ASXL1^aa1-587interacts with BRD4 to regulate gene expression. (A,B) Reciprocal IP and western blotting confirmed interaction of ASXL1^aa1-587 with BRD4 in the nuclear fraction of HEK293T cells transfected with EV, FLAG-tagged ASXL1^FL or FLAG-tagged ASXL1^aa1-587. Nuclear extractions were subjected to IP using indicated antibodies against FLAG or BRD4. (C) ChIP-qPCR on 32D cells expressing Flag-ASXL1^aa1-587 or an EV control. The antibodies used for ChIP are indicated. Normal immunoglobulin G (IgG) was used as a control. PCR was performed with primers specific for the Prdm16 promoter regions. (D) Drug screenings were performed on BM cells of Asxl1^Y588XTg or WT mice. Whole BM cells from WT mice were used as a control. The tests were done in triplicates using a 10-point 1:3 dilution series starting at a nominal test concentration of 10 μM (20 000-fold concentration range). DSSmod was calculated for all samples and the selective DSS_mod (sDSSmod) for each drug was calculated according to the formula sDSS_mod = DSS_mod (Asxl1^Y588XTg BM cells) − DSS_mod (WT BM cells). Positive values represent drugs that show higher specificity in affecting the survival of the Asxl1^Y588XTg BM cells; negative values represent drugs that show higher specificity in affecting WT BM cells. (E) CFU-C assay using whole BM cells with or without BET bromodomain inhibitors treatment (EP-11313 and JQ1). Whole BM cells from WT mice were used as a control. The experiment was performed in triplicate. The concentration of the drug used in colony assay is indicated on the x-axis. *P = 0.0407. Unpaired Student t test was used to assess statistical significance. (F) ChIP-qPCR was performed on 32D cells expressing ASXL1^aa1-587 with or without 24 hours of treatment with JQ1. The concentration of the drug was 0.1 μM. The antibodies used for ChIP are indicated. Normal IgG was used as a control. PCR was done using primers specific for the Prdm16 promoter region. *P < .05. Unpaired Student t test was used to assess statistical significance.