ARID3A acts in concert with GATA1 to activate megakaryocytic transcriptional programs. (A) Characterizing the molecular function of ARID3A in TAM/ML-DS leukemogenesis via sequential Gata1s acquisition and Arid3a repression. Gata1s FLCs were expanded for 3 weeks and then transduced with Arid3a cDNA, shArid3a #3 or their respective controls. RNA samples were obtained from each of the 4 different conditions and subjected to RNA-seq–based gene expression analysis (n = 2-3). (B) Normalized enrichment scores for up- or downregulated gene sets involved in hematopoietic differentiation, cell proliferation, and ML-DS progression. Gata1s FLCs were compared against wild-type FLCs (top); Arid3a and shArid3a Gata1s FLCs were compared against their respective Gata1s FLCs controls (LUC or sh-ctrl) after doxycycline induction (bottom). *P < .05; **P < .01; ***P < .001. (C) GSEA enrichment plots showing genes downregulated by the Gata1s mutation in FLCs, and their response to Arid3a modulation in Gata1s FLCs. (D) Venn diagram showing the number of genomic regions bound by ARID3A and/or GATA1s. The data were generated using CUT&RUN after the doxycycline-induced Arid3a expression in miR-125b-expressing Gata1s FLCs. Significantly enriched peaks were called using SEACR (sparse enrichment analysis for CUT&RUN.⁴⁰ (E) Heat maps depict the colocalization of ARID3A (green, left) and GATA1s (orange, middle) and chromatin accessibility (green, right) signals after doxycycline-induced Arid3a expression in miR-125b-expressing Gata1s FLCs. The data were generated by CUT&RUN (colocalization) and ATAC-seq (accessibility). Regions ±3 kb of the peak center are shown. Aggregate signals of single- and co-occupied regions are also provided (bottom; cobound [black] and ARID3A-bound only [green] are indicated). (F) Volcano plot showing the differential expression of ARID3A/GATA1s cobound genes after Arid3a knockdown in Gata1s FLCs.⁴⁰ Genes involved in megakaryocytic differentiation (green), significantly downregulated (blue) and upregulated (red) genes, and nonsignificantly changed genes (gray). (G) IGV snapshots of megakaryocytic genes, showing co-occupancy by ARID3A and GATA1 and chromatin accessibility (ATAC). The tracks display coverage (RPKM) (left). Scale and chromosome location are shown (top). n.s., not significant.