Journal: Nature Communications

Article Title: CBFA2T3-GLIS2 mediates transcriptional regulation of developmental pathways through a gene regulatory network

doi: 10.1038/s41467-024-53158-9

Figure Lengend Snippet: A Averaged signal-ranking plots of H3K27ac bound genes were identified from CUT&RUN-seq in two fusion-negative megakaryoblast samples and two fusion-positive megakaryoblast samples from secondary transplants. Super-enhancer (SE) cut-off values are displayed on the graphs. Source data are provided as a Source Data file. B Venn diagram showing the overlap between SEs in fusion-negative megakaryoblasts (blue) and fusion-positive megakaryoblasts (red). C Heatmaps of differentially expressed SEs in fusion-negative megakaryoblasts ( N = 222), fusion-positive megakaryoblasts ( N = 187), and fusion-positive megakaryoblasts that are also bound by the fusion ( N = 163). D H3K27ac binding profiles, fusion binding profiles, and corresponding RNA-sequencing tracks at the TESC , BCL2 , and TCF7L2 SE. The TESC SE, essential for megakaryoblast differentiation, is lost in fusion-positive megakaryoblasts. BCL2 , an anti-apoptotic gene, and TCF7L2 , a member of the WNT signaling pathway, are upregulated super-enhancers exclusive to fusion-positive megakaryoblasts (generated using SparK v2.6.2) .

Article Snippet: Cell-bound beads were collected and re-suspended in Antibody Buffer and 0.5 ug of one of the following antibodies: TY1 (GenScript A01004-40), ETO (Bethyl A303-509A), CtBP1 (ThermoFisher 10972-1-AP), p300 (ThermoFisher PA1848), H3K27ac (Active Motif 39034), and rabbit IgG (Epicypher 13-0042).

Techniques: Binding Assay, RNA Sequencing, Generated

Journal: Nature Communications

Article Title: CBFA2T3-GLIS2 mediates transcriptional regulation of developmental pathways through a gene regulatory network

doi: 10.1038/s41467-024-53158-9

Figure Lengend Snippet: A Co-immunoprecipitations using fusion-positive leukemic megakaryoblasts confirm the association of ETO, CtBP1, and p300 with the fusion. All immunoprecipitations were repeated twice, representative blots are shown. Source data are provided as a Source Data file. B CUT&RUN-seq has performed in fusion-positive megakaryoblasts from two secondary transplants and fusion negative megakaryoblasts cultured in vitro for ETO, CtBP1, and p300. Distribution of CUT&RUN-seq peak locations for ETO, CtBP1, and p300. CBFA2T3-GLIS2 is included as a comparison. TSS transcription start site, TTS transcription termination site, UTR untranslated region. Source data are provided as a Source Data file. C Summary of ETO, CtBP1, and p300 bound genes retained, gained, and lost in fusion-positive megakaryoblasts compared to fusion-negative megakaryoblasts. Source data are provided as a Source Data file. D Heatmaps of enrichment for CBFA2T3-GLIS2 (CG-TY), H3K27ac, ETO, CtBP1, and p300 at genes bound by the fusion. E Venn diagram showing the overlap of genes bound at the promoters of ETO, CtBP1, and p300 exclusively in fusion-positive megakaryoblasts and their overlap with fusion-bound genes (Supplementary Data ).

Article Snippet: Cell-bound beads were collected and re-suspended in Antibody Buffer and 0.5 ug of one of the following antibodies: TY1 (GenScript A01004-40), ETO (Bethyl A303-509A), CtBP1 (ThermoFisher 10972-1-AP), p300 (ThermoFisher PA1848), H3K27ac (Active Motif 39034), and rabbit IgG (Epicypher 13-0042).

Techniques: Cell Culture, In Vitro, Comparison

Journal: Nature Communications

Article Title: CBFA2T3-GLIS2 mediates transcriptional regulation of developmental pathways through a gene regulatory network

doi: 10.1038/s41467-024-53158-9

Figure Lengend Snippet: A Co-immunoprecipitations using fusion-positive leukemic megakaryoblasts confirm the association of ETO, CtBP1, and p300 with the fusion. All immunoprecipitations were repeated twice, representative blots are shown. Source data are provided as a Source Data file. B CUT&RUN-seq has performed in fusion-positive megakaryoblasts from two secondary transplants and fusion negative megakaryoblasts cultured in vitro for ETO, CtBP1, and p300. Distribution of CUT&RUN-seq peak locations for ETO, CtBP1, and p300. CBFA2T3-GLIS2 is included as a comparison. TSS transcription start site, TTS transcription termination site, UTR untranslated region. Source data are provided as a Source Data file. C Summary of ETO, CtBP1, and p300 bound genes retained, gained, and lost in fusion-positive megakaryoblasts compared to fusion-negative megakaryoblasts. Source data are provided as a Source Data file. D Heatmaps of enrichment for CBFA2T3-GLIS2 (CG-TY), H3K27ac, ETO, CtBP1, and p300 at genes bound by the fusion. E Venn diagram showing the overlap of genes bound at the promoters of ETO, CtBP1, and p300 exclusively in fusion-positive megakaryoblasts and their overlap with fusion-bound genes (Supplementary Data ).

Article Snippet: Cell-bound beads were collected and re-suspended in Antibody Buffer and 0.5 ug of one of the following antibodies: TY1 (GenScript A01004-40), ETO (Bethyl A303-509A), CtBP1 (ThermoFisher 10972-1-AP), p300 (ThermoFisher PA1848), H3K27ac (Active Motif 39034), and rabbit IgG (Epicypher 13-0042).

Techniques: Cell Culture, In Vitro, Comparison

Journal: Nature Communications

Article Title: CBFA2T3-GLIS2 mediates transcriptional regulation of developmental pathways through a gene regulatory network

doi: 10.1038/s41467-024-53158-9

Figure Lengend Snippet: A–E Co-immunoprecipitation using 293T cells transfected with empty vector (MIG), wild-type fusion, or a fusion containing one or more of the mutations outlined in Fig. . All immunoprecipitations were repeated twice, representative blots are shown. Source data for all blots are provided as a Source Data file. A Immunoprecipitation of ETO followed by staining with the TY-1 tag to detect CBFA2T3-GLIS2 and the reciprocal immunoprecipitation of CBFA2T3-GLIS2 via the TY-1 tag followed by staining for ETO is shown. B TY-1-tagged wild-type CBFA2T3-GLIS2 and one of three FLAG-tagged mutant constructs were co-transfected into 293T cells to evaluate the ability of the mutant constructs to dimerize with the wild-type fusion. Immunoprecipitation by TY-1 followed by staining for FLAG and the reciprocal immunoprecipitation of FLAG followed by staining for TY-1 is shown. C 293T cells were co-transfected with two mutant constructs, one FLAG-tagged and one TY-1-tagged, to verify the results shown in ( B ). Immunoprecipitation of TY-1-tagged NHR2 deletion mutant followed by staining for FLAG again revealed a reduction of dimerization. D TY-1-tagged wild-type and mutant fusion constructs were transfected into 293T cells. Cells were immunoprecipitated for CtBP1 followed by staining for the fusion via TY-1 and the reciprocal immunoprecipitation of the fusion followed by staining for CtBP1. E TY-1-tagged wild-type and mutant fusion constructs were transfected into 293T cells. Cells were immunoprecipitated for p300, followed by staining for the fusion via TY-1, and the reciprocal immunoprecipitation of the fusion followed by staining for p300. F , G Transplantation of immunodeficient NSG-SGM3 mice with primary megakaryoblasts transduced with the mutant fusion constructs ( N = 5 per mutant construct, N = 9 wild type in panel F and 12 wild type in panel G ). p < 0.0001 for NHR2 deletion and NHR1-2 (DL380,487AS) mutant constructs compared to wild type. p -values determined by log-rank test. Source data are provided as a Source Data file.

Article Snippet: Cell-bound beads were collected and re-suspended in Antibody Buffer and 0.5 ug of one of the following antibodies: TY1 (GenScript A01004-40), ETO (Bethyl A303-509A), CtBP1 (ThermoFisher 10972-1-AP), p300 (ThermoFisher PA1848), H3K27ac (Active Motif 39034), and rabbit IgG (Epicypher 13-0042).

Techniques: Immunoprecipitation, Transfection, Plasmid Preparation, Staining, Mutagenesis, Construct, Transplantation Assay, Transduction