Erden Tucer, M. | bioRxiv Science

bioRxiv2023

Transcriptomic Implications of Toxic Effects of Nanoparticles on Metabolic Pathways of Liver Cells

While nanoparticles find applications in various fields such as cosmetics, drug delivery, and medicine, they also exhibit significant drawbacks. The adverse effects remain inadequately comprehended and necessitate further analysis. This study focuses on assessing the impacts of diverse nanoparticles on HepaRG cell spheroids prior to conducting RNA-Seq analysis, cell viability assays were performed to determine the concentrations of NPs that induce toxicity while maintaining 80% cell viability--a concentration sufficient to cause toxicity without total cell death. To mimic the cellular microenvironment, HepaRG cell spheroids were generated and treated with four distinct nanoparticles of varying sizes. Subsequently, these spheroids underwent RNA-seq analysis to identify specific genes responsive to nanoparticle-induced toxicity. Our findings demonstrate that exposure to nanoparticles substantially modifies gene transcription. Notably, 38 differentially expressed genes were shared across all four types of nanoparticles. These genes were linked to distinct categories of Gene Ontology (GO) terms, thereby clarifying their functions and metabolic pathways through GO and KEGG pathway analyses. Remarkably, processes such as apoptosis, sensitivity to metal ions, hypoxia, and oxidative stress consistently exhibited significant enrichment across all nanoparticle types. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/568576v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@11098eaorg.highwire.dtl.DTLVardef@a766b1org.highwire.dtl.DTLVardef@46f90eorg.highwire.dtl.DTLVardef@18944d9_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Mediator-RNA Polymerase II Interactions Critical for Transcriptional Activation Are Mediated by the N-terminal Half of MED14 and C-terminal Domain of RPB1

Mediator is a large and evolutionarily conserved coactivator complex essential for RNA polymerase II (Pol II)-mediated gene regulation at multiple steps of the transcription process, including preinitiation complex (PIC) assembly and function. Here, we used the MultiBac baculovirus expression system to generate recombinant human core Mediator subcomplexes and subsequent biochemical approaches to dissect the mechanism by which Mediator facilitates direct recruitment of Pol II to core promoters. Our results highlight a pivotal role in this process for the N-terminal half (NTD) of the MED14 subunit. We show that a reconstituted 15-subunit human core Mediator complex that contains only the MED14-NTD is fully functional in facilitating both basal and activated (p53) transcription. This complex directly interacts with the C-terminal domain (CTD) of the RPB1 subunit of Pol II (RPB1 CTD) and is required for recruiting Pol II to core promoters. Moreover, recombinant RPB1 can completely reverse the human core Mediator-Pol II interaction. Notably, the human MED14-NTD region has secondary structure conservation with Schizosaccharomyces pombe. In addition, reanalysis of published cryo-EM structures of yeast Mediator-Pol II complexes strongly supports our conclusion. Thus, our analyses provide critical new insights into how Mediator binds to Pol II and recruits it to the promoters to facilitate transcription.

molecular biology↗