Fig. 5. A molecular mechanism of VGN50-mediated MYC-down-regulation.

a Cleavage under targets & release using nuclease (CUT&RUN). CUT&RUN was performed with BCBL-1 and BC-1 with the indicated antibodies. Enrichment of active histone modifications, and occupancies of RNAPII and SWI/SNF components at the MYC genomic region are shown as peaks. One of the biological duplicate samples for the BCBL-1 cell line is presented. BC-1 showed very similar peak distributions for these molecules. The MYC promoter, enhancer, and negative control regions used for qPCR analyses are indicated with shading. The positions of the MYC coding region, CASC11 coding region, and long non-coding RNA, PVT1, MIR1208 are shown in the panel. b VGN50 inhibits coactivator complex recruitment and c reduces H3K27Ac modification. qPCR is used to examine enrichment of DNA fragments in the presence of VGN50 or mutant peptide (24 μM). Peptide was applied 1 h prior to processing samples for CUT&RUN to replicate SLAM-seq experimental conditions. Non-specific IgG control was also included and enrichment with IgG control at each genomic region was set as 1 for b, and spiked-in luciferase plasmid DNA was used for additional internal control for c. d Formation of a larger protein complex with VGN50. Sucrose gradient sedimentation was used to monitor changes in protein complex formation in the presence of VGN50. The experiment flow chart is shown at the top of this panel. Fractions were collected from top (#1) to bottom (#23/24). Sample #24 without peptide is a smaller fraction of residual samples. Immunoblotting was performed on the odd number samples and the remaining fraction (#24 for no peptide sample) and probed with Flag-tagged SWI/SNF components (SMARCA4, SMARCC2, and SMARCB1). The position of each protein component based on molecular size is indicated. e KSHV K-Rta colocalizes with SWI/SNF components in reactivating BCBL-1 cells. TREx-BCBL-1 cells were triggered for reactivation by treatment with Dox and TPA for 28 h and stained with the indicated antibodies. Images were acquired with Keyence fluorescence microscopy. f A model for VGN50 molecular action. A model depicting the putative molecular action of VGN50 is presented. Coactivator complexes are dynamically assembled on enhancer and promoter regions via transcription factor binding. VGN50, an IDR fragment derived from an exceptionally potent viral transactivator, flexibly interacts with components of coactivators and traps them, thus reduces the available resources for cellular enhancers to activate MYC promoters.