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Abstract
The multi-subunit mammalian Mediator complex acts as an integrator of transcriptional regulation by RNA Polymerase II, and has emerged as a master coordinator of development and cell fate determination. We previously identified the Mediator subunit, MED28, as a cytosolic binding partner of merlin, the Neurofibromatosis 2 (NF2) tumor suppressor, and thus MED28 is distinct in having a cytosolic role as an NF2 interacting protein as well as a nuclear role as a Mediator complex subunit. Although limited in vitro studies have been performed on MED28, its in vivo function remains unknown. Employing a knockout mouse model, we describe for the first time the requirement for Med28 in the developing mouse embryo. Med28-deficiency causes peri-implantation lethality resulting from the loss of pluripotency of the inner cell mass accompanied by reduced expression of key pluripotency transcription factors Oct4 and Nanog. Further, overexpression of Med28 in mouse embryonic fibroblasts enhances the efficiency of their reprogramming to pluripotency. Cre-mediated inactivation of Med28 in induced pluripotent stem cells shows that Med28 is required for their survival. Intriguingly, heterozygous loss of Med28 results in differentiation of induced pluripotent stem cells into extraembryonic trophectoderm and primitive endoderm lineages. Our findings document the essential role of Med28 in the developing embryo as well as in acquisition and maintenance of pluripotency during reprogramming.
Highlights
Mediator is a large evolutionarily conserved protein complex comprising ~30 distinct subunits that plays a pivotal role in the regulation of eukaryotic mRNA synthesis
MED28 is an ~24 kDa protein expressed in many cell lines and tissues, which we identified previously as a binding partner of merlin, the Neurofibromatosis 2 (NF2) tumor suppressor [3]
As an essential coordinator of polymerase II (Pol II)-mediated transcription regulation, the Mediator complex is responsible for integrating signaling events to tissue-specific and cell-specific gene regulation and plays an essential role during development and as well as in disease processes [1, 22, 23]
Summary
Mediator is a large evolutionarily conserved protein complex comprising ~30 distinct subunits that plays a pivotal role in the regulation of eukaryotic mRNA synthesis. Our earlier study demonstrated that RNAi-mediated suppression of Med resulted in a significant induction of many genes involved in smooth muscle cell (SMC) differentiation, and Med suppression in the multipotent, mesenchymal-derived murine precursor cell line C2C12 caused transdifferentiation into SMCs [8]. A recent study employing a shRNA library to screen for regulators of transcription and chromatin necessary for maintaining murine embryonic stem (ES) cells identified many Mediator subunits including Med28 [9]. Mediator subunits Med and Med in complex with cohesin were shown to physically and functionally connect the enhancers and core promoters of active genes in murine ES cells [9]. Cre-mediated removal of Med from induced pluripotent stem cells (iPSCs) shows that Med is required for their survival and intriguingly, heterozygous loss of Med causes aberrant differentiation of iPSCs
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