Villeret, V. | bioRxiv Science

bioRxiv2023

Assessment of machine-learning predictions for MED25 ACID domain interactions with transactivation domains

Human Mediator complex subunit MED25 binds transactivation domains (TADs) present in various cellular and viral proteins using two binding interfaces found on opposite sides of its ACID domain, and referenced as H1 and H2. Here, we use and compare deep learning methods to characterize Human MED25-TADs interfaces and assess the predicted models to published experimental data. For the H1 interface, AlphaFold produces predictions with high reliability scores that agree well with experimental data, while the H2 interface predictions appear inconsistent, preventing reliable binding modes. Despite these limitations, we experimentally assess the validity of Lana-1 and IE62 MED25 interface predictions. AlphaFold predictions also suggest the existence of a unique hydrophobic pocket for Arabidopsis MED25 ACID domain.

biochemistry↗

bioRxiv2020

Mediator complex subunit Med19 binds directly GATA DNA-binding zinc finger and functions with Med1 in GATA-driven gene regulation in vivo

The evolutionarily-conserved multiprotein Mediator complex (MED) serves as an interface between DNA-bound transcription factors (TFs) and the RNA Polymerase II machinery. It has been proposed that each TF interacts with a dedicated MED subunit to induce specific transcriptional responses. However, binary MED subunit - TF partnerships are probably oversimplified models. Using Drosophila TFs of the GATA family - Pannier (Pnr) and Serpent (Srp) - as a model, we have previously established GATA cofactor evolutionarily-conserved function for the Med1 Mediator subunit. Here, we show that another subunit, Med19, is required for GATA-dependent gene expression and interacts physically with Pnr and Srp in cellulo, in vivo and in vitro through their conserved C-zinc finger (ZF), indicating general GATA co-activator functions. Interestingly, Med19 is critical for the regulation of all tested GATA target genes which is not the case for Med1, suggesting differential use of MED subunits by GATAs depending on the target gene. Lastly, despite their presumed distant position within the MED middle module, both subunits interact physically. In conclusion, our data shed new light first on the MED complex, engaging several subunits to mediate TF-driven transcriptional responses and second, on GATA TFs, showing that ZF DNA-binding domain also serves for transactivation.

developmental biology↗