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Mediator Med23-deficiency Enhances Neural Differentiation of Embryonic Stem Cells through Modulating BMP Signaling

ABSTRACT: Unraveling the mechanisms underlying early neural differentiation of ESCs is crucial to the cell-based therapies of neurodegenerate 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 embryonic stem cells (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 other Mediator subunit, Med1 or Med15, did not alter the neural differentiation of ESCs; and Med15 knockdown selectively inhibited endoderm differentiation, suggesting the specificity of cell fate control by distinctive Mediator subunits. Gene profiling revealed that Med23-depletion attenuated the BMP signaling in ESCs. Mechanistically, MED23 modulated Bmp4 expression by controlling the activity of ETS1 that is involved in the Bmp4 promoter-enhancer communication. Interestingly, Med23 knockdown in zebrafish embryos also enhanced the neural development at early embryogenesis, which could be reversible by coinjection 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. We used microarrays to detail the global gene expression after MED23 knockout

ORGANISM(S): Mus musculus

PROVIDER: GSE64500 | GEO | 2015/03/05

SECONDARY ACCESSION(S): PRJNA271133

REPOSITORIES: GEO

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Mediator Med23-deficiency Enhances Neural Differentiation of Embryonic Stem Cells through Modulating BMP Signaling

Project description:Unraveling the mechanisms underlying early neural differentiation of ESCs is crucial to the cell-based therapies of neurodegenerate diseases. Neural fate acquisition is proposed to be controlled by a âdefaultâ 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 embryonic stem cells (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 other Mediator subunit, Med1 or Med15, did not alter the neural differentiation of ESCs; and Med15 knockdown selectively inhibited endoderm differentiation, suggesting the specificity of cell fate control by distinctive Mediator subunits. Gene profiling revealed that Med23-depletion attenuated the BMP signaling in ESCs. Mechanistically, MED23 modulated Bmp4 expression by controlling the activity of ETS1 that is involved in the Bmp4 promoter-enhancer communication. Interestingly, Med23 knockdown in zebrafish embryos also enhanced the neural development at early embryogenesis, which could be reversible by coinjection 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. We used microarrays to detail the global gene expression after MED23 knockout We examined the gene expression level in WT and MED23 knockout ES cells in steady culture condition.

2015-03-05 | E-GEOD-64500 | biostudies-arrayexpress

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

Project description:The Mediator complex functions as a control center orchestrating diverse signalings, 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/adipocyte 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/adipocyte gene programs, suggesting its “Ying-Yang” function in directing adipogenesis vs. SMC differentiation. Examination of SRF enrichment in sictrl and si23 10T1/2 cells

2012-12-20 | E-GEOD-40369 | biostudies-arrayexpress

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

2012-12-20 | GSE40369 | GEO

Liver Med23 ablation improves glucose and lipid metabolism and prevent diet-induced obesity

Project description:Mediator complex function as an integrative hub for transcriptional regulation. Here we show that Mediator subunit MED23 regulate glucose and lipid metabolism via FOXO1 in liver. Here, we have generated a liver-specific Med23-knockout (LMKO) mouse and found that Med23-deletion in liver improved glucose and lipid metabolism, as well as insulin responsiveness, and prevented diet-induced obesity. Mechanistically, MED23 participated in gluconeogenesis and cholesterol synthesis by interacting with FOXO1. Disruption of this interaction by hepatic Med23-deletion impaired the Mediator and RNAP II recruitment and partially reduced the expression of the FOXO1 target genes. Remarkably, acute hepatic Med23 knockdown in db/db mice significantly improved insulin sensitivity. Overall, our data revealed Mediator MED23 as a critical regulator of glucose and lipid metabolism, suggesting novel therapeutic strategies against metabolic diseases.

2015-03-16 | GSE48966 | GEO

NANOG-OCT4-SOX2 Regulatory Module in Human Embryonic Stem Cells (dataset 1)

Project description:The transcription factors Nanog, Oct4 and Sox2 are the master regulators of pluripotency in mouse embryonic stem cells (mESCs), however, their functions in human ESCs (hESCs) have not been rigorously defined. Here we show that the requirements for NANOG, OCT4 and SOX2 in hESCs differ from those in mESCs. Both NANOG and OCT4 are required for self-renewal and repress differentiation. OCT4 controls both extraembryonic and epiblast-derived cell fates in a BMP4-dependent manner. OCT4-depleted hESCs commit to trophectoderm and primitive endoderm in the presence of BMP4, but undergo neuroectoderm differentiation in the absence of BMP4. NANOG represses neuroectoderm and neural crest commitment, but has little or no effect on the other lineages. We find that SOX2 is not required for self-renewal because it is redundant with SOX3, which is induced in SOX2-depleted hESCs. Simultaneous depletion of both SOX2 and SOX3 induces differentiation into the primitive streak. Unexpectedly, we identify significant variability in the usage of pluripotency factors by individual hESC lines, suggesting that the pluripotency network is remodelled to support a continuum of developmental states. Our study revises the general view of how NANOG, OCT4 and SOX2 orchestrate self-renewal in hESCs. Total RNA obtained from H1P (control)-, shNANOG (Nanog shRNA knockdown)-, shOCT4 (Oct4 shRNA knockdown)- or shSOX2 (Sox2 shRNA knockdown)-transduced hESCs for 8 days time course.

2012-04-05 | E-GEOD-34904 | biostudies-arrayexpress

Functional studies of the Yeast Mediator Tail Module Subunits

Project description:The yeast Mediator complex can be divided into three modules, designated Head, Middle and Tail. Tail comprises the Med2, Med3, Med5, Med15 and Med16 protein subunits, which are all encoded by genes that are individually non-essential for viability. In cells lacking Med16, Tail is displaced from Head and Middle. However, inactivation of MED5/MED15 and MED15/MED16 are synthetically lethal, indicating that Tail performs essential functions as a separate complex even when it is not bound to Middle and Head. We have used the N-Degron method to create temperature sensitive (ts) mutants in the Mediator tail subunits Med5, Med15 and Med16 to study the immediate effects on global gene expression when each subunit is individually inactivated, and when MED5/15 or MED15/16 are inactivated together. All Degron constructs were expressed from their normal chromosomal location under the control of their respective endogenous promoters. We isolated RNA from each strain as early as 45 minutes after changing from the permissive to the restrictive growth conditions to minimize possible secondary effects on gene expression that are not directly caused by the Degron construct(s).

2013-06-07 | E-GEOD-47712 | biostudies-arrayexpress

The Mediator complex subunit MED23 couples H2B mono-ubiquitination to transcriptional control and cell fate determination

Project description:The Mediator complex orchestrates multiple transcription factors with the Pol II apparatus for precise transcriptional control. However, its interplay with the surrounding chromatin remains poorly understood. Here, we analyze differential histone modifications between WT and MED23-/- (KO) cells and identify H2B mono-ubiquitination at lysine 120 (H2Bub) as a MED23-dependent histone modification. Using tandem affinity purification and mass spectrometry, we find that MED23 associates with the RNF20/40 complex, the enzyme for H2Bub, and show that this association is critical for the recruitment of RNF20/40 to chromatin. In a cell-free system, Mediator directly and substantially increases H2Bub on recombinant chromatin through its cooperation with RNF20/40 and the PAF complex. Integrative genome-wide analyses show that MED23 depletion specifically reduces H2Bub on a subset of MED23-contolled genes. Importantly, MED23-coupled H2Bub levels are oppositely regulated during myogenesis and lung carcinogenesis. In sum, these results establish a mechanistic link between the Mediator complex and a critical chromatin modification in coordinating transcription with cell growth and differentiation. To examine the enrichment of H2B ubiquitination, Pol II, H3K4me3, H3K79me3 in WT and KO MED23 MEF cells, we performed H2Bub ChIP-seq, Pol II ChIP-seq, H3K4me3 ChIP-seq and H3K79me3 ChIP-seq assays. 10 high-throughput sequencing data were deposited and WT, KO input data were controls for peak calling.

2015-09-16 | E-GEOD-59518 | biostudies-arrayexpress

A Med15 - Hrp1 complex associates with fission yeast Mediator

Project description:The multiprotein Mediator complex is an important regulator of RNA polymerase II-dependent genes in eukaryotic cell. In contrast to the situation in many other eukaryotes, the conserved Med15 protein is not a stable component of Mediator isolated from fission yeast. We now demonstrate that Med15 exists in a protein complex together with Hrp1, an ATP-dependent chromatin remodeling protein. The Med15/Hrp1 subcomplex is not a component of the core Mediator complex, but can interact with the repressive L-Mediator conformation. Deletion of MED15 and HRP1 cause similar effects on global steady-state levels of mRNA, but only MED15 is required for galactose-dependent activation of the inv1 gene. Hrp1 has been found in complex with other proteins and genome-wide analysis demonstrates that Med15 only associates with a distinct subset of Hrp1-bound gene promoters. Global analysis reveals that Hrp1-binding normally is associated with increased histone H3 density, but at promoters also bound by Med15, histone H3 density is instead increased. Our findings reveal that Med15 functions as a separate entity in fission yeast and indicate that the function and organization of the Mediator complex may differ significantly between eukaryotes. Keywords: ChIP-chip

2010-09-21 | GSE10079 | GEO

Mediator Complex Regulates Alternative mRNA Processing via the Med23 Subunit

Project description:Mediator complex is an integrative hub for transcriptional regulation. Here we show that Mediator regulates alternative mRNA processing via its Med23 subunit. Combining tandem affinity purification and mass spectrometry, we identified a number of mRNA processing factors that bind to a soluble recombinant Mediator subunit MED23 but not to several other Mediator components. One of these factors, hnRNP L, specifically interacts with MED23 in vitro and in vivo. Consistently, Mediator partially colocalizes with hnRNP L and the splicing machinery in the cell. Functionally Med23 regulates a subset of hnRNP L-targeted alternative splicing (AS) and alternative cleavage and polyadenylation (APA) events as shown by minigene reporters and exon array analysis. ChIP-seq analysis revealed that Med23 can regulate hnRNP L occupancy at their co-regulated genes. Taken together, these results demonstrate a crosstalk between Mediator and the splicing machinery, suggesting a novel mechanism for coupling mRNA processing to transcription. Examination of hnRNP L and H3K36me3 enrichment in sictrl and si23 Hela cells

2012-02-24 | E-GEOD-33887 | biostudies-arrayexpress

Mediator Complex Regulates Alternative mRNA Processing via the Med23 Subunit

Project description:Mediator complex is an integrative hub for transcriptional regulation. Here we show that Mediator regulates alternative mRNA processing via its Med23 subunit. Combining tandem affinity purification and mass spectrometry, we identified a number of mRNA processing factors that bind to a soluble recombinant Mediator subunit MED23 but not to several other Mediator components. One of these factors, hnRNP L, specifically interacts with MED23 in vitro and in vivo. Consistently, Mediator partially colocalizes with hnRNP L and the splicing machinery in the cell. Functionally Med23 regulates a subset of hnRNP L-targeted alternative splicing (AS) and alternative cleavage and polyadenylation (APA) events as shown by minigene reporters and exon array analysis. ChIP-seq analysis revealed that Med23 can regulate hnRNP L occupancy at their co-regulated genes. Taken together, these results demonstrate a crosstalk between Mediator and the splicing machinery, suggesting a novel mechanism for coupling mRNA processing to transcription. We performed an exon array experiment using HeLa cells expressing Med23, hnRNP L or control siRNAs which were established by a virus-mediated siRNA technology. Each sample was done in three biological replicates. Total RNA of these cell lines was processed and hybridized to the Affymetrix human exon array.

2012-02-24 | E-GEOD-33771 | biostudies-arrayexpress

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