Fig 1: Deubiquitylation of PIC components MED16 and MED24 is controlled by USP22 High‐throughput proteomic analysis of Mediator tail subunits MED16 and MED24 following depletion of USP22, with significance measured by Student’s t‐test. Specific ubiquitylated lysines detected are highlighted in red text.HCT116 cells were treated for 2 h with 100 nM Thapsigargin in the presence or absence of shRNA targeting USP22. Cells were harvested and fractionated, and fractions were subjected to immunoblotting with the indicated antibodies.In vitro deubiquitylation UbiTest of endogenously ubiquitylated MED24. HCT116 cells were treated with MG132 in the presence or absence of shRNA targeting USP22. Lysates were generated using buffer containing protease cocktail inhibitor, pan‐DUB inhibitor PR619, and the JAMM protease inhibitor o‐phenanthroline. Ubiquitylated proteins were purified on ubiquitin‐binding resin, and eluates were either undigested (lanes 1 and 2) or digested with USP2 to strip polyubiquitin (lanes 3 and 4) and reduce target proteins to unit length. Digestion reactions were subjected to immunoblotting with the indicated antibodies. Source data are available online for this figure.
Fig 2: USP22 is required for efficient recruitment of the Mediator tail module to ER stress response gene promoters and enhancersHCT116 cells were treated for 2 h with 100 nM Thapsigargin in the presence or absence of shRNA targeting USP22. Cells were cross‐linked, harvested, and subjected to high‐throughput ChIP‐sequencing for Mediator subunits MED1 (middle) and MED16 (tail). AGenome browser image of ChIP‐seq for middle and tail Mediator subunits and H3K4me3 HiChIP tracks before and after induction of ER stress at ER stress response gene BHLHE40. H3K4me1 and H3K27ac tracks are from the ENCODE Consortium (GEO accessions GSM945858 and GSM945853). ATF4/Xbp1s binding sites are from publicly available datasets (GEO accessions GSE69304 and GSE49952).BH3K4me3 HiChIP signal (log2) at promoter–enhancer loops before and after induction of ER stress or USP22 depletion at indicated bound group genes.C, DMean ChIP‐seq signal MED1 and MED16 surrounding gene promoters from gene groups defined in Fig 4.E, FLinear regression analysis of Mediator and USP22 recruitment to USP22‐bound target gene promoters following induction of ER stress.
Fig 3: SMRT sequencing reveals transcriptional elongation defects following CDK12 knockdown during AKI. (A) Integrative Genomics Viewer (IGV) representation shows the mapping of SMRT sequencing reads at the Med16, Smug1, Txnip, Cast, and Fgf1 genes. The presence of elongation defect due to CDK12 knockdown in mice treated with cisplatin as in number of short transcrips. (B) Three primers (located near the 5′ untranslated regions, coding sequence, 3′ untranslated regions) are labeled in blue, pink and red, respectively. Expression of Med16, Smug1, Txnip, Cast, and Fgf1 at selected regions in different groups was assessed by real-time PCR. Error bars indicate mean values of ±SD, n=3.
Fig 4: (A) Representative immunostaining micrographs show Med16, Smug1, Txnip, Cast, and Fgf1 expression in different groups, as indicated. (scale bar = 50 μm). (B) Representative western blots showing the expression of Med16, Smug1, Txnip, Cast, and Fgf1 in different groups both in vitro and in vivo. (C) Graphical representations of Med16, Smug1, Txnip, Cast, and Fgf1 levels in different groups, as indicated. *P<0.05 versus cisplatin alone or CDK12 knockdown with cisplatin (n=5).
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