Figure 2. Ectopic expression of ASXL1 truncations+BAP1 results in stable depletion of H2AK119Ub in the EML haematopoietic cell line.

(a) Using retroviral constructs, we generated EML cell lines that stably express ASXL1(1–479), BAP1, or both. A schematic representation of the retroviral constructs is shown to the left. Based on expression of retroviral reporters, green fluorescent protein (GFP) and Thy1.1, EML cells that express either ASXL1(1–479), BAP1, or both were purified by fluorescence-activated cell sorting (FACS). (b,c) EML cells were transduced with the indicated viral constructs, FACS sorted and expanded for 16 days in liquid culture in media supplemented with 100 ng ml⁻¹ SCF. (b) On day 16, nuclear lysates and acid-extracted histones prepared from these cells were subjected to western blotting with indicated antibodies. (c) On day 16, cells were subjected to intracellular staining with anti-H2AK119Ub and anti-H3K27me3 antibodies, and levels of H2AK119Ub and H3K27me3 were determined by flow cytometry. Geometric mean fluorescence intensities (gMFI) of H2AK119Ub and H3K27me3 stains were determined by subtracting gMFIs of no primary antibody controls from the absolute gMFIs. (d) EML cells transduced with either MiG-empty or ASXL1(1–479)+BAP1 were purified by FACS and expanded for 16 days in liquid culture. On day 16, total RNA was isolated, ribosome-depleted and subjected to RNA-seq. Differential expression and statistical significance were determined using DESeq. Highlighted in the plot and the table are genes that are normally expressed in the mast cell lineage that are upregulated in ASXL1(1–479)+BAP1-transduced EML cells.