Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: L3MBTL2 maintains MYCN-amplified neuroblastoma cell proliferation through silencing NRIP3 and BRME1 genes.

doi: 10.1111/gtc.13148

Figure Lengend Snippet: FIGURE 7 L3MBTL2-knockout reduces the level of H2AK119ub around the NRIP3 and BRME1 transcription start sites (TSSs). (a, b) Representative integrative genomics viewer (IGV) screenshots showing ChIP-seq-data-derived peaks of L3MBTL2, PCGF6, RING1B, H2AK119ub, H3K27me3, and MYCN on the NRIP1 and BRME1 loci. The blue boxes below each peak indicate peaks called using hiddenDomains with default parameters. The black boxes indicate the range used for ChIP-qPCR primer design. (c) Heatmap of PCGF6, MYCN, and H2AK119ub ChIP-seq signals in control (left) or L3MBTL2-knockout (right). PCGF6 ChIP-seq signal intensities in controls are sorted in descending order. (d) ChIP-qPCR showing changes in PCGF6, MYCN, and H2AK119ub binding around the NRIP3 and BRME1 promoter regions upon L3MBTL2 depletion. Numbers in parentheses indicate the primer sets used in Table S2. Data are presented as the mean ± SD, N = 3 (two-tailed Student's t-test).

Article Snippet: We employed the following primary antibodies: L3MBTL2 rabbit pAb (Active Motif), RING1B mouse mAb (Atsuta et al., 2004), PCGF6 rabbit pAb (Proteintech 24,103-1-AP), E2F6 rabbit pAb (LSBio LS-C352133), β-Tubulin mouse mAb (Boehringer Ingelheim, Ingelheim am Rhein, Germany), phospho-histone H2A.X (Ser139) mouse mAb (Millipore JBW301), ubiquityl-histone H2A (Lys119) rabbit mAb (CST, (D27C4) XP®, #8240), histone H3 rabbit pAb (Abcam ab1791, Bristol, United Kingdom), or DYKDDDDK Tag rabbit mAb (CST, #2368).

Techniques: Knock-Out, ChIP-sequencing, Derivative Assay, ChIP-qPCR, Control, Binding Assay, Two Tailed Test

Journal: The Journal of Clinical Investigation

Article Title: An epigenetic pathway regulates MHC-II expression and function in B cell lymphoma models

doi: 10.1172/JCI179703

Figure Lengend Snippet: ( A ) The protein levels of BAP1 in 4 uveal melanoma cell lines MP41, MP38, MP46, and MP65 were determined by Western blot. Representative blot from 2 biological repeats. ( B ) The Venn diagram shows the overlap between WT and catalytically dead BAP1 rescued genes in BAP1-null MP65 cell line. ( C ) The track example shows the expression levels of MHC-II cluster genes in MP65 BAP1 null cells restored by GFP, BAP1-WT, or BAP1-C91S. ( D ) The mRNA levels of MHC-II cluster genes were determined by real-time PCR in MP41, MP38, MP46, and MP65 cells. n = 3 technical replicates. Data are represented as mean ± SD. ( E ) Pathway analysis was performed with the genes that are selectively upregulated by WT BAP1 but not catalytically dead BAP1 in BAP1-KO HEK293T cells. ( F ) The mRNA levels of MHC-II cluster genes were determined by RNA-Seq in BAP1-WT and catalytic-dead KI HEK293T cells. BAP1 was depleted by CRISPR with 2 distinct sgRNAs in MDA-MB-231, KNS42, and MDA-MB-435s cell lines. ( G ) The protein levels of BAP1 were determined by Western blot. Representative blot from 2 biological repeats. ( H ) The mRNA levels of MHC-II cluster genes HLA-DMA , HLA-DMB , HLA-DPA1 , and HLA-DRB1 were determined by real-time PCR. n = 3 technical replicates. Data are represented as mean ± SD. ( I ) The track example shows the mRNA levels of MHC-II gene loci in BAP1-WT and BAP1-KO A20 cells. ( J ) The protein levels of cell-surface MHC-II molecule in BAP1-WT and -KO A20 cells were determined by FACS and analyzed by FlowJo software. ( K ) The protein levels of histone H2AK119Ub were determined by Western blot in both the WT A20 and 2 independent BAP1-KO clones. Representative blot from 2 biological repeats.

Article Snippet: H3K27me3 (catalog 9733), Histone H3 (catalog 4499S), H2AK119Ub (catalog 8240), IRF1 (catalog 8478S), RING1B (catalog 5694S), UTX (catalog 33510S), CD4 (catalog 25229S), MHC-II-FITC (catalog 42594S), MHC-II-APC (catalog 64776), and BAP1 (catalog 13271) antibodies were purchased from Cell Signaling Technology.

Techniques: Western Blot, Expressing, Real-time Polymerase Chain Reaction, RNA Sequencing, CRISPR, Software, Clone Assay

Journal: The Journal of Clinical Investigation

Article Title: An epigenetic pathway regulates MHC-II expression and function in B cell lymphoma models

doi: 10.1172/JCI179703

Figure Lengend Snippet: ( A ) The track example shows BAP1 occupancy at the MHC-II gene loci in MHC-II–positive cell line A20 cells and MHC-II–negative cell line LLC cells. ( B ) The heatmap shows the log 2 fold change of H2AK119Ub levels in BAP1-WT and 2 independent KO clones. All of the ChIP-Seq signals were centered on BAP1 peaks in BAP1-WT cells. ( C ) The track example shows the H2AK119Ub and H3K27me3 levels between BAP1-WT and -KO A20 cells (upper panel). The bottom panel shows the ATAC-Seq signal from both BAP1-WT and 2 independent KO clones at MHC-II gene loci. ( D ) Pathway analysis with the genes that are downregulated in BAP1-KO A20 cells. ( E ). The heatmap shows the log 2 fold change of UTX occupancy and H3K27me3 levels in BAP1-WT and -KO A20 cells centered on BAP1 peaks. ( F and G ) The track examples show the reduction of UTX occupancy and the increase of H3K27me3 levels at MHC-II cluster genes in BAP1-WT and -KO A20 cells.

Article Snippet: H3K27me3 (catalog 9733), Histone H3 (catalog 4499S), H2AK119Ub (catalog 8240), IRF1 (catalog 8478S), RING1B (catalog 5694S), UTX (catalog 33510S), CD4 (catalog 25229S), MHC-II-FITC (catalog 42594S), MHC-II-APC (catalog 64776), and BAP1 (catalog 13271) antibodies were purchased from Cell Signaling Technology.

Techniques: Clone Assay, ChIP-sequencing

Journal: The Journal of Clinical Investigation

Article Title: An epigenetic pathway regulates MHC-II expression and function in B cell lymphoma models

doi: 10.1172/JCI179703

Figure Lengend Snippet: ( A ) The MA plot (Bland-Altman plot) shows the up- or downregulated ATAC-Seq signal comparing BAP1-WT/KO cells. ( B ) The motif analysis with the downregulated ATAC-Seq peaks in BAP1-KO cells. ( C ) The heatmap shows the log 2 fold change of IRF1 chromatin binding in BAP1-WT and -KO cells. ( D ) The track example shows the chromatin occupancy of BAP1 and the protein levels of H2AK119Ub and H3K27me3 at the Irf1 gene locus. ( E ) The mRNA levels of Irf1 in A20 BAP1-WT and -KO cells were determined by RNA-Seq. ( F ) The protein levels of IRF1 in A20 BAP1-WT and -KO cells were determined by Western blot. Representative blot from 2 biological repeats. ( G ) The track example shows the chromatin occupancy of IRF1 at the Ciita promoter in both BAP1-WT and -KO cells. ( H ) The occupancy of IRF1 at the Ciita promoter region was quantified by ChIP-QPCR in BAP1-WT and -KO cells. n = 3 technical replicates. Data are represented as mean ± SD. ( I ) The track example shows the Ciita levels in BAP1-WT and -KO cells. ( J ) The track example shows the occupancy of CIITA at MHC-II cluster gene loci in BAP1-WT and -KO cells. ( K ) The Venn diagram shows the genes that are rescued by IRF1 in BAP1-KO cells. ( L ) The protein levels of MHC-II molecules in BAP1-WT, BAP1-KO, BAP1-KO-GFP, and BAP1-KO-Flag-IRF1 cells were determined by FACS analysis. ( M ) The pathway analysis with the total upregulated genes by IRF1 in A20 BAP1-KO cells. The BAP1-KO A20 cells were transduced with either GFP, WT BAP1, or catalytically dead BAP1. ( N ) The protein levels of BAP1 were determined by Western blot. Representative blot from 2 biological repeats. ( O ) The mRNA levels of Irf1 and Ciita were determined by real-time PCR. n = 3 technical replicates. Data are represented as mean ± SD.

Article Snippet: H3K27me3 (catalog 9733), Histone H3 (catalog 4499S), H2AK119Ub (catalog 8240), IRF1 (catalog 8478S), RING1B (catalog 5694S), UTX (catalog 33510S), CD4 (catalog 25229S), MHC-II-FITC (catalog 42594S), MHC-II-APC (catalog 64776), and BAP1 (catalog 13271) antibodies were purchased from Cell Signaling Technology.

Techniques: Binding Assay, RNA Sequencing, Western Blot, ChIP-qPCR, Transduction, Real-time Polymerase Chain Reaction

Journal: The Journal of Clinical Investigation

Article Title: An epigenetic pathway regulates MHC-II expression and function in B cell lymphoma models

doi: 10.1172/JCI179703

Figure Lengend Snippet: ( A ) The A20 BAP1-KO cells were treated with various concentrations of PRC1 inhibitor RB-3 for 6 days. The protein levels of H2AK119Ub and RING1B were determined by Western blot. Representative blot from 2 biological repeats. ( B ) The log 2 fold change heatmap shows the H2AK119Ub levels in A20 BAP1-KO cells treated with either DMSO or RB-3. ( C ) The A20 BAP1-WT or -KO cells were treated with either DMSO or RB-3 for 6 days. The gene-expression profiles for each condition were determined by RNA-Seq. ( D ) The Venn diagram shows the overlap between BAP1 targeted genes and RB-3 rescued genes. ( E ) The pathway analysis with the 860 BAP1 targeted genes that were restored by RB-3 treatment. ( F ) The A20 BAP1-WT cells were treated with DMSO for 6 days, and the BAP1-KO cells were treated with either DMSO or RB-3 (5 μM) for 6 days. The mRNA levels of MHC-II cluster genes in each treatment were determined by RNA-Seq. ( G ) The BAP1-KO A20 cells were treated with either DMSO or RB-3 (10 μM) for 6 days. The protein levels of MHC-II were determined by FACS analysis. ( H ) The A20 BAP1-WT cells were treated with DMSO for 6 days, and the BAP1-KO cells were treated with either DMSO or various concentrations of RB-3 for 6 days. The IRF1 protein levels were determined by Western blot. Representative blot from 2 biological repeats. ( I and J ) The mRNA levels of Irf1 ( I ) and Ciita mRNA ( J ) were determined by real-time PCR. n = 3 technical replicates. Data are represented as mean ± SD. ( K and L ) The BAP1-KO cells were treated with RB-3 (10 μM) or/and GSK126 (5 μM) for 6 days. The protein levels of H2AK119Ub and H3K27me3 were determined by Western blot. ( K ) Representative blot from 2 biological repeats. The protein levels of cell surface MHC-II molecules were determined by FACS analysis ( L ).

Article Snippet: H3K27me3 (catalog 9733), Histone H3 (catalog 4499S), H2AK119Ub (catalog 8240), IRF1 (catalog 8478S), RING1B (catalog 5694S), UTX (catalog 33510S), CD4 (catalog 25229S), MHC-II-FITC (catalog 42594S), MHC-II-APC (catalog 64776), and BAP1 (catalog 13271) antibodies were purchased from Cell Signaling Technology.

Techniques: Western Blot, Gene Expression, RNA Sequencing, Real-time Polymerase Chain Reaction

Journal: The Journal of Clinical Investigation

Article Title: An epigenetic pathway regulates MHC-II expression and function in B cell lymphoma models

doi: 10.1172/JCI179703

Figure Lengend Snippet: ( A ) The cell growth rate of A20 BAP1-WT and -KO cells was determined by cell counting assay. ( B ) 2 × 10 6 of BAP1-WT and BAP1-KO clones were inoculated into the right flank of 6-week-old nude mice. The tumor size was measured every day for 1 week after inoculation. ( C ) Images of representative tumor tissue samples from each mouse were taken at the end of the experiment. ( D ) 5 × 10 6 of BAP1-WT and BAP1-KO clones were inoculated into the right flank of 6-week-old BALB/c mice. The tumor size was measured every day for 2 weeks after the inoculation. Data are represented as mean ± SEM. A 2-tailed unpaired Student’s t test was used for statistical analysis. ** P < 0.01; * P < 0.05. ( E ) When each tumor reached 1 cm 3 , the mouse was euthanized, and the survival probability was shown. ( F ) Tumor tissue from both BAP1-WT and BAP1-KO was harvested and subjected to scRNA-Seq. The UMAP analysis identifies different clusters of cell populations within the tumor tissue based on the gene-expression profiles. ( G ) The bar plot shows the percentage of each cell cluster in the tumor tissues. ( H ) The violin plot shows the expression levels of Irf1 , Ciita , and MHC-II cluster genes in BAP1-WT and -KO tumor cell population. ( I ) Immune cells from the first clustering were isolated (T cells and macrophages) and reclustered with UMAP algorithm. ( J ) The percentages of each type of immune cells were calculated in BAP1-WT and -KO A20 tumors and are shown in the bar plot. ( K ) The IHC staining was performed with CD4-specific antibody in BAP1-WT and -KO tumor tissues. Scale bars: 10 μm.

Article Snippet: H3K27me3 (catalog 9733), Histone H3 (catalog 4499S), H2AK119Ub (catalog 8240), IRF1 (catalog 8478S), RING1B (catalog 5694S), UTX (catalog 33510S), CD4 (catalog 25229S), MHC-II-FITC (catalog 42594S), MHC-II-APC (catalog 64776), and BAP1 (catalog 13271) antibodies were purchased from Cell Signaling Technology.

Techniques: Cell Counting, Clone Assay, Gene Expression, Expressing, Isolation, Immunohistochemistry

Journal: The Journal of Clinical Investigation

Article Title: An epigenetic pathway regulates MHC-II expression and function in B cell lymphoma models

doi: 10.1172/JCI179703

Figure Lengend Snippet: ( A ) The box plot shows the Spearman’s correlation between the expression levels of human MHC-II genes and BAP1, EZH2, RNF2, PCGF6, or KDM2B in B cell lymphoma patients (TCGA, PanCancer Atlas). ( B – E ) The scatter plot shows the positive correlation between HLA-DMA and ( B ), EZH2 ( C ), PCGF6 ( D ), or KDM2B ( E ) in B cell lymphoma patient samples. ( F ) The schematic model shows how the BAP1 complex and the PRC1 establish a dynamic epigenetic state at MHC-II cluster gene loci by precisely regulating histone H2AK119Ub level and IRF1/CIITA axis which is critical for MHC-II gene expression. ( G ) The epigenetic balance between BAP1 and PRC1 plays an essential role in “hot” and “cold” tumor switching and tumor immune response.

Article Snippet: H3K27me3 (catalog 9733), Histone H3 (catalog 4499S), H2AK119Ub (catalog 8240), IRF1 (catalog 8478S), RING1B (catalog 5694S), UTX (catalog 33510S), CD4 (catalog 25229S), MHC-II-FITC (catalog 42594S), MHC-II-APC (catalog 64776), and BAP1 (catalog 13271) antibodies were purchased from Cell Signaling Technology.

Techniques: Expressing, Gene Expression

Journal: The Journal of Clinical Investigation

Article Title: An epigenetic pathway regulates MHC-II expression and function in B cell lymphoma models

doi: 10.1172/JCI179703

Figure Lengend Snippet: ( A ) The A20 BAP1-KO cells were treated with various concentrations of PRC1 inhibitor RB-3 for 6 days. The protein levels of H2AK119Ub and RING1B were determined by Western blot. Representative blot from 2 biological repeats. ( B ) The log 2 fold change heatmap shows the H2AK119Ub levels in A20 BAP1-KO cells treated with either DMSO or RB-3. ( C ) The A20 BAP1-WT or -KO cells were treated with either DMSO or RB-3 for 6 days. The gene-expression profiles for each condition were determined by RNA-Seq. ( D ) The Venn diagram shows the overlap between BAP1 targeted genes and RB-3 rescued genes. ( E ) The pathway analysis with the 860 BAP1 targeted genes that were restored by RB-3 treatment. ( F ) The A20 BAP1-WT cells were treated with DMSO for 6 days, and the BAP1-KO cells were treated with either DMSO or RB-3 (5 μM) for 6 days. The mRNA levels of MHC-II cluster genes in each treatment were determined by RNA-Seq. ( G ) The BAP1-KO A20 cells were treated with either DMSO or RB-3 (10 μM) for 6 days. The protein levels of MHC-II were determined by FACS analysis. ( H ) The A20 BAP1-WT cells were treated with DMSO for 6 days, and the BAP1-KO cells were treated with either DMSO or various concentrations of RB-3 for 6 days. The IRF1 protein levels were determined by Western blot. Representative blot from 2 biological repeats. ( I and J ) The mRNA levels of Irf1 ( I ) and Ciita mRNA ( J ) were determined by real-time PCR. n = 3 technical replicates. Data are represented as mean ± SD. ( K and L ) The BAP1-KO cells were treated with RB-3 (10 μM) or/and GSK126 (5 μM) for 6 days. The protein levels of H2AK119Ub and H3K27me3 were determined by Western blot. ( K ) Representative blot from 2 biological repeats. The protein levels of cell surface MHC-II molecules were determined by FACS analysis ( L ).

Article Snippet: H3K27me3 (catalog 9733), Histone H3 (catalog 4499S), H2AK119Ub (catalog 8240), IRF1 (catalog 8478S), RING1B (catalog 5694S), UTX (catalog 33510S), CD4 (catalog 25229S), MHC-II-FITC (catalog 42594S), MHC-II-APC (catalog 64776), and BAP1 (catalog 13271) antibodies were purchased from Cell Signaling Technology.

Techniques: Western Blot, Gene Expression, RNA Sequencing, Real-time Polymerase Chain Reaction

Journal: The Journal of Clinical Investigation

Article Title: An epigenetic pathway regulates MHC-II expression and function in B cell lymphoma models

doi: 10.1172/JCI179703

Figure Lengend Snippet: ( A ) The MA plot (Bland-Altman plot) shows the up- or downregulated ATAC-Seq signal comparing BAP1-WT/KO cells. ( B ) The motif analysis with the downregulated ATAC-Seq peaks in BAP1-KO cells. ( C ) The heatmap shows the log 2 fold change of IRF1 chromatin binding in BAP1-WT and -KO cells. ( D ) The track example shows the chromatin occupancy of BAP1 and the protein levels of H2AK119Ub and H3K27me3 at the Irf1 gene locus. ( E ) The mRNA levels of Irf1 in A20 BAP1-WT and -KO cells were determined by RNA-Seq. ( F ) The protein levels of IRF1 in A20 BAP1-WT and -KO cells were determined by Western blot. Representative blot from 2 biological repeats. ( G ) The track example shows the chromatin occupancy of IRF1 at the Ciita promoter in both BAP1-WT and -KO cells. ( H ) The occupancy of IRF1 at the Ciita promoter region was quantified by ChIP-QPCR in BAP1-WT and -KO cells. n = 3 technical replicates. Data are represented as mean ± SD. ( I ) The track example shows the Ciita levels in BAP1-WT and -KO cells. ( J ) The track example shows the occupancy of CIITA at MHC-II cluster gene loci in BAP1-WT and -KO cells. ( K ) The Venn diagram shows the genes that are rescued by IRF1 in BAP1-KO cells. ( L ) The protein levels of MHC-II molecules in BAP1-WT, BAP1-KO, BAP1-KO-GFP, and BAP1-KO-Flag-IRF1 cells were determined by FACS analysis. ( M ) The pathway analysis with the total upregulated genes by IRF1 in A20 BAP1-KO cells. The BAP1-KO A20 cells were transduced with either GFP, WT BAP1, or catalytically dead BAP1. ( N ) The protein levels of BAP1 were determined by Western blot. Representative blot from 2 biological repeats. ( O ) The mRNA levels of Irf1 and Ciita were determined by real-time PCR. n = 3 technical replicates. Data are represented as mean ± SD.

Article Snippet: H3K27me3 (catalog 9733), Histone H3 (catalog 4499S), H2AK119Ub (catalog 8240), IRF1 (catalog 8478S), RING1B (catalog 5694S), UTX (catalog 33510S), CD4 (catalog 25229S), MHC-II-FITC (catalog 42594S), MHC-II-APC (catalog 64776), and BAP1 (catalog 13271) antibodies were purchased from Cell Signaling Technology.

Techniques: Binding Assay, RNA Sequencing, Western Blot, ChIP-qPCR, Transduction, Real-time Polymerase Chain Reaction

Journal: The Journal of Clinical Investigation

Article Title: An epigenetic pathway regulates MHC-II expression and function in B cell lymphoma models

doi: 10.1172/JCI179703

Figure Lengend Snippet: ( A ) The A20 BAP1-KO cells were treated with various concentrations of PRC1 inhibitor RB-3 for 6 days. The protein levels of H2AK119Ub and RING1B were determined by Western blot. Representative blot from 2 biological repeats. ( B ) The log 2 fold change heatmap shows the H2AK119Ub levels in A20 BAP1-KO cells treated with either DMSO or RB-3. ( C ) The A20 BAP1-WT or -KO cells were treated with either DMSO or RB-3 for 6 days. The gene-expression profiles for each condition were determined by RNA-Seq. ( D ) The Venn diagram shows the overlap between BAP1 targeted genes and RB-3 rescued genes. ( E ) The pathway analysis with the 860 BAP1 targeted genes that were restored by RB-3 treatment. ( F ) The A20 BAP1-WT cells were treated with DMSO for 6 days, and the BAP1-KO cells were treated with either DMSO or RB-3 (5 μM) for 6 days. The mRNA levels of MHC-II cluster genes in each treatment were determined by RNA-Seq. ( G ) The BAP1-KO A20 cells were treated with either DMSO or RB-3 (10 μM) for 6 days. The protein levels of MHC-II were determined by FACS analysis. ( H ) The A20 BAP1-WT cells were treated with DMSO for 6 days, and the BAP1-KO cells were treated with either DMSO or various concentrations of RB-3 for 6 days. The IRF1 protein levels were determined by Western blot. Representative blot from 2 biological repeats. ( I and J ) The mRNA levels of Irf1 ( I ) and Ciita mRNA ( J ) were determined by real-time PCR. n = 3 technical replicates. Data are represented as mean ± SD. ( K and L ) The BAP1-KO cells were treated with RB-3 (10 μM) or/and GSK126 (5 μM) for 6 days. The protein levels of H2AK119Ub and H3K27me3 were determined by Western blot. ( K ) Representative blot from 2 biological repeats. The protein levels of cell surface MHC-II molecules were determined by FACS analysis ( L ).

Article Snippet: H3K27me3 (catalog 9733), Histone H3 (catalog 4499S), H2AK119Ub (catalog 8240), IRF1 (catalog 8478S), RING1B (catalog 5694S), UTX (catalog 33510S), CD4 (catalog 25229S), MHC-II-FITC (catalog 42594S), MHC-II-APC (catalog 64776), and BAP1 (catalog 13271) antibodies were purchased from Cell Signaling Technology.

Techniques: Western Blot, Gene Expression, RNA Sequencing, Real-time Polymerase Chain Reaction

Journal: The Journal of Clinical Investigation

Article Title: An epigenetic pathway regulates MHC-II expression and function in B cell lymphoma models

doi: 10.1172/JCI179703

Figure Lengend Snippet: ( A ) The cell growth rate of A20 BAP1-WT and -KO cells was determined by cell counting assay. ( B ) 2 × 10 6 of BAP1-WT and BAP1-KO clones were inoculated into the right flank of 6-week-old nude mice. The tumor size was measured every day for 1 week after inoculation. ( C ) Images of representative tumor tissue samples from each mouse were taken at the end of the experiment. ( D ) 5 × 10 6 of BAP1-WT and BAP1-KO clones were inoculated into the right flank of 6-week-old BALB/c mice. The tumor size was measured every day for 2 weeks after the inoculation. Data are represented as mean ± SEM. A 2-tailed unpaired Student’s t test was used for statistical analysis. ** P < 0.01; * P < 0.05. ( E ) When each tumor reached 1 cm 3 , the mouse was euthanized, and the survival probability was shown. ( F ) Tumor tissue from both BAP1-WT and BAP1-KO was harvested and subjected to scRNA-Seq. The UMAP analysis identifies different clusters of cell populations within the tumor tissue based on the gene-expression profiles. ( G ) The bar plot shows the percentage of each cell cluster in the tumor tissues. ( H ) The violin plot shows the expression levels of Irf1 , Ciita , and MHC-II cluster genes in BAP1-WT and -KO tumor cell population. ( I ) Immune cells from the first clustering were isolated (T cells and macrophages) and reclustered with UMAP algorithm. ( J ) The percentages of each type of immune cells were calculated in BAP1-WT and -KO A20 tumors and are shown in the bar plot. ( K ) The IHC staining was performed with CD4-specific antibody in BAP1-WT and -KO tumor tissues. Scale bars: 10 μm.

Article Snippet: H3K27me3 (catalog 9733), Histone H3 (catalog 4499S), H2AK119Ub (catalog 8240), IRF1 (catalog 8478S), RING1B (catalog 5694S), UTX (catalog 33510S), CD4 (catalog 25229S), MHC-II-FITC (catalog 42594S), MHC-II-APC (catalog 64776), and BAP1 (catalog 13271) antibodies were purchased from Cell Signaling Technology.

Techniques: Cell Counting, Clone Assay, Gene Expression, Expressing, Isolation, Immunohistochemistry

Journal: The Journal of Clinical Investigation

Article Title: An epigenetic pathway regulates MHC-II expression and function in B cell lymphoma models

doi: 10.1172/JCI179703

Figure Lengend Snippet: ( A ) The box plot shows the Spearman’s correlation between the expression levels of human MHC-II genes and BAP1, EZH2, RNF2, PCGF6, or KDM2B in B cell lymphoma patients (TCGA, PanCancer Atlas). ( B – E ) The scatter plot shows the positive correlation between HLA-DMA and ( B ), EZH2 ( C ), PCGF6 ( D ), or KDM2B ( E ) in B cell lymphoma patient samples. ( F ) The schematic model shows how the BAP1 complex and the PRC1 establish a dynamic epigenetic state at MHC-II cluster gene loci by precisely regulating histone H2AK119Ub level and IRF1/CIITA axis which is critical for MHC-II gene expression. ( G ) The epigenetic balance between BAP1 and PRC1 plays an essential role in “hot” and “cold” tumor switching and tumor immune response.

Article Snippet: H3K27me3 (catalog 9733), Histone H3 (catalog 4499S), H2AK119Ub (catalog 8240), IRF1 (catalog 8478S), RING1B (catalog 5694S), UTX (catalog 33510S), CD4 (catalog 25229S), MHC-II-FITC (catalog 42594S), MHC-II-APC (catalog 64776), and BAP1 (catalog 13271) antibodies were purchased from Cell Signaling Technology.

Techniques: Expressing, Gene Expression

Journal: Protein & Cell

Article Title: Tumor-derived neomorphic mutations in ASXL1 impairs the BAP1-ASXL1-FOXK1/K2 transcription network

doi: 10.1007/s13238-020-00754-2

Figure Lengend Snippet: C-terminally truncated ASXL1 mutants retain BAP1 binding, but impair or lose interaction with HCF-1 and FOXK1/2 . (A) Cartoon representation of the immunoprecipitation–Mass Spectrometry (IP-MS) assay for identification of interacting proteins of full-length ASXL1 (ASXL1 FL -Flag) and tumor-derived mutant ASXL1 (ASXL1 N646 -Flag). (B) Gene Ontology (GO) biological process enrichment analysis of proteins that lose the interaction with C-terminally truncated ASXL1 mutant as identified by IP-MS. (C) Localization of ASXL1 FL -Flag and ASXL1 N646 -Flag in HEK293T cells. (D) IP-MS identification of interaction between ASXL1 N646 -Flag and endogenous proteins of BAP1, HCF-1, FOXK1, and FOXK2 in U2OS cells, as described above in (A). PSM, the total number of identified peptide spectra matched for the protein. (E) Interaction between different ASXL1 truncations and endogenous HCF-1, FOXK1, FOXK2 and BAP1. See also Fig. S2A. (F) Domain mapping of ASXL1 reveals the residues in ASXL1 which are essential for FOXK1, FOXK2, HCF-1 binding. See also Fig. S3. (G) Interaction between full-length ASXL1 and endogenous HCF-1, FOXK1 and FOXK2 in BAP1 knockdown cells. (H) Interaction between BAP1 and endogenous HCF-1, FOXK1 and FOXK2 in ASXL1 knockdown cells. (I) In vitro pull-down assay shows ASXL1 interacts with FOXK1 directly

Article Snippet: All the antibodies used in the study were purchased commercially, including Anti-Flag tag (Sigma, catalog F7425), anti-Myc (HuaBio, catalog EM31105), anti-BAP1 (Santa Cruz Biotechnology, catalog sc-28383 for IP and WB; GST, catalog 78105 for ChIP), anti-ASXL1 (Abcam, catalog ab228009; Abcam, catalog ab221455), anti-FOXK1 (Abcam, catalog ab18196), anti-FOXK2 (Abcam, ab5298), anti-HCF-1 (GST, catalog 50708), anti-H2AK119 mono-ubiquitination (Abcam, catalog ab193203), anti-H3K27me3 (Abcam, catalog ab6002), anti-Histone H2A (Abcam, catalog ab18255), anti-Histone H3 (CST, catalog 9715), anti-TXNIP (Abcam, catalog ab188865), anti-HIF-1α (Abcam, catalog ab216842), anti-STAT3 (CST, catalog 12640), anti-p-STAT3 (CST, catalog 9145), anti-JAK2 (Abcam, catalog ab108596), anti-p-JAK2 (Abcam, catalog ab32101), anti-GAL4 (Santa Cruz Biotechnology, catalog sc-510), anti-ACTIN (Genscript, catalog A00702).

Techniques: Binding Assay, Immunoprecipitation, Mass Spectrometry, Derivative Assay, Mutagenesis, In Vitro, Pull Down Assay

Journal: Protein & Cell

Article Title: Tumor-derived neomorphic mutations in ASXL1 impairs the BAP1-ASXL1-FOXK1/K2 transcription network

doi: 10.1007/s13238-020-00754-2

Figure Lengend Snippet: C-terminally truncated ASXL1 mutants lose interaction with multiple DNA-binding proteins . (A) RNA-seq analysis reveals differentially expressed genes by deletion of mutant ASXL1 in K562 cells ( ASXL1 +/N590 vs. ASXL1 +/− ). (B) Principal component analysis (PCA) of RNA-sequencing results among the indicated groups of K562 cells. (C) Heatmap of 231 genes differentially expressed in the indicated groups of K562 cells, as identified by RNA-seq and analyzed by two-way ANOVA analysis. (D) Venn diagrams depicting a significant proportion of genes differentially affected by deletion of mutant ASXL1 are direct target genes of FOXK1 or FOXK2 (fold change > 1.4). (E) Venn diagrams depicting overlap between the indicated groups of K562 cells, including the overlap between down-regulated genes by mutant ASXL1 ( n = 1,159) and down-regulated genes by deletion of either FOXK1 ( n = 2,197) or FOXK2 ( n = 2,358), as determined by RNA-seq analysis. (F) Schematic representation of the mammalian two-hybrid system. (G and H) A summary of DNA-binding proteins which selectively bind with full-length ASXL1, but not ASXL1 N646 , identified by the mammalian two-hybrid system and verified by Western blot. Some representative Western blot results are shown in (H). See also Fig. S7B–E

Article Snippet: All the antibodies used in the study were purchased commercially, including Anti-Flag tag (Sigma, catalog F7425), anti-Myc (HuaBio, catalog EM31105), anti-BAP1 (Santa Cruz Biotechnology, catalog sc-28383 for IP and WB; GST, catalog 78105 for ChIP), anti-ASXL1 (Abcam, catalog ab228009; Abcam, catalog ab221455), anti-FOXK1 (Abcam, catalog ab18196), anti-FOXK2 (Abcam, ab5298), anti-HCF-1 (GST, catalog 50708), anti-H2AK119 mono-ubiquitination (Abcam, catalog ab193203), anti-H3K27me3 (Abcam, catalog ab6002), anti-Histone H2A (Abcam, catalog ab18255), anti-Histone H3 (CST, catalog 9715), anti-TXNIP (Abcam, catalog ab188865), anti-HIF-1α (Abcam, catalog ab216842), anti-STAT3 (CST, catalog 12640), anti-p-STAT3 (CST, catalog 9145), anti-JAK2 (Abcam, catalog ab108596), anti-p-JAK2 (Abcam, catalog ab32101), anti-GAL4 (Santa Cruz Biotechnology, catalog sc-510), anti-ACTIN (Genscript, catalog A00702).

Techniques: DNA Binding Assay, RNA Sequencing Assay, Mutagenesis, Western Blot

Journal: Protein & Cell

Article Title: Tumor-derived neomorphic mutations in ASXL1 impairs the BAP1-ASXL1-FOXK1/K2 transcription network

doi: 10.1007/s13238-020-00754-2

Figure Lengend Snippet: Tumor-derived ASXL1 mutant interferes with the BAP1-ASXL1-FOXK1/K2 axis to down-regulate tumor suppressor genes . (A) The mRNA expression of tumor suppressor genes in the indicated groups of K562 cells, as determined by RNA-seq analysis. (B–F) The occupancy of FOXK1, FOXK2, ASXL1, EZH2 and SUZ12 at the promoter regions of indicated genes, analyzed by using the Cistrome Data Browser and UCSC genome browser. Moreover, BAP1 and H2AK119Ub enrichment at promoters of these genes were detected by ChIP-qPCR in ASXL1 +/N590 knockout K562 cells and FOXK1 and/or FOXK2 knockout K562 pools. IgG was included as negative control for ChIP-qPCR. According to ChIP-Seq retrieved from the GTRD database ( https://gtrd.biouml.org/ ), CMTM7 is not a direct target gene of FOXK1 and FOXK2, although BAP1 is enriched at the promoter region of CMTM7. This gene thus is included as a negative control. Asterisks denote statistical significance with two-tailed Student’s t -test or one-way ANOVA (B–F). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 for the indicated comparison; ns = not significant

Article Snippet: All the antibodies used in the study were purchased commercially, including Anti-Flag tag (Sigma, catalog F7425), anti-Myc (HuaBio, catalog EM31105), anti-BAP1 (Santa Cruz Biotechnology, catalog sc-28383 for IP and WB; GST, catalog 78105 for ChIP), anti-ASXL1 (Abcam, catalog ab228009; Abcam, catalog ab221455), anti-FOXK1 (Abcam, catalog ab18196), anti-FOXK2 (Abcam, ab5298), anti-HCF-1 (GST, catalog 50708), anti-H2AK119 mono-ubiquitination (Abcam, catalog ab193203), anti-H3K27me3 (Abcam, catalog ab6002), anti-Histone H2A (Abcam, catalog ab18255), anti-Histone H3 (CST, catalog 9715), anti-TXNIP (Abcam, catalog ab188865), anti-HIF-1α (Abcam, catalog ab216842), anti-STAT3 (CST, catalog 12640), anti-p-STAT3 (CST, catalog 9145), anti-JAK2 (Abcam, catalog ab108596), anti-p-JAK2 (Abcam, catalog ab32101), anti-GAL4 (Santa Cruz Biotechnology, catalog sc-510), anti-ACTIN (Genscript, catalog A00702).

Techniques: Derivative Assay, Mutagenesis, Expressing, RNA Sequencing Assay, Knock-Out, Negative Control, ChIP-sequencing, Two Tailed Test

Journal: Protein & Cell

Article Title: Tumor-derived neomorphic mutations in ASXL1 impairs the BAP1-ASXL1-FOXK1/K2 transcription network

doi: 10.1007/s13238-020-00754-2

Figure Lengend Snippet: Tumor-derived ASXL1 mutant regulates glucose metabolism, oxygen sensing, and JAK-STAT3 signaling pathways . (A) The mRNA expression of indicated genes in FOXK1 and/or FOXK2 knockout K562 cell pools, as determined by qRT-PCR. (B) The mRNA expression of indicated genes in ASXL1 +/N590 and ASXL1 +/− K562 clones, as determined by qRT-PCR. (C) TXNIP protein level in ASXL1 +/N590 and ASXL1 +/− clones, as determined by Western blot. (D) Glucose uptake in ASXL1 +/N590 and ASXL1 +/− clones, as measured using a Glucose Assay Kit as described in the “ ” section. (E) Relative concentration of lactate, ATP, and ADP in ASXL1 +/N590 and ASXL1 +/− K562 clones, as measured by LC–MS/MS as described in the “ ” section. (F) The protein levels of HIF-1α in ASXL1 +/N590 and ASXL1 +/− K562 clones under the culture conditions of normoxia or hypoxia (1% and 8% O 2 , exposure for 48 h) . (G) The mRNA expression of HIF-1α target genes in ASXL1 +/N590 and ASXL1 +/− K562 clones under the culture conditions of normoxia or hypoxia (1% O 2 , exposure for 48 h). (H) JAK2, STAT3, and their site-specific phosphorylation levels in ASXL1 +/N590 and ASXL1 +/− clones. (I) The mRNA expression of STAT3 target genes in ASXL1 +/N590 and ASXL1 +/− clones, as determined by qRT-PCR. Asterisks denote statistical significance with two-tailed Student’s t -test (B, D, E, G, I) or one-way ANOVA (A). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 for the indicated comparison; ns = not significant

Article Snippet: All the antibodies used in the study were purchased commercially, including Anti-Flag tag (Sigma, catalog F7425), anti-Myc (HuaBio, catalog EM31105), anti-BAP1 (Santa Cruz Biotechnology, catalog sc-28383 for IP and WB; GST, catalog 78105 for ChIP), anti-ASXL1 (Abcam, catalog ab228009; Abcam, catalog ab221455), anti-FOXK1 (Abcam, catalog ab18196), anti-FOXK2 (Abcam, ab5298), anti-HCF-1 (GST, catalog 50708), anti-H2AK119 mono-ubiquitination (Abcam, catalog ab193203), anti-H3K27me3 (Abcam, catalog ab6002), anti-Histone H2A (Abcam, catalog ab18255), anti-Histone H3 (CST, catalog 9715), anti-TXNIP (Abcam, catalog ab188865), anti-HIF-1α (Abcam, catalog ab216842), anti-STAT3 (CST, catalog 12640), anti-p-STAT3 (CST, catalog 9145), anti-JAK2 (Abcam, catalog ab108596), anti-p-JAK2 (Abcam, catalog ab32101), anti-GAL4 (Santa Cruz Biotechnology, catalog sc-510), anti-ACTIN (Genscript, catalog A00702).

Techniques: Derivative Assay, Mutagenesis, Expressing, Knock-Out, Quantitative RT-PCR, Clone Assay, Western Blot, Glucose Assay, Concentration Assay, Liquid Chromatography with Mass Spectroscopy, Two Tailed Test

Journal: Protein & Cell

Article Title: Tumor-derived neomorphic mutations in ASXL1 impairs the BAP1-ASXL1-FOXK1/K2 transcription network

doi: 10.1007/s13238-020-00754-2

Figure Lengend Snippet: Tumor-derived ASXL1 mutants promote leukemia cell growth through increased cell cycle progression and decreased apoptosis . (A and B) Cell proliferation of ASXL1 +/N590 and ASXL1 +/− K562 clones, as well as ASXL1 +/N646 and ASXL1 +/− Kasumi-1 clones under normoxia or hypoxia (1% O 2 ) condition for the indicated time periods. (C) The ratios of G0/G1-phase and G2/M-phase in ASXL1 +/N590 and ASXL1 +/− clones under hypoxia (1% O 2, 24 h). (D) Cell apoptosis and viability in ASXL1 +/N590 and ASXL1 +/− clones upon serum starvation. The indicated K562 cells were maintained in RPMI-1640 medium without FBS for 48 h. (E) Apoptosis-related markers in ASXL1 +/N590 and ASXL1 +/− clones upon serum starvation. Cells were cultured as mentioned above in (D). (F) ASXL1 +/N590 and ASXL1 +/− K562 clones were treated with 2-MeOE2 (HIF-1α inhibitor) for 24 h, and the cytotoxicity was determined by CCK-8 assay as described in the “ ” section. (G) Working model. C-terminally truncated ASXL1 mutant is expressed at a much higher protein level than wild-type ASXL1, and loses interaction with transcription factors such as FOXK1 and FOXK2, but still interacts with BAP1. Thus, the mutant ASXL1 protein inhibits the interaction between BAP1 and wild-type ASXL1 in a dominant-negative manner, reduces BAP1 enrichment and increases H2AK119 mono-ubiquitination at the promoters of FOXK1/K2 target genes, and impairs the function of BAP1-ASXL1-FOXK1/K2 axis to regulate target genes and leukemia cell growth. Asterisks denote statistical significance with two-tailed Student’s t -test (A, B, C, D, F). * P < 0.05; *** P < 0.001 for the indicated comparison; ns = not significant

Article Snippet: All the antibodies used in the study were purchased commercially, including Anti-Flag tag (Sigma, catalog F7425), anti-Myc (HuaBio, catalog EM31105), anti-BAP1 (Santa Cruz Biotechnology, catalog sc-28383 for IP and WB; GST, catalog 78105 for ChIP), anti-ASXL1 (Abcam, catalog ab228009; Abcam, catalog ab221455), anti-FOXK1 (Abcam, catalog ab18196), anti-FOXK2 (Abcam, ab5298), anti-HCF-1 (GST, catalog 50708), anti-H2AK119 mono-ubiquitination (Abcam, catalog ab193203), anti-H3K27me3 (Abcam, catalog ab6002), anti-Histone H2A (Abcam, catalog ab18255), anti-Histone H3 (CST, catalog 9715), anti-TXNIP (Abcam, catalog ab188865), anti-HIF-1α (Abcam, catalog ab216842), anti-STAT3 (CST, catalog 12640), anti-p-STAT3 (CST, catalog 9145), anti-JAK2 (Abcam, catalog ab108596), anti-p-JAK2 (Abcam, catalog ab32101), anti-GAL4 (Santa Cruz Biotechnology, catalog sc-510), anti-ACTIN (Genscript, catalog A00702).

Techniques: Derivative Assay, Clone Assay, Cell Culture, CCK-8 Assay, Mutagenesis, Dominant Negative Mutation, Two Tailed Test