Effect of over expression of MED13 IDR in human osteosarcoma U2OS cell line

The eukaryotic transcriptional Mediator comprises a large Core (cMED) and a dissociable CDK8 kinase module (CKM). cMED recruits RNA polymerase II (Pol II) and promotes pre-initiation complex formation in a manner repressed by the CKM through mechanisms presently unknown. Herein we report cryo-electron microscopy structures of the complete human Mediator and its CKM. Combined with biochemical and functional analyses, these structures provide a mechanistic framework to explain the basis for CKM-mediated repression of cMED function. The CKM binds to cMED at multiple sites through both MED12 and a large intrinsically disordered region (IDR) in MED13. The MED13 IDR blocks Pol II/MED26 recruitment onto cMED by direct occlusion of their corresponding binding sites, leading to functional repression of cMED-dependent transcription. Notably, the CKM is anchored to the cMED Hook, positioning CDK8 downstream and proximal to the transcription start site, which sheds new light on its stimulatory function in post-initiation events. To investigate the function of MED13 IDR in transcription initiation, we over-expressed MED13 IDR using lentivirus based system in U2OS cell line. We then performed gene expression profiling analysis using data obtained from RNA-seq. Comparative gene expression profiling analysis of RNA-seq data for triplicate of MED13 IDR and control cells.