Mediator Requirement for Both Recruitment and Postrecruitment Steps in Transcription Initiation

Molecular Biology Institute and Department of Microbiology, Immunology and Molecular Genetics, University of California Los Angeles, 611 Young Drive East, Los Angeles, California 90095, USA.
Molecular Cell (Impact Factor: 14.02). 04/2005; 17(5):683-94. DOI: 10.1016/j.molcel.2005.02.010
Source: PubMed


Mediator complexes are required for activators to stimulate Pol II preinitiation complex assembly on an associated promoter. We show here that for the mouse Egr1 gene, controlled largely by MAP kinase phosphorylation of the ELK1 transcription factor, the MED23 Mediator subunit that interacts with phospho-ELK1 is also required to stimulate Pol II initiation at a step subsequent to preinitiation complex assembly. In Med23-/- cells, histone acetylation, methylation, and chromatin remodeling complex association at the Egr1 promoter were equivalent to that of wild-type cells, yet Egr1 induction was greatly reduced. MAP kinase activation stimulated Pol II and GTF promoter binding. However, the difference in factor binding between wild-type and mutant cells was much less than the difference in transcription, and Pol II remained localized to the promoter in mutant cells. These results indicate that an interaction with MED23 stimulates initiation by promoter bound Pol II in addition to Pol II and GTF recruitment.

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Available from: Hiroshi Handa
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    • "We therefore developed an RNF20/40 ChIP assay to analyze binding of RNF20/40 to the MED23-targeted Egr1 gene locus using a mixture of antibodies specific to RNF20 and RNF40. As indicated in Fig 2I, MED23 deficiency reduced the recruitment of RNF20/40 by approximately threefold at the Egr1 promoter region, which coincided with our previous finding that MED23 deficiency reduces Mediator recruitment to a similar degree at the Egr1 gene promoter (Wang et al, 2005). We next tested if the differential recruitment of RNF20/40 in WT and KO MEFs affects association of RNF20/40 with chromatin. "
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    ABSTRACT: 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-controlled 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. © 2015 The Authors.
    Full-text · Article · Sep 2015 · The EMBO Journal
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    • "ChIP assays were performed as described previously (Wang et al., 2005), and immunoprecipitated DNA was quantified by real-time PCR. The following antibodies were used: anti-ETS1 (Santa Cruz, sc-350X; 1:100); anti-Pol II (Santa Cruz, sc-899; 1:100); anti-P300 (Santa Cruz, sc-585; 1:100); anti-H3K4me1 (Abcam, ab8895; 1:500) and anti- H3K27ac (Abcam, ab4729; 1:500). "
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    ABSTRACT: 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. © 2015. Published by The Company of Biologists Ltd.
    Full-text · Article · Jan 2015 · Development
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    • "Mouse livers (0.2 g) for ChIP assays were freshly isolated and washed with pre-cooled phosphate-buffered saline (PBS). The tissue was homogenized with homogenizer (IKA) with a final concentration of 1% formaldehyde in DMEM and the homogenate was rotated on a shaker for 15 min at room temperature followed by the addition of glycine to a final concentration of 0.125 M. The following procedures were performed as described previously67. DNA pulled down by ChIP was analyzed by Q-PCR with SYBR Premix Ex TaqTM(TAKARA). "
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    ABSTRACT: Mediator complex is a molecular hub integrating signaling, transcription factors, and RNA polymerase II (RNAPII) machinery. Mediator MED23 is involved in adipogenesis and smooth muscle cell differentiation, suggesting its role in energy homeostasis. Here, through the generation and analysis of a liver-specific Med23-knockout mouse, we found that liver Med23 deletion improved glucose and lipid metabolism, as well as insulin responsiveness, and prevented diet-induced obesity. Remarkably, acute hepatic Med23 knockdown in db/db mice significantly improved the lipid profile and glucose tolerance. Mechanistically, MED23 participates in gluconeogenesis and cholesterol synthesis through modulating the transcriptional activity of FOXO1, a key metabolic transcription factor. Indeed, hepatic Med23 deletion impaired the Mediator and RNAPII recruitment and attenuated the expression of FOXO1 target genes. Moreover, this functional interaction between FOXO1 and MED23 is evolutionarily conserved, as the in vivo activities of dFOXO in larval fat body and in adult wing can be partially blocked by Med23 knockdown in Drosophila. Collectively, our data revealed Mediator MED23 as a novel regulator for energy homeostasis, suggesting potential therapeutic strategies against metabolic diseases.Cell Research advance online publication 16 September 2014; doi:10.1038/cr.2014.120.
    Full-text · Article · Sep 2014 · Cell Research
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