catalytic dead mutant  (Addgene inc)

Journal: iScience

Article Title: Targeting histone methylation to reprogram the transcriptional state that drives survival of drug-tolerant myeloid leukemia persisters

doi: 10.1016/j.isci.2022.105013

Figure Lengend Snippet:

Article Snippet: MSCV JMJD3 mutant (KDM6B-H1390A) , Addgene , Cat#21214.

Techniques: Recombinant, Modification, Protease Inhibitor, Gene Expression, Western Blot, Reverse Transcription, DNA Library Preparation, Control, Sequencing, Mutagenesis, Software

Journal: iScience

Article Title: Targeting histone methylation to reprogram the transcriptional state that drives survival of drug-tolerant myeloid leukemia persisters

doi: 10.1016/j.isci.2022.105013

Figure Lengend Snippet:

Article Snippet: MSCV-JMJD3 , Addgene , Cat#21212.

Techniques: Recombinant, Modification, Protease Inhibitor, Gene Expression, Western Blot, Reverse Transcription, DNA Library Preparation, Control, Sequencing, Mutagenesis, Software

Journal: Nucleic Acids Research

Article Title: KDM6B promotes PARthanatos via suppression of O 6 -methylguanine DNA methyltransferase repair and sustained checkpoint response

doi: 10.1093/nar/gkac471

Figure Lengend Snippet: Identification of KDM6B as a key regulator of PARP-1-dependent cell death. ( A ) The scheme of the CRISPR screening. ( B ) Top 18 hits including KDM6B were identified from the screening. Red, >105 000 reads. Pink, > 200 reads. Purple, >10 reads. Gray, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$ \ge$\end{document} 1 read. ( C ) Genotyping of KDM6B KO2 HeLa cells. ( D ) Immunoblot analysis of scrambled control (SC), KDM6B KO, and C-terminal WT-KDM6B (WT-C) as well as its H1390A mutant (mut-C) rescue HeLa cells. Numbers indicate the signal intensity. ( E and F ) Representative cell death images in SC, KDM6B KO2, and rescued HeLa cells 24 h after the treatment with DMSO or MNNG (50 μM, 15 min) (E). PI-positive cells are quantified in (F) (mean ± SEM, n = 4–7). Scale bar, 20 μm. PI/H, propidium iodide/Hoechst staining. TL, transmission light. **** P < 0.0001 by two-way ANOVA Sidak's multiple comparisons test. ( G and H ) Representative colony survival in SC, KDM6B KO2, and rescued HeLa cells treated with vehicle, MNNG (2 μM), TMZ (250 μM), Cyclophosphamide (CP, 500 μM), and Carmustine (25 μM) for 10 days (G). Colony numbers are quantified in H (mean ± SEM, n = 2–12). * P < 0.05; *** P < 0.001; **** P < 0.0001 versus DMSO by two-way ANOVA Tukey's multiple comparisons test.

Article Snippet: Full-length and C-terminal truncated KDM6B cDNA was amplified by PCR from KDM6B plasmids (Addgene, #21212 and #21214) and subcloned into pLVX-Ubc-FLAG vector.

Techniques: CRISPR, Western Blot, Control, Mutagenesis, Staining, Transmission Assay

Journal: Nucleic Acids Research

Article Title: KDM6B promotes PARthanatos via suppression of O 6 -methylguanine DNA methyltransferase repair and sustained checkpoint response

doi: 10.1093/nar/gkac471

Figure Lengend Snippet: KDM6B regulates PARP-1 activation and MGMT expression. ( A and B ) Volcano plots of KDM6B target genes in HeLa cells. Log 2 FC represents the fold change of mRNA expression in SC/KDM6B KO. n = 2. ( C ) Venn diagram of KDM6B rescued target genes in HeLa cells. ( D ) Immunoblot analysis of PAR and DNA damage-related proteins in SC and KDM6B KO2 HeLa cells treated with vehicle (−) or MNNG for indicated time. ( E ) RT-qPCR analysis of indicated genes in SC and KDM6B KO2 HeLa cells (mean ± SEM, n = 3–6). ** P < 0.01 by unpaired two-tailed Student's t test. ( F ) RNA-seq analysis of MGMT expression in SC, KDM6B KO2, and rescued HeLa cells (mean ± SEM, n = 4). **** P < 0.0001 by one-way ANOVA Sidak's multiple comparisons test. ( G ) Immunoblot analysis of MGMT in SC, KDM6B KO2, and rescued HeLa cells. ( H ) RT-qPCR analysis of MGMT expression in HeLa cells expressing EV, WT KDM6B, or catalytically mutant (mut) KDM6B (mean ± SEM, n = 6–12). * P < 0.05; **** P < 0.0001 vs . EV by one-way ANOVA Dunnett's multiple comparisons test. ( I ) Immunoblot analysis of indicated proteins in HeLa cells expressing full-length WT or H1390A KDM6B. ( J ) Representative KDM6B and MGMT immunostaining images in HeLa cells expressing WT or catalytically mutant KDM6B. Scale bar, 20 μm. ( K ) Immunoblot analysis of MGMT in HeLa cells treated with or without GSK-J4 for 72 h. ( L and M ) Representative DNA PAGE gels of methylated and unmethylated MGMT promoters in WT and KDM6B KO2 HeLa cells (L). DNA intensity is quantified in M (mean ± SEM, n = 4). **** P < 0.0001 by two-way ANOVA Sidak's multiple comparisons test. ( N ) In vitro MGMT activity assay. The assay strategy is shown on the top. Immunoblot analysis of biotin is shown at the bottom. Numbers indicate the signal intensity.

Article Snippet: Full-length and C-terminal truncated KDM6B cDNA was amplified by PCR from KDM6B plasmids (Addgene, #21212 and #21214) and subcloned into pLVX-Ubc-FLAG vector.

Techniques: Activation Assay, Expressing, Western Blot, Quantitative RT-PCR, Two Tailed Test, RNA Sequencing, Mutagenesis, Immunostaining, Methylation, In Vitro, Activity Assay

Journal: Nucleic Acids Research

Article Title: KDM6B promotes PARthanatos via suppression of O 6 -methylguanine DNA methyltransferase repair and sustained checkpoint response

doi: 10.1093/nar/gkac471

Figure Lengend Snippet: Pharmacological inhibition or deletion of MGMT sensitizes KDM6BKO cells to alkylating agents. ( A ) Immunoblot analysis of MGMT in SC and KDM6B KO2 HeLa cells 6 h after the treatment of MNNG (50 μM, 15 min) and/or BG (200 μM). ( B and C ) Representative cell death images in SC and KDM6B KO2 HeLa cells 72 h after treatment with MNNG and/or BG (B). PI-positive cells are quantified in C (mean ± SEM, n = 3). **** P < 0.0001 by two-way ANOVA Tukey's multiple comparisons test. ns, not significant. ( D ) Immunoblot analysis of MGMT in SC, KDM6B KO2, MGMT KO, and KDM6B/MGMT DKO HeLa cells 4 h after MNNG treatment. Numbers indicate the signal intensity. ( E and F ) Representative cell images in SC, KDM6B KO2, MGMT KO, KDM6B/MGMT DKO HeLa cells 24 h after MNNG treatment (E). Cell death is quantified in F (mean ± SEM, n = 3). **** P < 0.0001 by two-way ANOVA Sidak's multiple comparisons test. ( G, H ) Representative images (G) and quantification (H) of AIF nuclear translocation in SC and KDM6B KO cells in the presence or absence of BG (200 μM, pretreatment 24 h) and PARP inhibitor Olaparib (10 μM, 30 min pretreatment) at 4 h post MNNG (50 μM, 15 min) treatment. Red, AIF staining; Blue, DAPI staining; Purple, overlay of AIF and DAPI in the nuclei (mean ± SEM, n = 5). **** P < 0.0001 by two-way ANOVA Tukey's multiple comparisons test. Scale bar, 10 μm.

Article Snippet: Full-length and C-terminal truncated KDM6B cDNA was amplified by PCR from KDM6B plasmids (Addgene, #21212 and #21214) and subcloned into pLVX-Ubc-FLAG vector.

Techniques: Inhibition, Western Blot, Translocation Assay, Staining

Journal: Nucleic Acids Research

Article Title: KDM6B promotes PARthanatos via suppression of O 6 -methylguanine DNA methyltransferase repair and sustained checkpoint response

doi: 10.1093/nar/gkac471

Figure Lengend Snippet: MGMT regulates O 6 MeG levels and O 6 MeG-triggered PARP-1 activation. (A and B) Representative images of O 6 MeG staining ( A ) and quantification ( B ) in SC, KDM6B-KO2, MGMT (MT) KO, KDM6B/MGMT DKO cells with or without MGMT inhibitor BG (200 μM) or PARP inhibitor Olaparib (10 μM) at 0 min, 2 h and 6 h after MNNG treatment (25 μM, 15 min). * P < 0.05, *** P < 0.001, **** P < 0.0001 by one-way ANOVA Sidak's multiple comparisons test. Scale bar, 20 μm. ( C ) Immunoblot analysis of PARP-1 activation and γH2AX in SC and MGMT KO HeLa cells treated with vehicle (−) or MNNG (25 μM, 15 min) for indicated time. ( D ) Immunoblot analysis of PARP-1 activation and γH2AX in SC, KDM6B KO2, MGMT KO, KDM6B/MGMT DKO HeLa cells treated with vehicle (−) or MNNG (50 μM, 15 min) for indicated time. Numbers on the blot indicate the relative signal intensity.

Article Snippet: Full-length and C-terminal truncated KDM6B cDNA was amplified by PCR from KDM6B plasmids (Addgene, #21212 and #21214) and subcloned into pLVX-Ubc-FLAG vector.

Techniques: Activation Assay, Staining, Western Blot

Journal: Nucleic Acids Research

Article Title: KDM6B promotes PARthanatos via suppression of O 6 -methylguanine DNA methyltransferase repair and sustained checkpoint response

doi: 10.1093/nar/gkac471

Figure Lengend Snippet: Cell cycle distribution in SC and KDM6B KO cells following alkylating agent MNNG treatment. Cells were treated with 50 μM MNNG for 15 min, stained with EdU/PI, and analyzed by flow cytometry at 2, 4, 6, 8, 15 and 22 h post the treatment. ( A ) Representative flow cytometry images. ( B ) Cell cycle distribution was quantified (mean ± SEM, n = 3). **** P < 0.0001 vs. identical cell cycle in DMSO group, # P < 0.0001 versus S phase in KDM6B KO cells at the identical timepoint, by two-way ANOVA Tukey's multiple comparisons test.

Article Snippet: Full-length and C-terminal truncated KDM6B cDNA was amplified by PCR from KDM6B plasmids (Addgene, #21212 and #21214) and subcloned into pLVX-Ubc-FLAG vector.

Techniques: Staining, Flow Cytometry

Journal: Nucleic Acids Research

Article Title: KDM6B promotes PARthanatos via suppression of O 6 -methylguanine DNA methyltransferase repair and sustained checkpoint response

doi: 10.1093/nar/gkac471

Figure Lengend Snippet: KDM6B KO promotes sustained Chk1 activation for DNA repair following alkylating agent treatment. ( A ) Immunoblot analysis of checkpoint response and DNA damage in SC and KDM6B KO2 HeLa cells 0–6 h after MNNG treatment. ( B and C ) Effects of MGMT and MGMT C145A mutant overexpression (B) and MGMT KO (C) on checkpoint response in SC and KDM6B KO HeLa cells 1 h and 4 h after MNNG treatment. Asterisk indicates endogenous MGMT and the top band in the MGMT blot designates ectopic MGMT (B). ( D ) Effects of checkpoint inhibition by GDC0575 (50 nM) on checkpoint response and DNA damage in SC, KDM6B KO2, and KDM6B/MGMT DKO HeLa cells 1 h and 6 h after MNNG treatment. ( E and F ) Effects of checkpoint inhibition by GDC0575 (50 nM) on MNNG-induced cell death in SC, KDM6B KO2, and KDM6B/MGMT DKO HeLa cells 24 h after MNNG treatment. Representative cell death images are in SC, KDM6B KO2, KDM6B/MGMT DKO HeLa cells 24 h after MNNG treatment (E). Cell death is quantified in F (mean ± SEM, n = 3). **** P < 0.0001 by two-way ANOVA Tukey's multiple comparisons test. ( G ) Immunoblot analysis of checkpoint response in SC and KDM6B KO2 HeLa cells 0–6 h after hydroxyurea (HU, 2 mM) treatment.

Article Snippet: Full-length and C-terminal truncated KDM6B cDNA was amplified by PCR from KDM6B plasmids (Addgene, #21212 and #21214) and subcloned into pLVX-Ubc-FLAG vector.

Techniques: Activation Assay, Western Blot, Mutagenesis, Over Expression, Inhibition

Journal: Nucleic Acids Research

Article Title: KDM6B promotes PARthanatos via suppression of O 6 -methylguanine DNA methyltransferase repair and sustained checkpoint response

doi: 10.1093/nar/gkac471

Figure Lengend Snippet: XRCC1 regulates MNNG-induced DNA damage and cell death. ( A ) Immunoblot analysis of XRCC1 overexpression (OE) and DNA damage in HeLa cells 4 h after MNNG treatment (50 μM, 15 min). SC, scrambled control. ( B ) Immunoblot analysis of XRCC1 KO (mixed clones) and DNA damage in HeLa cells 4 h after MNNG treatment. ( C ) Immunoblot analysis of XRCC1 expression and DNA damage in SC, KDM6B KO and KDM6B/XRCC1 (6B/XR) DKO (mixed clones) HeLa cells 4 h after MNNG treatment. ( D and E ) Effects of XRCC1 on MNNG-induced cell death in SC, XRCC1 OE, XRCC1 KO, KDM6B KO, and KDM6B/XRCC1 (6B/XR) DKO HeLa cells 24 or 72 h after MNNG treatment. Representative cell death images are shown in (D). Cell death is quantified in (E) (mean ± SEM, n = 3). **** P < 0.0001 by two-way ANOVA Sidak's multiple comparisons test.

Article Snippet: Full-length and C-terminal truncated KDM6B cDNA was amplified by PCR from KDM6B plasmids (Addgene, #21212 and #21214) and subcloned into pLVX-Ubc-FLAG vector.

Techniques: Western Blot, Over Expression, Control, Clone Assay, Expressing

Journal: Nucleic Acids Research

Article Title: KDM6B promotes PARthanatos via suppression of O 6 -methylguanine DNA methyltransferase repair and sustained checkpoint response

doi: 10.1093/nar/gkac471

Figure Lengend Snippet: KDM6B is a key cell death regulator of PARthanatos induced by alkylating agents. KDM6B regulates two sequential DNA repair processes including MGMT direct DNA repair and checkpoint associated repair. Loss of KDM6B on one hand enhances MGMT direct DNA repair to promote cell survival, on the other hand triggers sustained Chk1 activation and increases checkpoint associated DNA repair to fix DNA evading from the first step MGMT repair process leading to cell survival. High levels of KDM6B suppress both DNA repair processes leading to cell death. Blocking MGMT and checkpoint associated repair re-sensitizes cancer cells to alkylating agents.

Article Snippet: Full-length and C-terminal truncated KDM6B cDNA was amplified by PCR from KDM6B plasmids (Addgene, #21212 and #21214) and subcloned into pLVX-Ubc-FLAG vector.

Techniques: Activation Assay, Blocking Assay

Journal: Nature Communications

Article Title: KDM6B promotes activation of the oncogenic CDK4/6-pRB-E2F pathway by maintaining enhancer activity in MYCN-amplified neuroblastoma

doi: 10.1038/s41467-021-27502-2

Figure Lengend Snippet: a – c The expression of KDM6B , KDM6A , and UTY in normal human trunk neural crest (GSE14340) and four different neuroblastoma cohorts (Versteeg GSE16476, Delattre GSE14880, Hiyama GSE16237, Lastowska GSE13136). y-Axis represents the normalized log2 expression value. n = 5 for NCC, n = 88 for Versteeg, n = 64 for Delattre, n = 51 for Hiyama, n = 30 for Lastowska. Data are represented as mean ± SD. **** p < 0.0001, two-tailed, unpaired t test. d – f The expression of KDM6B , KDM6A , and UTY in three different neuroblastoma RNA-seq cohorts. The RNA-seq data of St Jude ( d ) was downloaded from https://pecan.stjude.cloud . The RNA-seq data of TARGET ( e ) and SEQC ( f ) datasets were downloaded from R2 ( https://hgserver1.amc.nl/cgi-bin/r2/main.cgi ). y -Axis represents the Fragment Per Kilobase of transcript per Million (FPKM) mapped reads. n = 160 for St Jude dataset, n = 161 for TARGET dataset, n = 498 for SEQC dataset. Data are represented as mean ± SD. **** p < 0.0001, two-tailed, unpaired t test. g The epigenetic landscapes consisting of histone marks and transcription factor binding distinguish KDM6B from KDM6A in primary neuroblastoma tissues with MYCN amplification or without MYCN amplification ( https://pecan.stjude.cloud/proteinpaint/study/mycn_nbl_2018 ). h Crystal violet staining of colonies after BE2C and SK-N-AS cells were transfected with four different siRNA oligos to knockdown KDM6B for 7 days. siCtrl = siRNA control oligo, NT = no treatment. i Western blot analysis with indicated antibodies to assess MYCN or C-MYC expression after 3-day transfection of 4 different siRNA to knockdown KDM6B in BE2C and SK-N-AS. The blots are representative of three independent experiments. j BE2C and MSCV-KDM6B overexpressing (KDM6B-OE) BE2C cells were transfected with siRNA control (siCtrl) and siRNA targeting the endogenous 3′ untranslated region (3′UTR) of KDM6B (siKDM6B-3′UTR). Four days later, cells were stained with crystal violet. k Quantification of cell density of each group ( n = 3) using imageJ. Data are represented as mean ± SD. Shown are individual biological replicates. **** p < 0.0001, two-tailed, unpaired t test. l BE2C and MSCV-KDM6B overexpressing (KDM6B-OE) BE2C cells were transfected with siRNA control (siCtrl) and siRNA siKDM6B-3′UTR. Three days later, cells were subject to immunoblotting with indicated antibodies. The blots are representative of three independent experiments. Source data are provided as a “Source data” file.

Article Snippet: To overexpression KDM6B, MSCV-JMJD3 (Addgene#21212) were packaged into retroviral particles, which were transduced into BE2C cells and selected with puromycin for stable expression.

Techniques: Expressing, Two Tailed Test, RNA Sequencing, Binding Assay, Amplification, Staining, Transfection, Knockdown, Control, Western Blot

Journal: Nature Communications

Article Title: KDM6B promotes activation of the oncogenic CDK4/6-pRB-E2F pathway by maintaining enhancer activity in MYCN-amplified neuroblastoma

doi: 10.1038/s41467-021-27502-2

Figure Lengend Snippet: a Quantitative comparison of all chemical and genetic perturbation gene sets ( n = 3403) from the MSigDB by gene set enrichment analysis (GSEA) for increased (left) and reduced (right) expression of global genes caused by KDM6B knockdown. Data are presented as a scatterplot of normalized p value (right y -axis)/false discovery q value (left y -axis) vs. normalized enrichment score (NES) ( x -axis) for each evaluated gene set. The gene sets circled in red color indicate cell cycle, pRB-E2F, and MYC pathway gene sets. p Value calculated by one-sided Fisher’s exact test. The FDR is calculated by comparing the distribution of normalized enrichment scores from many different genesets. b Two examples of GSEA from a show that genes downregulated by depletion of KDM6B are enriched with Rb1 knockout and E2F targets. p Value calculated by one-sided Fisher’s exact test. The FDR is calculated by comparing the distribution of normalized enrichment scores from many different genesets. c Heatmap shows the gene list from the E2F targets ( b ). d – f Similar analysis for GSK-J4 treatment as shown in ( a – c ). Source data are provided as a “Source data” file.

Article Snippet: To overexpression KDM6B, MSCV-JMJD3 (Addgene#21212) were packaged into retroviral particles, which were transduced into BE2C cells and selected with puromycin for stable expression.

Techniques: Comparison, Expressing, Knockdown, Knock-Out

Journal: Nature Communications

Article Title: KDM6B promotes activation of the oncogenic CDK4/6-pRB-E2F pathway by maintaining enhancer activity in MYCN-amplified neuroblastoma

doi: 10.1038/s41467-021-27502-2

Figure Lengend Snippet: Transcription factor binding motif enrichment for KDM6B target genes (top 15).

Article Snippet: To overexpression KDM6B, MSCV-JMJD3 (Addgene#21212) were packaged into retroviral particles, which were transduced into BE2C cells and selected with puromycin for stable expression.

Techniques: Binding Assay

Journal: Nature Communications

Article Title: KDM6B promotes activation of the oncogenic CDK4/6-pRB-E2F pathway by maintaining enhancer activity in MYCN-amplified neuroblastoma

doi: 10.1038/s41467-021-27502-2

Figure Lengend Snippet: a ATAC-seq was performed after BE2C cells were treated with 2.5 μM of GSK-J4 for 24 and 48 h. Summary of peak calling number of ATAC-seq (cut-off p < 0.05, log2 fold change ≧ 0.5) including the upregulated and downregulated nucleosome free regions, and the annotated locations of the peaks at defined promoter and enhancer regions. p Value obtained by one-sided Empirical Bayes Statistics test. b Snapshot of AKT1 locus using Integrative Genomic Viewer (IGV) for ATAC-seq, H3K27Ac ChIP-seq, and RNA-seq. The ATAC-seq analysis shows the downregulation of two peaks at the 5′ promoter region of AKT1 and one peak at the non-coding RNA LINC00638 . The RNA-seq results showed that the AKT1 transcript was downregulated by 48 h of GSK-J4 treatment and KDM6B knockdown. H3K27Ac in BE2C cells was referenced to GSM2113518 . c Snapshot of E2F8 lo c us using Integrative Genomic Viewer (IGV) for ATAC-seq, H3K27Ac ChIP-seq, and RNA-seq. The ATAC-seq shows the downregulation of three peaks at the enhancer region of E2F8 , next to the NAV2 gene locus. The RNA-seq results showed that E2F8 transcript was downregulated by KDM6B inhibition while the adjacent NAV2 expression is barely detectable. d Cartoon indicates the rationale of footprinting analysis. The DNA motifs bound by transcription factors such as E2F1 protect the cut from transposase Tn5, while the adjacent open chromatin gives rise to a high signal of nucleosome free region after ATAC-seq analysis. After GSK-J4 treatment, the open chromatin was repressed and consequently reducing the reads of free DNA. e Footprinting plot shows the reduction of open chromatin at predicted binding motifs of E2Fs and MYC after BE2C cells were treated with GSK-J4 for 24 and 48 h. Source data are provided as a “Source data” file.

Article Snippet: To overexpression KDM6B, MSCV-JMJD3 (Addgene#21212) were packaged into retroviral particles, which were transduced into BE2C cells and selected with puromycin for stable expression.

Techniques: ChIP-sequencing, RNA Sequencing, Knockdown, Inhibition, Expressing, Footprinting, Binding Assay

Journal: Nature Communications

Article Title: KDM6B promotes activation of the oncogenic CDK4/6-pRB-E2F pathway by maintaining enhancer activity in MYCN-amplified neuroblastoma

doi: 10.1038/s41467-021-27502-2

Figure Lengend Snippet: a , b Heatmap indicating the CUT&Tag signal intensity of H3K27me3 ( a ) and H3K4me1 ( b ) around differentially expressed genes (from TSS-5kb to TES + 5 kb). CUT&Tag experiments were done with two biological replicates in KDM6B knockdown and GSK-J4 treatment, respectively. Differentially expressed genes were identified by cutoffs (log2FC > 0.5) from RNA-seq. TSS transcription start site, TES transcription end site. The p -value is obtained from the limma moderated t -statistic, Benjamini and Hochberg’s method used to control the false discovery rate. c The signals of H3K27me3 and H3K4me1 (CUT&Tag), H3K27Ac (ChIP-seq) , and ATAC-seq between the genomic locus of MYCN , GACAT3 , and FAM49A , snapshot using IGV program. d , e Motif analysis of H3K27me3 ( d ) and H3K4me1 ( e ) peaks by using HOMER known motif search. Top ten most significant motifs from each sub-group were visualized as a heatmap. The heatmap scale indicates −log 10 ( p value). p -Value obtained by HOMER scoring function of one-sided cumulative hypergeometric distribution. f The Hi–C data of BE2C neuroblastoma cells (data extracted from the St Jude Cloud) showing that MYCN and its adjacent FAM49A locus reside in the same topologically associated domain (TAD). The Arc indicates the chromatin interactions, which were generated from Jurkat ChIA-PET SMC1 (Mango) ( https://proteinpaint.stjude.org/ ). Source data are provided as a “Source data” file.

Article Snippet: To overexpression KDM6B, MSCV-JMJD3 (Addgene#21212) were packaged into retroviral particles, which were transduced into BE2C cells and selected with puromycin for stable expression.

Techniques: Knockdown, RNA Sequencing, Control, ChIP-sequencing, Hi-C, Generated, ChIA Pet Assay

Journal: Nature Communications

Article Title: KDM6B promotes activation of the oncogenic CDK4/6-pRB-E2F pathway by maintaining enhancer activity in MYCN-amplified neuroblastoma

doi: 10.1038/s41467-021-27502-2

Figure Lengend Snippet: a Genes downregulated by KDM6B knockdown were compared with gene profiles induced with chemical compounds from Library of Integrated Network-Based Cellular Signatures (LINCS) . The top chemical signature hits, which are basically overrepresented by palbociclib, are highlighted in pink dots. The y -axis indicates the combined score. The x -axis indicates adjusted p value. n = 18,947 for gene signatures. p Value is calculated from one-sided Fisher exact test. Combined score is computed by taking the log of p -value from the Fisher exact test and multiplying that by the z -score of the deviation from the expected rank. b Same analysis performed as in ( a ) for GSK-J4 treatment. n = 22231 for gene signatures. c The 89-gene signature derived from palbociclib treatment of 4 neuroblastoma cell lines was included in gene sets for GSEA analysis of KDM6B knockdown. p Value calculated by one-sided Fisher’s exact test. The FDR is calculated by comparing the distribution of normalized enrichment scores from many different genesets. d The 89-gene signature derived from palbociclib treatment was included in gene sets for GSEA analysis of GSK-J4. p Value calculated by one-sided Fisher’s exact test. The FDR is calculated by comparing the distribution of normalized enrichment scores from many different genesets. e BE2C cells were seeded with low numbers in six-well plate and treated with different concentrations of GSK-J4 or/and palbociclib for 10 days. The cell colonies were stained with crystal violet. f Bliss index for the combination of GSK-J4 and palbociclib. Positive scores indicate synergy, negative scores indicate antagonism. g Western blot to assess the expression of CDK4 and CDK6 that were transduced into BE2C cells. The blots are representative of three independent experiments. h The BE2C parental and CDK4/6 overexpressing cells were seeded with low numbers in six-well plate and treated with different concentrations of GSK-J4 or palbociclib for 8 days. The cell colonies were stained with crystal violet. i , j BE2C and CDK4 or CDK6 overexpressing BE2C cells (BE2C-CDK4-OE, BE2C-CDK6-OE) were treated with different concentrations of palbociclib ( i ) or GSK-J4 ( j ). EC50 was calculated using Prestoblue assay. n = 4 for each dose. Data are represented as mean ± SD. k Western blot to assess the expression of pRB after inducible knockout in BE2C cells. The blots are representative of three independent experiments. l Photos taken under microscope (10×) show Rb1 knockout leads to resistance to palbociclib. Scale bar = 100 μM. The photos are representative of three independent experiments. m The BE2C wildtype and Rb1 knockout cells were seeded with low numbers in six-well plate and treated with different concentrations of GSK-J4 or/and palbociclib for 8 days. The cell colonies were stained with crystal violet. Source data are provided as a “Source data” file.

Article Snippet: To overexpression KDM6B, MSCV-JMJD3 (Addgene#21212) were packaged into retroviral particles, which were transduced into BE2C cells and selected with puromycin for stable expression.

Techniques: Knockdown, Derivative Assay, Staining, Western Blot, Expressing, Prestoblue Assay, Knock-Out, Microscopy

Journal: Nature Communications

Article Title: KDM6B promotes activation of the oncogenic CDK4/6-pRB-E2F pathway by maintaining enhancer activity in MYCN-amplified neuroblastoma

doi: 10.1038/s41467-021-27502-2

Figure Lengend Snippet: a Upon stimulation by mitogens, KDM6B is recruited to chromatin to maintain the low levels of H3K27me3 at the distal regulatory enhancer regions marked by H3K4me1 overlapping with the CTCF-/BORIS-binding sites, which loops and physically interacts with E2F that binds at the promoter of target genes, together with associated transcriptional machinery including RNA polymerase II and mediators, driving the MYCN and E2F transcriptome. b When inhibited by CDK4/6 inhibitor, the inhibitory pRB complexes with HDAC to suppress gene transcription. When KDM6B is inhibited by GSK-J4, the H3K27me3 will accumulate at the distal regions to displace the H3K4me1 modifier KMT2 and evicts the transcription activators of promoter–enhancer, leading to the downregulation of MYCN and E2F transcriptome. c A network composed by MYCN, E2F, EZH2, and KDM6B regulates the cell proliferation and differentiation of neuroblastoma.

Article Snippet: To overexpression KDM6B, MSCV-JMJD3 (Addgene#21212) were packaged into retroviral particles, which were transduced into BE2C cells and selected with puromycin for stable expression.

Techniques: Binding Assay