Fig 2: Pharmacologic inhibition of PRC1.1 component USP7 upregulates HLA-I in MCPyV+ MCC.(A) Dependency data from the Cancer Dependency Map (DepMap) (59, 60) were stratified based on TP53 mutation status (TP53-mut [n = 532] vs. TP53-WT [n = 235]). Left: Pearson’s correlation coefficients with corresponding P values and FDRs of the top genes that are codependent with USP7 in TP53-mutated lines, with PRC1.1 genes highlighted (see Supplemental Methods). Right: Graphical comparison of dependency of USP7 with PRC1.1 genes PCGF1 and RING1 in TP53-WT (blue) and TP53-mut cell lines (red). The x- and y-axes display gene effect scores determined by CERES, an algorithm which estimates gene-dependency levels from CRISPR-Cas9 survival screens” (60) (B) Flow cytometry experiments measuring surface HLA-I in MCC lines treated with USP7 inhibitor XL177A or control compound XL177B, performed in technical triplicate. One-way ANOVA was performed, followed by Welch’s 2-tailed t tests comparing XL177A and XL177B MFIs, normalized to DMSO (see Methods). *P < 0.05; **P < 0.01; NS, P = 0.05. (C) HLA I flow cytometry to assess the effect of USP7 inhibitors in MKL-1 p53-WT control lines (left) or p53-KO lines (right; lines 1–3 refer to 3 different single-cell p53-KO clones). Cells were treated with 100 nM XL177A (red), XL177B (black), or DMSO (light gray). For statistical analysis, 2-way ANOVA was performed, followed by post hoc Tukey’s multiple-comparison tests (see Methods). (D) Heatmap of peptide abundances within the HLA-I–presented peptidomes of MCC-301 cells treated with XL177A (red) or XL177B (black), compared with untreated cells (gray) (n = 2 replicates). Only peptides that were significantly differentially expressed between any 2 treatment groups (determined by 2-sample, 2-tailed t test) are shown. (E) Frequency of peptides presented on each HLA allele in MCC-301 cells treated with XL177A or XL177B, compared with untreated cells.

Fig 3: The FRRUC is required for H2A-ubiquitylation at sites of DNA damage.(A) U-2OS cells were transfected with the indicated siRNAs. Cells were fixed 30 min after laser microirradiation and stained with antibodies to ?H2A.X (orange) and H2AK119Ub1 (green). Scale bar represents 5 µm. The graph represents the mean intensity of the H2AK119Ub1 signal on the ?H2A.X track, normalized to the mean values of the siCTRL sample. At least 28 laser stripes were analyzed per condition from 2 or three independent experiments. A white dash line denotes the border of each nucleus. Error bars represent S.E.M. Differences relative to siCTRL were calculated using one-way ANOVA. ****p=0.0001, ***p=0.001, **p=0.01, *p=0.05, ns = not significant. (B) U-2OS cells engineered to express FLAG-HA-tagged RNF68, RNF68(?RING) or RNF68(Y252D) were transfected with siRNAs targeting the 3’ UTR of RNF68 (siRNF68 seq2). Cells were fixed 30 min after laser microirradiation and stained with antibodies to ?H2A.X (orange) and H2AK119Ub1 (green). Scale bar represents 5 µm. The graph represents the mean intensity of the H2AK119Ub1 signal on the ?H2A.X track, normalized to the mean values of the siCTRL sample. At least 30 laser stripes were analyzed per condition from 2 or three independent experiments. A white dash line denotes the border of each nucleus. Error bars represent S.E.M. Differences relative to siCTRL were calculated using one-way ANOVA. ****p=0.0001, ***p=0.001, **p=0.01, *p=0.05, ns = not significant. (C) Left: Schematic of DSB generation by ER-mCherry-LacI-FokI-DD at lac operator (LacO) repeats integrated into the genome of U-2OS DSB reporter cells. The ER-mCherry-LacI-FokI-DD is stabilized by Shield1 and targeted to the nucleus by 4-OHT. The qChIP primer sets around the induced DSBs are labeled as p1-p2. ER, estrogen receptor; DD, destabilization domain. The ER-mCherry-LacI-FokI-DD nuclease was induced for 1 hr using Shield-1 and 4-OHT. Right: qChIP was performed in FokI reporter U-2OS cells with and without FokI-induced DSBs using antibodies to H2A and ubiquitylated H2A. To determine the relative amount of mono-ubiquitylated H2A, H2AK119Ub1 values were normalized on total H2A values. Error bars represent S.E.M from =3 independent experiments. P values were calculated using one-way ANOVA. ****p=0.0001, ***p=0.001, **p=0.01, *p=0.05, ns = not significant.

Fig 4: Pcgf1 knockout ES cells are generated by CRSPR/Cas9 technology.(A) Schematic diagram of two sgRNAs targeting sites in Pcgf1. The sgRNA-targeting sequences were highlighted in blue, and the PAM sequences were labeled in red. PCR products using primers F and R were cloned into plasmid and sequenced. Note that a DNA fragment of 659 bp was deleted. Cas9 mediated a DNA double-stranded breaks ~3 bp upstream of the PAM (red triangle). (B) Genotyping analysis of Pcgf1 knockout ES cells (using the F and R primers). (C) The Pcgf1 mRNA level in ES cells was analysed by RT-PCR (M, DNA marker). (D) Western blot analysis demonstrated the loss of Pcgf1 protein in ES cells. Pcgf1 was detected as a 30 kDa band in WT extracts but not in extracts from Pcgf1-/- ES cells.

Fig 5: The PRC1.1 complex implicated as a suppressor of HLA-I in a genome-wide CRISPR screen.(A) Gene-level ranking of positive (left) and negative (right) CRISPR-KO screen hits, according to STARS, a gene-ranking algorithm for genetic screens (39). Inset: GSEA analysis of screen hits. (B) Flow cytometry for surface HLA-I in MCC-301 PRC1.1 KO lines (PCGF1, USP7, and BCORL1). Data visualized with biexponential scaling. (C) Western blot for PCGF1 (top) and USP7 (bottom) in WT MCC-301, a control sgRNA MCC-301 line, or the indicated knockout line. (D) Top: Volcano plot showing LFC in gene expression in an MCC-301 PCGF1-KO line compared with a control sgRNA line. Bottom: GSEA plot demonstrating enrichment of PRC2 target genes upon PCGF1 knockout. (E) Western blot of TAP1 in PCGF1-KO and control sgRNA lines at varying IFN-? concentrations. (F) RNA-Seq analysis of HLA-I genes, PRC1.1, PRC2, and ST-MYCL-EP400 in a cohort of 51 MCC tumors. Left: Unsupervised hierarchical clustering heatmap by Euclidian distance. Top track: Tumor purity scores for each tumor, generated by ESTIMATE (53). Pearson’s correlation coefficients between each PRC2, PRC1.1, or ST-MYCL-EP400 component and each class I gene were calculated, and the bar charts (right) show the number of Pearson’s coefficients that were less than –0.3. (G) UCSC Genome Browser view of USP7 and PCGF1 with ChIP-Seq tracks for MAX (red), EP400 (blue), MCPyV ST antigen (pink), and activating histone marks H3K4me3 and H3K27ac (black). The “-1” and “-2” suffixes refer to 2 different antibodies used for each protein. (H) ChIP-qPCR targeting the USP7 and PCGF1 promoters, using MKL-1 chromatin immunoprecipitated with a MAX (left) or EP400 (right) antibody (n = 3). P values were calculated by 1-way ANOVA followed by post hoc Dunnett’s multiple-comparison test. (I) Protein expression of USP7, PCGF1, and MYCL in MKL-1 cells transduced with the indicated doxycycline-inducible shRNAs. (J) Schematic of putative interactions between MCPyV viral antigens and screen hits MYCL and PRC1.1.