k562 aml homo sapiens  (ATCC)

Journal: iScience

Article Title: The USP7-TRIM27 axis mediates non-canonical PRC1.1 function and is a druggable target in leukemia

doi: 10.1016/j.isci.2021.102435

Figure Lengend Snippet: LC-MS/MS-based identification of new PRC1.1 subunits (A) LC-MS/MS analysis of KDM2B-GFP, GFP-RING1B, and PCGF1-GFP pull outs in K562 cells showing 20 overlapping interaction partners including PRC1.1 subunits (in red) and new interaction partners such as USP7 and TRIM27 (in blue). (B) Schematic overview of PRC1.1 subunit composition. PRC1.1 preferentially binds to non-methylated CpG islands via the CxxC domain of KDM2B. The ubiquitination of histone H2A on lysine 119 is mediated by RING1A/B. (C) Schematic representation of KDM2B deletion mutants and the endogenously expressed long form (LF) and short form (SF; lacking the JmjC domain) of KDM2B. (D) Western blots showing expression of GFP-fusions at near to endogenous KDM2B expression levels. (E) Table showing LC-MS/MS data of PRC1.1 subunits and other PcG proteins co-precipitating with wild-type and mutant forms of KDM2B-GFP. Numbers indicate the average total spectra counts, as measured in triplicate, corrected for maximally identifiable peptides based on in silico protein digests. (F) Volcano plot showing MaxQuant-based label-free quantification of triplicate measurements of KDM2B LF-GFP and KDM2B Δ(LRR)-GFP pull outs. PRC1.1 subunits are indicated in red and USP7 and TRIM27 in blue. (G) Western blot analysis of independent pull outs of (mutant) KDM2B-GFP with input (I, 1/90 th ), non-bound (NB, 1/90 th ), and bound (B, 1/3 rd ) fractions loaded and stained with antibodies against GFP, PCGF1, RING1B, USP7, and TRIM27. (H) PCGF1-GFP pull outs with input (I, 1/90 th ), non-bound (NB, 1/90 th ), and bound (B, 1/3 rd ) and stained with antibodies directed against GFP, KDM2B, RING1B, USP7, and TRIM27. See also Figure S1 and , , and .

Article Snippet: K562 (AML) (Homo-sapiens) , ATCC , Cat# CCL-243.

Techniques: Liquid Chromatography with Mass Spectroscopy, Methylation, Ubiquitin Proteomics, Western Blot, Expressing, Mutagenesis, In Silico, Quantitative Proteomics, Staining

Journal: iScience

Article Title: The USP7-TRIM27 axis mediates non-canonical PRC1.1 function and is a druggable target in leukemia

doi: 10.1016/j.isci.2021.102435

Figure Lengend Snippet: GFP-USP7 predominantly interacts with PRC1.1 (A) Confocal images of fixed K562 GFP-USP7 cells stained with DAPI. Scale bars represent 25 μm. (B) Western blots showing relative expression of GFP-USP7 versus endogenous USP7 in K562 cells and efficient precipitation of GFP-USP7 using GFP-Trap beads. Input (I, 1/90 th ), non-bound (NB, 1/90 th ), and bound (B, 1/3 rd ) are indicated. (C) Volcano plot showing enrichment of GFP-USP7-specific interaction partners in GFP and GFP-USP7 pull outs. Pull outs were performed in triplicate on K562 GFP and GFP-USP7 cells, and samples were analyzed using LC-MS/MS, followed by data analysis using MaxQuant and Perseus software. Statistical analysis was performed using Student's t test (false discovery rate (FDR) < 0.01; fold change (FC) > 10). PRC1.1, PRC1.2/1.4, and PRC1.6 subunits are indicated in red, blue, and purple respectively. (D) Volcano plot showing enrichment of previously identified USP7 interaction partners (orange), including the MRN-MDC1 complex (green). Statistical analysis was performed using Student's t test (false discovery rate (FDR) < 0.01; fold change (FC) > 10). (E) Intensity-based absolute quantification (iBAQ) value-based calculation of relative stoichiometry values relative to BCOR. (F) Western blot analysis of independent pull outs of GFP, PCGF1-GFP, PCGF2-GFP, PCGF4-GFP, GFP-RING1B, and GFP-CBX2 where input (I, 1/90 th ), non-bound (NB, 1/90 th ), and bound (B1/3 rd ) fractions are loaded and stained with USP7 and RING1B antibodies. See also .

Article Snippet: K562 (AML) (Homo-sapiens) , ATCC , Cat# CCL-243.

Techniques: Staining, Western Blot, Expressing, Liquid Chromatography with Mass Spectroscopy, Software, Quantitative Proteomics

Journal: iScience

Article Title: The USP7-TRIM27 axis mediates non-canonical PRC1.1 function and is a druggable target in leukemia

doi: 10.1016/j.isci.2021.102435

Figure Lengend Snippet: USP7 deubiquitinase activity is essential for PRC1.1 integrity (A) Purification of His-tagged ubiquitinated proteins under denaturing conditions in GFP-RING1B and PCGF1-GFP cells treated with DMSO or P22077 for 24 h (30 μM) followed by western blot analysis. (B) Volcano plot showing differential interaction of GFP-RING1B (left) and PCGF1-GFP (right; both indicated in green) with PRC1.1 subunits (highlighted in orange) as measured by label-free quantification (LFQ) of LC-MS/MS data in triplicate. The UBB protein is indicated in blue. Statistical analysis was performed using Student's t test (false discovery rate (FDR) < 0.1; fold change (FC) > 2). (C) Intensity-based absolute quantification (iBAQ) value ratios of several identified PRC1.1 proteins as identified in GFP-RING1B (left) and PCGF1-GFP (right) pull outs from P22077- or DMSO-treated cells. Data are shown as mean ± SD (n = 2 or 3). (D) Western blot of GFP pull outs on PCGF1-GFP and GFP-RING1B in the absence (−) or presence (+) of P22077 (72 h, 30 μM) probed with antibodies for GFP, RING1B, and PCGF1. Input (I, 1/90 th ), non-bound (NB, 1/90 th ), and bound (B, 1/3 rd ) fractions are shown. (E) Mean fluorescent intensity (MFI) analysis of K562 cells expressing either GFP-RING1B, PCGF1-GFP, or KDM2B-GFP treated with DMSO or P22077 for 72 h. See also .

Article Snippet: K562 (AML) (Homo-sapiens) , ATCC , Cat# CCL-243.

Techniques: Activity Assay, Purification, Western Blot, Quantitative Proteomics, Liquid Chromatography with Mass Spectroscopy, Expressing

Journal: iScience

Article Title: The USP7-TRIM27 axis mediates non-canonical PRC1.1 function and is a druggable target in leukemia

doi: 10.1016/j.isci.2021.102435

Figure Lengend Snippet: USP7 inhibition induces loss of PRC1.1 occupancy and H2AK119ub at target loci (A) ChIP-qPCRs on K562, K562 GFP-RING1B, and K562 PCGF1-GFP cells, treated with DMSO or P22077 (72 h, 30 μM), using antibodies against KDM2B or GFP. (B and C) ChIP-qPCRs on K562 cells using antibodies directed against H2AK119ub (B), H3K4me3 (C), on several PRC1.1 loci. Error bars represent SD of technical qPCR replicates. (D and E) ChIP-qPCRs on DMSO- or P22077-treated K562 cells for 4 h, 8 h, and 16 h using antibodies against H2AK119ub (D), GFP (reading out KDM2B-GFP and GFP-RING1B), and H3K27ac (E) on various PRC1.1 loci. Error bars represent SD of technical qPCR replicates. (F) ChIP-qPCRs on K562 cells treated with either DMSO or FT671 (48 h, 10 μM) using antibodies against KDM2B and H2AK119ub. Error bars represent SD of three biological replicate experiments. See also Figure S2 .

Article Snippet: K562 (AML) (Homo-sapiens) , ATCC , Cat# CCL-243.

Techniques: Inhibition

Journal: iScience

Article Title: The USP7-TRIM27 axis mediates non-canonical PRC1.1 function and is a druggable target in leukemia

doi: 10.1016/j.isci.2021.102435

Figure Lengend Snippet: Genome-wide loss of H2AK119ub upon USP7 inhibition (A–D) ChIP-seq on K562 cells treated with either DMSO or FT671 (24 h, 10 μM) using antibodies against H2AK119ub, H3K4me3, H3K36me3, or H3K27me3. (A) Venn diagram depicting overlapping peaks −/+ 5kb from the transcription start site (TSS). (B) Density plots displaying epimarks around the TSS or across the gene body until + 5kb after the transcription end site (TES). (C) Heat maps of the H2AK119ub signal around the TSS. (D) Representative screenshots of epimarks at four PRC1.1 loci. (E) Non-canonical PRC1.1 or canonical PRC1 peaks shared between six primary AML CD34 + patient samples ( van den Boom et al., 2016 ) were overlaid with USP7 peaks from CUTTL1 cells ( Jin et al., 2019 ). (F) Representative screens shots of PRC1.1 and PRC1 loci depicting USP7 and KDM2B binding and H3K27me3 levels. See also Figure S3 .

Article Snippet: K562 (AML) (Homo-sapiens) , ATCC , Cat# CCL-243.

Techniques: Genome Wide, Inhibition, ChIP-sequencing, Binding Assay

Journal: iScience

Article Title: The USP7-TRIM27 axis mediates non-canonical PRC1.1 function and is a druggable target in leukemia

doi: 10.1016/j.isci.2021.102435

Figure Lengend Snippet: Loss of TRIM27 partially rescues USP7 inhibitor sensitivity (A) Knockdown efficiencies of two independent TRIM27 shRNAs in K562 cells. Error bars represent SD from technical triplicates. (B) KDM2B-GFP and H2AK119ub ChIP-qPCRs with error bars representing SD based on three independent experiments, statistical analysis was performed using Student's t test; ∗p < 0.05 and ∗∗p < 0.01. (C) Western blot analysis of KDM2B-GFP pull outs on K562 cells expressing SCR or TRIM27 (#1) shRNAs using the indicated antibodies. Input (I, 1/90 th ), non-bound (NB, 1/90 th ), and bound (B, 1/3 rd ) as indicated. (D) H2AK119ub ChIP-qPCR on K562 cells expressing SCR or TRIM27 (#2) shRNAs and treated with DMSO or FT671 (48 h, 10μM). Error bars represent SD based on three independent experiments; statistical analysis was performed using Student's t test; ∗p < 0.05 and ∗∗p < 0.01. (E) Cumulative cell proliferation of K562 cells expressing SCR or TRIM27 (#1 or #2) shRNAs and treated with DMSO or FT671. Error bars represent SD based on two independent experiments. (F) Knockdown efficiencies of two independent USP7 shRNAs in K562 cells. Error bars represent SD from technical triplicates. (G) Western blot analysis of KDM2B-GFP pull outs on K562 cells expressing SCR or USP7 (#1 and #2) shRNAs using the indicated antibodies. Input (I, 1/90 th ), non-bound (NB, 1/90 th ), and bound (B, 1/3 rd ) as indicated. (H) Endogenous KDM2B and H2AK119ub ChIP-qPCRs on K562 cells expressing SCR of USP7 (#1 and #2) shRNAs. Error bars represent SD based on three independent experiments; statistical analysis was performed using Student's t test; ∗p < 0.05 and ∗∗p < 0.01. (I) Western blot analysis of THP-1 doxycycline-inducible CRISPR/Cas9 USP7 knockout cells treated with doxycycline for 3 days and subsequent culture for 4 days and stained with the indicated antibodies. (J) Endogenous KDM2B and H2AK119ub ChIP-qPCRs on doxycycline-treated cells (day 6). Error bars represent SD based on three independent experiments; statistical analysis was performed using Student's t test; ∗p < 0.05 and ∗∗p < 0.01. (K) Graphical abstract of effects of USP7 inhibition versus USP7 or TRIM27 knockdown. See also Figure S4 .

Article Snippet: K562 (AML) (Homo-sapiens) , ATCC , Cat# CCL-243.

Techniques: Knockdown, Western Blot, Expressing, ChIP-qPCR, CRISPR, Knock-Out, Staining, Inhibition

Journal: iScience

Article Title: The USP7-TRIM27 axis mediates non-canonical PRC1.1 function and is a druggable target in leukemia

doi: 10.1016/j.isci.2021.102435

Figure Lengend Snippet: USP7 inhibition leads to transcriptional changes of PRC1.1 target genes (A) Unsupervised clustering of RNA-seq data from three independent experiments where K562 cells were treated with DMSO or FT671 (10μM, 48 h) samples. (B) Venn diagram showing overlap of significantly up- and downregulated genes (Student's t test, p < 1x10 −6 ) with previously identified PRC1.1 target genes. (C) GSEA analysis showing enrichment score against ranked RNA-seq data. RNA-seq data were compared with indicated gene sets. (D) Gene ontology analyses of regulatory processes (RP) of all up- and downregulated genes in FT671-treated cells. (E) Gene ontology analyses of regulatory processes (RP) of up- and downregulated genes in FT671-treated cells that are also targeted by PRC1.1. (F) Screens shots of our ChIP-seq tracks ( van den Boom et al., 2016 ) for H2AK119ub, H3K27me3, PCGF1, PCGF2, PCGF4, CBX2, RING1A, RING1B, and KDM2B (all GFP-fusions) in K562 cells. ChIP-seq tracks for H3K4me3, H3K36me3, RNAPII, H3K27ac, EZH2, SUZ12 (all K562), and USP7 (CUTLL1) were downloaded from ENCODE/Broad. In addition, endogenous KDM2B, H2AK119ub, H3K27me3, and H3K4me3 in two primary AML patient cell samples are shown. See also Figure S5 .

Article Snippet: K562 (AML) (Homo-sapiens) , ATCC , Cat# CCL-243.

Techniques: Inhibition, RNA Sequencing, ChIP-sequencing

Journal: iScience

Article Title: The USP7-TRIM27 axis mediates non-canonical PRC1.1 function and is a druggable target in leukemia

doi: 10.1016/j.isci.2021.102435

Figure Lengend Snippet:

Article Snippet: K562 (AML) (Homo-sapiens) , ATCC , Cat# CCL-243.

Techniques: Control, Recombinant, Blocking Assay, Plasmid Preparation, Software, Purification, SYBR Green Assay