Transcription factors exert their regulatory potential on RNA polymerase II machinery through a multiprotein complex called Mediator complex or Mediator. The Mediator complex integrates regulatory signals from cell regulatory cascades with the regulation by transcription factors. The Mediator complex consists of 25 subunits in Saccharomyces cerevisiae and 30 or more subunits in multicellular eukaryotes. Mediator subunit 28 (MED28), along with MED30, MED23, MED25 and MED26, belong to presumably evolutionarily new subunits that seem to be absent in unicellular eukaryotes and are likely to have evolved together with multicellularity and cell differentiation. Previously, we have shown that an originally uncharacterized predicted gene, F28F8.5, is the true MED28 orthologue in Caenorhabditis elegans (mdt-28) and showed that it is involved in a spectrum of developmental processes. Here, we studied the proteomic interactome of MDT-28 edited as GFP::MDT-28 using Crispr/Cas9 technology or MDT-28::GFP expressed from extrachromosomal arrays in transgenic C. elegans exploiting the GFPTRAP system and mass spectrometry. The results show that MDT-28 associates with the Head module subunits MDT-6, MDT-8, MDT-11, MDT-17, MDT20, MDT-22, and MDT-30 and the Middle module subunit MDT-14. The analyses also identified additional proteins as preferential MDT-28 interactants, including chromatin-organizing proteins, structural proteins and enzymes. The results provide evidence for MDT-28 engagement in the Mediator Head module and support the possibility of physical (direct or indirect) interaction of MDT-28 with additional proteins, reflecting the transcription-regulating potential of primarily structural and enzymatic proteins at the level of the Mediator complex.
Mediator is a multiprotein complex that connects regulation mediated by transcription factors with RNA polymerase II transcriptional machinery and integrates signals from the cell regulatory cascades with gene expression. One of the Mediator subunits, Mediator complex subunit 28 (MED28), has a dual nuclear and cytoplasmic localization and function. In the nucleus, MED28 functions as part of Mediator and in the cytoplasm, it interacts with cytoskeletal proteins and is part of the regulatory
cascades including that of Grb2. MED28 thus has the potential to bring cytoplasmic regulatory interactions towards the centre of gene expression regulation. In this study, we identified MDT-28, the nematode orthologue of MED28, as a likely target of lysine acetylation using bioinformatic prediction of post-translational modifications. Lysine acetylation was experimentally confirmed using anti-acetyl lysine antibody on immunoprecipitated GFP::MDT-28 ex-pressed in synchronized C. elegans. Valproic acid (VPA), a known inhibitor of lysine deacetylases, enhanced the lysine acetylation of GFP::MDT-28. At the subcellular level, VPA decreased the nuclear localization of GFP::MDT-28 detected by fluorescence-lifetime imaging microscopy (FLIM). This indicates that the nuclear pool of MDT-28 is regulated by a mechanism sensitive to VPA and provides an indirect support for a variable relative proportion of MED28 orthologues with other Mediator subunits. and Corresponding author: Markéta Kostrouchová