Journal: Science advances

Article Title: BAP1 regulates epigenetic switch from pluripotency to differentiation in developmental lineages giving rise to BAP1-mutant cancers.

doi: 10.1126/sciadv.aax1738

Figure Lengend Snippet: Fig. 1. Loss of Bap1 during Xenopus development produces a distinctive phenotype. (A) Representative embryos analyzed at late gastrula (stage 12) following injection into one blastomere at the two-cell stage of escalating doses (7.5, 10, and 20 ng) of a morpholino targeting the 5′UTR of bap1 mRNA (Bap1MO) or a bap1 base pair mismatch control morpholino (Bap1MO-Ctrl). Below each bright-field image is a corresponding fluorescence image demonstrating fluorescein isothiocyanate as a lineage tracer for the injected side (green color). Depletion of Bap1 produces gastrulation failure, as evidenced by incomplete blastopore closure (arrows). Arrowheads indicate injected side. Panels show dorsal view, anterior down. (B) Summary of results of experiments described in (A), showing that depletion of Bap1 produces gastrulation failure ranging from mild (yellow) to severe (red) in a dose-dependent manner. (C) Representative embryos treated as above with 7.5 ng of Bap1MO, which eventually completed gastrula- tion and developed axial foreshortening and bending (arrow) starting at early tail bud stages, compared to uninjected sibling embryos. Panels show lateral view, anterior left, except the lower right panel, which shows dorsal view, anterior down. (D) Representative embryos treated as above with 7.5 ng of Bap1MO, which eventually completed gastrulation and were evaluated at stage 37 or 45, showing microphthalmia or anophthalmia (black arrows) starting at late tail bud stages, and proliferation of morphologi- cally immature melanoblasts with altered migration pattern (red arrows) starting at late tail bud and early tadpole stages. Panels show lateral view (except panels labeled dorsal view), anterior left. Arrowheads indicate the injected side. (E) Transverse sections through the head of a representative early–tadpole stage embryo stained with hematoxylin and eosin, following injection into one blastomere (D1.2) at the 16-cell stage with 7.5 ng of Bap1MO, showing disruption of eye development on the side injected with Bap1MO (right side, arrowhead), compared to normal eye development on the uninjected control side (left side). Dotted line indicates midline. (F) Normal histologic appearance of the eye at early–tadpole stage embryo [same orientation as (E)], with ocular structures indicated. RPE, retinal pigment epithelium. (G to I) Represent ative eyes at late tail bud/early–tadpole stage embryos showing mild (G), moderate (H), and severe (I) ocular malformation associated with injection of 7.5 ng of Bap1MO into blastomere D1.2 (which gives rise to retina, lens, and other eye structures) at the 16-cell stage. In severe cases, such as the example in (I), retinal tissue does not form, and the eye remains filled with yolk platelets (pink). (J) Whole-mount in situ hybridization (WISH) of indicated eye markers in embryos injected with 7.5 ng of Bap1MO and a lineage tracer into D1.2 at the 16-cell stage and analyzed at the indicated stages, showing aberrant development of ocular tissues in the absence of Bap1. Markers include ventx2 (dorsal retina, unaffected), pax2 (ventral optic stalk), mitf and dct [retinal pigment epithelium and uveal melanocytes (UMCs)], rx1 (ciliary marginal zone and photoreceptor), otx2 and rbpms (retinal ganglion cell layer), and -crystallin (lens). Panels show lateral view, anterior side left. Uninj., uninjected. St., stage. Scale bars, 250 m.

Article Snippet: UMCs with knockout of BAP1 were created by clonally isolating UMCs stably transduced with lentiviral particles encoding spCAS9 (Addgene plasmid no. 50661) and guide RNA against BAP1 (Addgene plasmid no. 64114) directing the CRISPR-mediated deletion of the first exon of the BAP1 gene.

Techniques: Injection, Control, Fluorescence, Migration, Labeling, Staining, Disruption, In Situ Hybridization

Journal: Science advances

Article Title: BAP1 regulates epigenetic switch from pluripotency to differentiation in developmental lineages giving rise to BAP1-mutant cancers.

doi: 10.1126/sciadv.aax1738

Figure Lengend Snippet: Fig. 2. Bap1 loss deregulates expression of pluripotency and lineage commitment genes. (A to P) Representative embryos injected with 7.5 ng of the Bap1MO morpholino into one blastomere at the two-cell stage (arrowheads indicate injected side) and then fixed and analyzed for mRNA expression of the indicated developmental genes by WISH at the specified stages (gastrula, stage 12; midneurula, stages 14 to 17; early tail bud, stage 24). Bap1-depleted embryos fail to silence pluripotency factors such as vent1/2 (orthologs of mammalian Nanog) and oct25 (ortholog of mammalian Oct4) (A to C) and fail to activate lineage commitment factors such as fzd7 (dorsal mesoderm and ectoderm), vegT and bra (axial mesoderm), myoD (muscle), keratin1 (non-neural ectoderm), sox2 (neural ectoderm), rx1 (early eye field), zic1 and msx1 (neural fold/prospective neural crest), and foxD3 and sox10 (neural crest) (D to N). Bap1 loss results in a failure of neural crest cell migration in sox10-expressing cells compared to the uninjected control (O and P). Panels show the dorsal-caudal view (A, B, and D to F), the dorsal view (C and G to N), or the lateral view (O and P), anterior side left. Dotted lines indicate midline. Arrowheads indicate injected side. Scale bar, 250 m.

Article Snippet: UMCs with knockout of BAP1 were created by clonally isolating UMCs stably transduced with lentiviral particles encoding spCAS9 (Addgene plasmid no. 50661) and guide RNA against BAP1 (Addgene plasmid no. 64114) directing the CRISPR-mediated deletion of the first exon of the BAP1 gene.

Techniques: Expressing, Injection, Migration, Control

Journal: Science advances

Article Title: BAP1 regulates epigenetic switch from pluripotency to differentiation in developmental lineages giving rise to BAP1-mutant cancers.

doi: 10.1126/sciadv.aax1738

Figure Lengend Snippet: Fig. 3. Loss of Bap1 abrogates the assembly of H3K27ac at promoters of key genes regulating lineage commitment and differentiation. (A) Heat map demon- strating the top 1000 most differentially expressed genes by RNA-seq between embryos at one-cell stage with 15 ng of either Bap1MO or Bap1MO-Ctrl and collected at late gastrulation (stage 12), when morphologic effects of Bap1 loss are first evident. (B) Gene set enrichment analysis (GSEA) plots demonstrating the most highly significant pathways represented by the differentially expressed genes associated with Bap1 loss. FDR, false discovery rate. (C) Heat maps of ChIP-seq data demonstrating global genomic occupancy of the indicated histone marks across all annotated genes in embryos treated as above with either Bap1MO or Bap1MO-Ctrl. TSS, transcription start site. (D) Violin plots summarizing ChIP-seq data restricted to differentially expressed genes. (E) ChIP-seq and RNA-seq tracks of representative lineage commitment genes that fail to assemble H3K27ac at promoters and to activate mRNA expression in Bap1-deficient embryos.

Article Snippet: UMCs with knockout of BAP1 were created by clonally isolating UMCs stably transduced with lentiviral particles encoding spCAS9 (Addgene plasmid no. 50661) and guide RNA against BAP1 (Addgene plasmid no. 64114) directing the CRISPR-mediated deletion of the first exon of the BAP1 gene.

Techniques: RNA Sequencing, ChIP-sequencing, Expressing

Journal: Science advances

Article Title: BAP1 regulates epigenetic switch from pluripotency to differentiation in developmental lineages giving rise to BAP1-mutant cancers.

doi: 10.1126/sciadv.aax1738

Figure Lengend Snippet: Fig. 4. Hdac4 is a key mediator of the Bap1-deficient phenotype. (A) Representative embryos injected at the one-cell stage with 7.5 ng of Bap1MO with or without 16 ng of a morpholino directed against Hdac4 (Hdac4MO) and analyzed at midneurula stage (stage 15, dorsal view, anterior to left) and early tail bud stage (stage 26, lateral view, anterior to left). (B) Summary of results at stages 15 and 26, showing substantial rescue of the Bap1-deficient phenotype with Hdac4MO. (C) Representative embryos treated as above and analyzed by WISH, demonstrating that failed induction of the indicated developmental genes in Bap1-deficient embryos is rescued by Hdac4MO. Caudal view, dorsal up. (D) ChIP–quantitative polymerase chain reaction (qPCR) for indicated gene promoters following ChIP for H3K27ac, confirming that failure to assemble H3K27ac at promoters in Bap1-deficient embryos can be rescued by depletion of Hdac4. Scale bars, 250 m.

Article Snippet: UMCs with knockout of BAP1 were created by clonally isolating UMCs stably transduced with lentiviral particles encoding spCAS9 (Addgene plasmid no. 50661) and guide RNA against BAP1 (Addgene plasmid no. 64114) directing the CRISPR-mediated deletion of the first exon of the BAP1 gene.

Techniques: Injection, Real-time Polymerase Chain Reaction

Journal: Cancer cytopathology

Article Title: Analysis of early pleural fluid samples in patients with mesothelioma: A case series exploration of morphology, BAP1, and CDKN2A status with implications for the concept of mesothelioma in situ in cytology.

doi: 10.1002/cncy.22548

Figure Lengend Snippet: Figure 1. All initial pleural fluid specimens were comprised of predominantly dispersed mesothelial cells, (A) which in some cases, were present in moderate numbers with some size variation (arrow) and occasionally enlarged nucleoli, in keeping with an atypical mesothelial population (Papanicolaou stain). (B) The accompanying cell block revealed a similar population of cells, which were confirmed to be mesothelial in origin (calretinin immunohistochemistry [IHC]) and, in retrospect, (C) demonstrated loss of nuclear BRCA1-associated protein 1 (BAP1) staining (arrow) (BAP1 IHC). (D) Fluorescence in situ hybridization on this case (patient 1) identified no homozygous deletion of cyclin-dependent kinase inhibitor 2A (CDKN2A) (2 red CDKN2A signals were accompanied by at least 1 green chromosome 9 centromere signal per abnormal nucleus). (E) A diagnostic biopsy taken 2 years later revealed epithelioid mesothelioma (H&E stain) with (F) BAP1 loss (BAP1 IHC). (G) Fluorescence in situ hybridization analysis performed on this sample demonstrated homozygous deletion of CDKN2A (indicated by the loss of 2 CDKN2A red signals in abnormal nuclei with at least 1 retained chromosome 9 green signal, as indicated by the arrows).

Article Snippet: BAP1 IHC staining was performed using the anti- BAP1 C4 monoclonal antibody from Santa Cruz Biotechnology at a dilution of 1:50, with an UltraView Universal DAB Detection Kit on a Ventana BenchMark ULTRA platform.

Techniques: Papanicolaou Stain, Blocking Assay, Immunohistochemistry, Staining, Fluorescence, In Situ Hybridization, Diagnostic Assay

Journal: Cancer cytopathology

Article Title: Analysis of early pleural fluid samples in patients with mesothelioma: A case series exploration of morphology, BAP1, and CDKN2A status with implications for the concept of mesothelioma in situ in cytology.

doi: 10.1002/cncy.22548

Figure Lengend Snippet: Figure 2. Five of the initial samples carried a benign diagnosis, such as that of patient 8 shown here. (A) The smear contained dispersed mesothelial cells, including some enlarged forms (long arrows; the short arrow indicates mesothelial cells within normal size limits) among macrophages, neutrophils, and lymphocytes (DiffQuik preparation). (B) The cell block contained a similar population of cells with the retrospectively performed BRCA1-associated protein 1 (BAP1) demonstrating intact nuclear staining (arrow) (BAP1 immunohistochemistry [IHC]). (C,D) The diagnostic sample demonstrated large clusters of malignant mesothelial cells with irregular, hyperchromatic nuclei, prominent nucleoli and moderate volumes of dense cytoplasm (Papanicolaou stain). (E) Mesothelial origin was confirmed by cytokeratin 5/6 IHC, among other stains, and (F) BAP1 was noted to be lost on the retrospectively performed stain (BAP1 IHC).

Article Snippet: BAP1 IHC staining was performed using the anti- BAP1 C4 monoclonal antibody from Santa Cruz Biotechnology at a dilution of 1:50, with an UltraView Universal DAB Detection Kit on a Ventana BenchMark ULTRA platform.

Techniques: Biomarker Discovery, Blocking Assay, Staining, Immunohistochemistry, Diagnostic Assay, Papanicolaou Stain