Fig 2: Irreversible inhibition of intracellular Hsp90. a Schematic illustration of reaction mechanism of NASA-based covalent inhibitor. b Molecular structures of PU-H71 (non-covalent inhibitor) and NASA-based covalent inhibitor 12. c In-gel fluorescence analysis of fluorescein labelling of Hsp90 with 11 in SKBR3 cells after inhibitor washout. After cells were incubated with various concentrations of PU-H71 or 12 for 3 h, the cells were washed with medium and further incubated with 11 for 3 h. The cells were washed, lysed and analysed by in-gel fluorescence imaging. Error bars represent s.d., n = 3. d Viability of the cells 69 h after PU-H71 or 12 washout. Data represent mean values ± standard error of the mean (s.e.m.) for three (PU-H71) or six (compound 12) independent experiments. e Western blotting analysis of the destabilization of client proteins induced by inhibiting Hsp90 chaperone activity, and f the normalised band intensities. Cells were treated with PU-H71 or 12 for 3 h, followed by washing with media and further incubation for 21 h. The protein band intensity was normalised to DMSO control (lane 1). β-actin is a control as a non-Hsp90 client protein. Error bars represent s.d., n = 3

Fig 3: WDR82 is required for HUSH accumulation on chromatin(A) Heatmap showing odds ratio values for overlap between H3K4me3, H3K9me3, MPP8, TASOR, and WDR82 ChIP-seq peaks. Peaks were called using MACS2 with broad peak settings. Statistics were calculated by BEDTools fisher. The number of peaks for respective proteins is indicated at the bottom.(B) Heatmaps of WDR82 ChIP-seq coverage in WT mESCs, centered and sorted as in Figure 3A. The x axis represents distance from MPP8 ChIP peak in kb.(C) Western blot of WT and TASOR-AGH mESC total cell lysates. Control and WDR82 KO clones from each cell line are shown. Membranes were probed with antibodies against WDR82, L1-ORF1p, and HSP90 as a loading control.(D) Heatmaps showing TASOR (left) or MPP8 (middle) differential ChIP-seq coverage in WDR82 KO – WT backgrounds compared with reanalyzed ChIP-seq coverage for TRIM28 (right). Signal is centered and sorted as in Figure 3A. The x axis represents distance from MPP8 ChIP peak in kb.(E) Aggregate plot showing TASOR ChIP-seq coverage (y axis) relative to the distance from HUSH ChIP peak (x axis) in WT (red) and WDR82 KO (blue) mESCs.(F) Top: browser tracks of TASOR, MPP8 and WDR82 ChIP-seq in WT and WDR82 KO mESCs. Bottom: browser tracks of PRO-seq in WT (orange) and WDR82 KO (blue) mESCs for the positive (+) and negative (−) DNA strands. The arrow marks the observed shifts in ChIP-seq and PRO-seq signals in WDR82 KO samples.(G) Aggregate plot showing TASOR ChIP-seq coverage individually scaled to span (0,1) interval in each sample (y axis) to visualize changes in plot shape, relative to the distance from HUSH ChIP peak (x axis) in WT (red) and WDR82 KO (blue) mESCs.(H) Heatmaps showing subtraction of PRO-seq coverage (WDR82 KO – WT), for sense (S, left) and antisense (AS, right) strands. Heatmaps were centered and sorted as in Figure 3A. The x axis represents distance from MPP8 ChIP peak in kb.

Fig 4: Controls for HUSH ablation in landing pad cells.a) MPP8 western blot in WT, and a subset of MPP8 targeted, and Control targeted populations. Membranes were probed with antibodies against MPP8 and ACTB as a loading control. b) Scatterplot of corrected eGFP and mTagBFP2 signal for all promoters tested in WT, ΔMPP8 and control edited backgrounds. ΔMPP8 and control edited measurements were done in triplicates in population of cells. c) MPP8 western blot in WT, and clonal ΔMPP8 lines. Membranes were probed with antibodies against MPP8 and HSP90 as a loading control. d) H3K9me3 ChIP-qPCR percent input recovery at sites labeled in (3B) for Ef1a insert lines in clonal WT, clonal ΔMPP8, population ΔMPP8 and control edited populations. n=2, mean ± s.e.