Journal: Genome Biology

Article Title: MBD5 and MBD6 stabilize the BAP1 complex and promote BAP1-dependent cancer

doi: 10.1186/s13059-022-02776-x

Figure Lengend Snippet: Endogenous MBD5 and MBD6 are stable components in the BAP1 complex. A HEK293T cells were infected by lentivirus expressing GFP, GFP-tagged MBD5, or GFP-tagged-MBD6. The protein levels of GFP, GFP-MBD5, and GFP-MBD6 were determined by western blot, n =2. B The GFP-fusion proteins were purified from HEK293T cells stably expressing GFP, GFP-MBD5, or GFP-MBD6 defined in A . The purified proteins were subjected to mass spectrometry analysis. Peptide numbers of subunits within the BAP1 complex pulled down by GFP-tagged proteins are shown. C Immunoprecipitation (IP) of endogenous BAP1 from HEK293T cells followed by immunoblot (IB) for BAP1, MBD5, and MBD6. IgG was used as a negative control, n = 2. D IP of endogenous MBD5 or MBD6 from HEK293T cells followed by IB for MBD5, MBD6, BAP1, ASXL1, and ASXL2. IgG was used as a negative control, n = 2. E Nuclear extract from HEK293T cells was subjected to size exclusion (SE) chromatography and then protein levels of BAP1, MBD5, and MBD6 were determined by western blot analysis, n = 2. F The average plots shown represent the chromatin occupancy of H3K27Ac, H3K4me1, H3K4me3, and H3K27me3 centered on MBD5 peaks ( F ) or MBD6 peaks ( G ). H Sorted and centered heatmaps generated from ChIP-seq data analyses show the occupancy of BAP1, MBD5, and MBD6 in HEK293T cells. All rows are centered on BAP1 peaks based on the ranking of signals. I The average plot shows the co-occupancy between BAP1, MBD5, and MBD6 centered on BAP1 peaks. J The representative tracks show H3K27Ac, H3K4me1, and H3K4me3 levels at BAP1, MBD5, and MBD6 occupied loci in HEK293T cells. K A Venn diagram representation of the overlapped ChIP-seq peaks between BAP1, MBD5, and MBD6

Article Snippet: BAP1 (#13271S), ASXL2 (#71257), H3K27ac (#8173S), H3K4me1 (#5326S), H3K4me3 (#9751), H3K27me3 (#9733), and histone H3 (#4499S) antibodies were purchased from Cell Signaling Technology.

Techniques: Infection, Expressing, Western Blot, Purification, Stable Transfection, Mass Spectrometry, Immunoprecipitation, Negative Control, Chromatography, Generated, ChIP-sequencing

Journal: Genome Biology

Article Title: MBD5 and MBD6 stabilize the BAP1 complex and promote BAP1-dependent cancer

doi: 10.1186/s13059-022-02776-x

Figure Lengend Snippet: The ASXL subunits link MBD5, MBD6 to BAP1 via their C-terminal PHD fingers. A IP of endogenous MBD5 or MBD6 from wild-type or BAP1 knockout HEK293T cells followed by IB for BAP1, ASXL1, and ASXL2. IgG was used as a negative control, n = 2. B IP of endogenous MBD5 or MBD6 from ASXL1/2/3 triple-knockout (TKO) HEK293T cells followed by IB for BAP1, ASXL1, ASXL2, MBD5, and MBD6. IgG was used as a negative control, n = 2. C Schematic diagram depicting the truncations of human ASXL1 protein. D HEK293T cells were transfected with plasmids expressing GFP, GFP-tagged full-length, or truncated ASXL1. The levels of GFP-tagged proteins were determined by western blot analysis, n = 2. E , F HEK293T cells were co-transfected with plasmids expressing each fragment of ASXL1 and either Halo-tagged MBD5 ( E ) or MBD6 ( F ). Then, the cells were subjected to co-IP assay using GFP-trap agarose followed by western blotting using antibodies against BAP1 and Halo-tag, n = 2. G Schematic diagram depicting the truncations of C-terminus of human ASXL1 protein. H HEK293T cells were transfected with plasmids expressing either GFP, GFP-tagged full-length, or GFP-tagged truncated C-terminus of ASXL1 and the protein levels of GFP-tagged proteins were determined by western blot analysis, n = 2. I , J HEK293T cells were co-transfected with plasmids expressing GFP-tagged truncated C-terminus of ASXL1 and either Halo-tagged MBD6 ( I ) or MBD5 ( J ). They were subjected to co-IP assay using an antibody against GFP followed by western blotting using antibodies against Halo-tag, n = 2. K CLUSTALW alignment shows the similarity between PHD fingers of ASXL1/2/3. L HEK293T cells were transfected with GFP-tagged PHD fingers of ASXL1/2/3 followed by IP of GFP and IB of MBD5, MBD6, and BAP1, n = 2. M The GFP-fusion proteins were purified from HEK293T cells transduced with GFP-tagged PHD finger of ASXL1/2/3. The purified proteins were subjected to mass spectrometry analysis. Peptide numbers of representative proteins were shown. N Schematic diagram depicting the PHD finger depletion of human ASXL1/2 proteins targeted by sgRNA. O , P IP of MBD6 from wild-type cells and ASXL1-PHD finger deleted ( O ) or ASXL2-PHD finger deleted ( P ) cells followed by IB of MBD6 and ASXL1 ( O ) or ASXL2 ( P ), n = 2

Article Snippet: BAP1 (#13271S), ASXL2 (#71257), H3K27ac (#8173S), H3K4me1 (#5326S), H3K4me3 (#9751), H3K27me3 (#9733), and histone H3 (#4499S) antibodies were purchased from Cell Signaling Technology.

Techniques: Knock-Out, Negative Control, Triple Knockout, Transfection, Expressing, Western Blot, Co-Immunoprecipitation Assay, Purification, Transduction, Mass Spectrometry

Journal: Genome Biology

Article Title: MBD5 and MBD6 stabilize the BAP1 complex and promote BAP1-dependent cancer

doi: 10.1186/s13059-022-02776-x

Figure Lengend Snippet: MBD5 and MBD6 evolutionally contributes to the stability of the BAP1 complex. A Alignment by CLUSTALW analysis shows the similarity between MBD domains of human MBD5 and MBD6. B HEK293T cells were co-transfected with plasmids expressing GFP-tagged PHD finger of ASXL1 and each Halo-tagged truncated MBD domain of MBD6. Then the cells were subjected to co-IP assay using GFP-trap agarose followed by IB for Halo-tag, n = 2. C HEK293T cells were transfected with plasmids expressing Halo-tagged MBD6-WT, MBD6-Δ12aa, or MBD6-K61A/C66A together with GFP-tagged ASXL1 followed by IP of GFP and IB for Halo-tag, n = 2. D HEK293T cells were transduced with lentivirus expressing GFP, GFP-tagged MBD6-WT, MBD6-Δ12aa, or MBD6-K61A/C66A. The protein levels of ASXL1, ASXL2, and BAP1 were determined by western blot. HSP90 was used as an internal control, n = 2. E , F HEK293T cells stably expressing GFP, GFP-tagged MBD5, or GFP-tagged MBD6 were treated with CHX (50 μg/ml) for indicated time durations. The whole cell lysate was collected at each time point, and the total protein level of BAP1 was determined by western blot, n =2 ( E ) and quantified by ImageJ ( F ). G The computational BLAST with the 12-aa from human MBD5 and MBD6 in Drosophila protein database. H Schematic diagram depicting human MBD5 and MBD6 and isoforms of Drosophila SBA. I The GFP-fusion proteins were purified from Drosophila S2 cells transduced with the GFP-tagged MBD domain of SBA. The purified proteins were subjected to mass spectrometry analysis. Peptide numbers of representative proteins pulled down by the GFP-MBD domain of SBA were shown. J Drosophila S2 cells were subjected to co-IP assay using an antibody against Calypso followed by IB for ASX and SBA, n = 2. K Drosophila S2 cells were subjected to co-IP assay using an antibody against SBA followed by IB for SBA and ASX, n = 2. L Drosophila S2 cells were subjected to co-IP assay using an antibody against ASX followed by IB for ASX and SBA, n = 2. M The western blot shows protein levels of SBA, ASX, and Calypso in Drosophila S2 cells treated with dsRNA targeting SBA. Histone H3 was used as internal control, n = 2. N Schema of the Drosophila PR-DUB complex and the human BAP1 complex

Article Snippet: BAP1 (#13271S), ASXL2 (#71257), H3K27ac (#8173S), H3K4me1 (#5326S), H3K4me3 (#9751), H3K27me3 (#9733), and histone H3 (#4499S) antibodies were purchased from Cell Signaling Technology.

Techniques: Transfection, Expressing, Co-Immunoprecipitation Assay, Transduction, Western Blot, Stable Transfection, Purification, Mass Spectrometry

Journal: Genome Biology

Article Title: MBD5 and MBD6 stabilize the BAP1 complex and promote BAP1-dependent cancer

doi: 10.1186/s13059-022-02776-x

Figure Lengend Snippet: Characterization of MBD6 occupancy in SCLC cells. A The representative tracks show chromatin occupancy of MBD6, ASXL1/2/3, and BAP1 binding sites in the human SCLC cell line NCI-H1963. B The bar plot shows the feature distribution of MBD6, ASXL1/2/3, and BAP1 peaks in NCI-H1963 cells. C Sorted and centered heatmaps generated from ChIP-seq and ATAC-seq data analyses show the occupancy of MBD6, ASXL1/2/3, BAP1, H3K27Ac, H3K4me1, H3K4me3, and H3K27me3 in NCI-H1963 cells. All signals were centered on MBD6 peaks, which were classified into three clusters, based on k-means clustering according to MBD6, H3K4me1, H3K4me3, and H3K27Ac ChIP-seq. D Representative tracks showing the occupancy MBD6, BAP1, ASXL1-3, and histone marks. E Pathway analysis was performed using ChIPseeker with genes nearest to MBD6 three cluster peaks defined in C . F Motif enrichment analysis of MBD6 three clusters’ peaks defined in C from NCI-H1963 cells. G The box plot shows the log2-fold-change of H3K4me1, H3K4me3, and H3K27Ac signals versus input at MBD6/BAP1/ASXL1, MBD6/BAP1/ASXL2, or MBD6/BAP1/ASXL3 co-occupied genomic regions

Article Snippet: BAP1 (#13271S), ASXL2 (#71257), H3K27ac (#8173S), H3K4me1 (#5326S), H3K4me3 (#9751), H3K27me3 (#9733), and histone H3 (#4499S) antibodies were purchased from Cell Signaling Technology.

Techniques: Binding Assay, Generated, ChIP-sequencing

Journal: Genome Biology

Article Title: MBD5 and MBD6 stabilize the BAP1 complex and promote BAP1-dependent cancer

doi: 10.1186/s13059-022-02776-x

Figure Lengend Snippet: MBD6 is essential for BAP1-dependent gene expression in SCLC. The average plot ( A ) and representative tracks ( B ) show the global reduction of BAP1 in MBD6-depleted SCLC cells by two distinct sgRNAs. C Gene Set Enrichment Analysis (GSEA) of BAP1 gene signature enrichment in MBD6-depleted conditions. D The log2 fold-change heatmap shows the reduction of BAP1 peaks at MBD6 loci in cells transduced with two distinct MBD6-specific sgRNAs. E The log2 fold-change heatmap shows the change of expression levels of genes nearest to MBD6 peaks after MBD6 (left two lanes) and BAP1 (right two lanes) depletion by CRISPR, n =2. F NCI-H1963 cells were transduced with either non-targeting sgRNA or two distinct sgRNA specific to ASXL1/2/3 or BAP1. The protein levels of BAP1, ASXL1, ASXL2, and ASXL3 were determined by western blot. Tubulin was used as an internal control, n = 2. G The log2 fold-change gene expression heatmap shows BAP1/MBD6 co-targeted genes in ASXL1/2/3-depleted conditions by CRISPR knockout, n =2. H Pathway analysis by Metascape of genes that are commonly downregulated upon BAP1, MBD6, or ASXL3 depletion in NCI-H1963 cells. I The mRNA levels of NEUROG2 and CDK18 were determined by real-time qPCR in NCI-H1963 cells transduced with either non-targeting sgRNA, two different BAP1-specific sgRNAs, or two different ASXL1/2/3 sgRNAs

Article Snippet: BAP1 (#13271S), ASXL2 (#71257), H3K27ac (#8173S), H3K4me1 (#5326S), H3K4me3 (#9751), H3K27me3 (#9733), and histone H3 (#4499S) antibodies were purchased from Cell Signaling Technology.

Techniques: Expressing, Transduction, CRISPR, Western Blot, Knock-Out