapeutic target in ACC prevention and treatment.

Mediator Med23 deficiency enhances neural differentiation of murine embryonic stem cells through modulating BMP signaling.

Unraveling the mechanisms underlying early neural differentiation of embryonic stem cells (ESCs) is crucial to developing cell-based therapies of neurodegenerative diseases. Neural fate acquisition is proposed to be controlled by a 'default' mechanism, for which the molecular regulation is not well understood. In this study, we investigated the functional roles of Mediator Med23 in pluripotency and lineage commitment of murine ESCs. Unexpectedly, we found that, despite the largely unchanged pluripotency and self-renewal of ESCs, Med23 depletion rendered the cells prone to neural differentiation in different differentiation assays. Knockdown of two other Mediator subunits, Med1 and Med15, did not alter the neural differentiation of ESCs. Med15 knockdown selectively inhibited endoderm differentiation, suggesting the specificity of cell fate control by distinctive Mediator subunits. Gene profiling revealed that Med23 depletion attenuated BMP signaling in ESCs. Mechanistically, MED23 modulated Bmp4 expression by controlling the activity of ETS1, which is involved in Bmp4 promoter-enhancer communication. Interestingly, med23 knockdown in zebrafish embryos also enhanced neural development at early embryogenesis, which could be reversed by co-injection of bmp4 mRNA. Taken together, our study reveals an intrinsic, restrictive role of MED23 in early neural development, thus providing new molecular insights for neural fate determination.

Disruption of Mediator rescues the stunted growth of a lignin-deficient Arabidopsis mutant.

Lignin is a phenylpropanoid-derived heteropolymer important for the strength and rigidity of the plant secondary cell wall. Genetic disruption of lignin biosynthesis has been proposed as a means to improve forage and bioenergy crops, but frequently results in stunted growth and developmental abnormalities, the mechanisms of which are poorly understood. Here we show that the phenotype of a lignin-deficient Arabidopsis mutant is dependent on the transcriptional co-regulatory complex, Mediator. Disruption of the Mediator complex subunits MED5a (also known as REF4) and MED5b (also known as RFR1) rescues the stunted growth, lignin deficiency and widespread changes in gene expression seen in the phenylpropanoid pathway mutant ref8, without restoring the synthesis of guaiacyl and syringyl lignin subunits. Cell walls of rescued med5a/5b ref8 plants instead contain a novel lignin consisting almost exclusively of p-hydroxyphenyl lignin subunits, and moreover exhibit substantially facilitated polysaccharide saccharification. These results demonstrate that guaiacyl and syringyl lignin subunits are largely dispensable for normal growth and development, implicate Mediator in an active transcriptional process responsible for dwarfing and inhibition of lignin biosynthesis, and suggest that the transcription machinery and signalling pathways responding to cell wall defects may be important targets to include in efforts to reduce biomass recalcitrance.

Activating RNAs associate with Mediator to enhance chromatin architecture and transcription.

Recent advances in genomic research have revealed the existence of a large number of transcripts devoid of protein-coding potential in multiple organisms. Although the functional role for long non-coding RNAs (lncRNAs) has been best defined in epigenetic phenomena such as X-chromosome inactivation and imprinting, different classes of lncRNAs may have varied biological functions. We and others have identified a class of lncRNAs, termed ncRNA-activating (ncRNA-a), that function to activate their neighbouring genes using a cis-mediated mechanism. To define the precise mode by which such enhancer-like RNAs function, we depleted factors with known roles in transcriptional activation and assessed their role in RNA-dependent activation. Here we report that depletion of the components of the co-activator complex, Mediator, specifically and potently diminished the ncRNA-induced activation of transcription in a heterologous reporter assay using human HEK293 cells. In vivo, Mediator is recruited to ncRNA-a target genes and regulates their expression. We show that ncRNA-a interact with Mediator to regulate its chromatin localization and kinase activity towards histone H3 serine 10. The Mediator complex harbouring disease- displays diminished ability to associate with activating ncRNAs. Chromosome conformation capture confirmed the presence of DNA looping between the ncRNA-a loci and its targets. Importantly, depletion of Mediator subunits or ncRNA-a reduced the chromatin looping between the two loci. Our results identify the human Mediator complex as the transducer of activating ncRNAs and highlight the importance of Mediator and activating ncRNA association in human disease.

Mediator MED23 plays opposing roles in directing smooth muscle cell and adipocyte differentiation.

The Mediator complex functions as a control center, orchestrating diverse signaling, gene activities, and biological processes. However, how Mediator subunits determine distinct cell fates remains to be fully elucidated. Here, we show that Mediator MED23 controls the cell fate preference that directs differentiation into smooth muscle cells (SMCs) or adipocytes. Med23 deficiency facilitates SMC differentiation but represses adipocyte differentiation from the multipotent mesenchymal stem cells. Gene profiling revealed that the presence or absence of Med23 oppositely regulates two sets of genes: the RhoA/MAL targeted cytoskeleton/SMC genes and the Ras/ELK1 targeted growth/adipogenic genes. Mechanistically, MED23 favors ELK1-SRF binding to SMC gene promoters for repression, whereas the lack of MED23 favors MAL-SRF binding to SMC gene promoters for activation. Remarkably, the effect of MED23 on SMC differentiation can be recapitulated in zebrafish embryogenesis. Collectively, our data demonstrate the dual, opposing roles for MED23 in regulating the cytoskeleton/SMC and growth/adipogenic gene programs, suggesting its "Ying-Yang" function in directing adipogenesis versus SMC differentiation.

Wwp2 is essential for palatogenesis mediated by the interaction between Sox9 and mediator subunit 25.

Sox9 is a direct transcriptional activator of cartilage-specific extracellular matrix genes and has essential roles in chondrogenesis. Mutations in or around the SOX9 gene cause campomelic dysplasia or Pierre Robin Sequence. However, Sox9-dependent transcriptional control in chondrogenesis remains largely unknown. Here we identify Wwp2 as a direct target of Sox9. Wwp2 interacts physically with Sox9 and is associated with Sox9 transcriptional activity via its nuclear translocation. A yeast two-hybrid screen using a cDNA library reveals that Wwp2 interacts with Med25, a component of the Mediator complex. The positive regulation of Sox9 transcriptional activity by Wwp2 is mediated by the binding between Sox9 and Med25. In zebrafish, morpholino-mediated knockdown of either wwp2 or med25 induces palatal malformation, which is comparable to that in sox9 mutants. These results provide evidence that the regulatory interaction between Sox9, Wwp2 and Med25 defines the Sox9 transcriptional mechanisms of chondrogenesis in the forming palate.