Fig. 2: Linear ubiquitination machinery regulates the transcriptional activity of KSHV RTA by modulating its binding to target gene promoters. | Nature Communications

Fig. 2: Linear ubiquitination machinery regulates the transcriptional activity of KSHV RTA by modulating its binding to target gene promoters.

From: Linear ubiquitination regulates the KSHV replication and transcription activator protein to control infection

Fig. 2: Linear ubiquitination machinery regulates the transcriptional activity of KSHV RTA by modulating its binding to target gene promoters.

a, b iSLK/rKSHV.219 cells with OTULIN overexpression (a) or knockdown (b) were treated as indicated. RT-qPCR was performed to quantify the expression levels of KSHV lytic genes RTA, ORF59, ORF9, and ORF8.1. a, n = 2 biological replicates and 2 technical replicates, * P = 0.02288, 0.03410, 0.00229, 0.02574 (Cntrl vs. Tet/VPA) for RTA, ORF59, ORF9, ORF8.1; #, P = 0.02796, 0.03488, 0.00233, 0.03357 (OTULIN+Tet/VPA vs. Tet/VPA) for RTA, ORF59, ORF9, ORF8.1. b, n = 2 biological replicates and 2 technical replicates, * P = 0.03772, 0.03042, 0.00704 (siCntrl vs. siCntrl+Tet/VPA) for RTA, ORF59, ORF8.1; #, P = (0.03734, 0.02794, 0.03573), (0.03299, 0.02145), (0.00646, 0.01030), (0.00027, 0.00276) (siCntrl vs. siOTULIN) for RTA, ORF59, ORF9, ORF8.1. c OTULIN inhibits RTA transcriptional activities in reporter assays. HEK293T cells were co-transfected with luciferase reporter plasmid pOri-Lyt-Luc, a Renilla luciferase control plasmid, together with or without RTA, in the presence of increased amount of OTULIN or OTULINC129A, then harvested and their luciferase activities were quantified. n = 3 biological replicates and 2 technical replicates. d OTULIN inhibits, while HOIP enhances, RTA binding to the Ori-Lyt promoter. HEK293T cells were co-transfected with the reporter plasmid pOri-Lyt-Luc, RTA-Flag, and with Myc-OTULIN, Myc-HOIP, or Myc-OTULINC129A as indicated. Cells were collected for ChIP assay using anti-Flag magnetic agarose beads. Precipitated Ori-Lyt promoter DNAs were quantified using qPCR. n = 2 biological replicates and 2 technical replicates. e, f OTULIN inhibits, while HOIP enhances, RTA binding to its target gene promoters. iSLK/rKSHV.219 cells were transfected with Myc-OTULIN, Myc-OTULINC129A, or Myc-HOIP followed by treatment with Tet plus VPA. The cells were collected and subjected to ChIP assay using an anti-RTA antibody. The quantities of PAN (e) or ORF57 (f) promoter DNA in the precipitates were determined by qPCR, e, f, n = 2 biological replicates and 2 technical replicates. Data are displayed as mean ± s.d., two-sided t test. Source data are provided as a Source Data file.

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