Investigation of the effect of MED8 sumoylation on carboxy-terminal domain of RPB1

Abstract

Posttranslational modifications by the Small Ubiquitin-like Modifier (SUMO) in a dynamic and reversible process that plays a crucial role in biology by modulating protein structure, function, and dynamics. Numerous in vitro and in vivo studies revealed that sumoylation supports diverse cellular processes including development, differentiation, maintenance of cellular identity, DNA repair and transcription. Mediator, a highly conserved multi-subunit complex, also plays an essential role in transcription. It supports gene transcription by acting as a bridge between signal-specific transcription factors at enhancers and the pre-initiation complex (PIC) at gene promoter. Importantly, it also supports PIC assembly and promotes phosphorylation of Serine 5 in the C-terminal domain (CTD) of RNA polymerase II by TFIIH. Interestingly, a recent study from our laboratory revealed that multiple subunits of the Mediator complex are sumoylated during murine adipocyte differentiation, which suggests that Mediator sumoylation could regulate adipogenic transcription. This thesis investigates the sumoylation of the subunit MED8, focusing particularly on lysine 112 (K112). Indeed, this residue is highly conserved amongst species and positioned at the site of CTD phosphorylation during transcriptional initiation. The project´s primary objectives were to confirm MED8 as a SUMO target in HeLa cells and to evaluate the influence of MED8-K112 sumoylation on CTD phosphorylation at Serine 5. To fulfill these objectives, we constructed plasmids to express both wild-type MED8 and several MED8 mutants. These constructs subsequently used to assess MED8 sumoylation in HeLa cells and analyze CTD phosphorylation levels by western blot. These findings reveal that MED8 is a SUMO target in HeLa cells and that preventing sumoylation at the K112 residue enhances RNA Pol II CTD phosphorylation at Serine 5. The conservation of lysine 112 suggests a novel, general mechanism in transcriptional regulation. Further studies are needed to elucidate the specific molecular mechanisms underlying the regulatory effects of MED8 sumoylation and its implications for transcriptional control.