ARID3A interacts with SMAD2/3 and promotes TGFβ pathway activation. (A) Experimental design for isolating ARID3A-containing protein complexes from CMK cells. (B) Volcano plot showing enriched proteins in LC-MS/MS after ARID3A pulldown, compared with the IgG control (n = 2). Significantly-enriched proteins (red); and ARID3A (green) and SMAD2 (blue). (C) Western blot confirming the coimmunoprecipitation of SMAD2 and SMAD3 and ARID3A. (D) Venn diagram showing the number of genomic regions bound by ARID3A, SMAD2 and/or SMAD3. The data were obtained via CUT&RUN, after the doxycycline-induced Arid3a expression in miR-125b-expressing Gata1s FLCs. Significantly enriched peaks were called using SEACR.⁴⁰ (E) Heat maps depicting the colocalization of ARID3A (green, left), SMAD2/3 (blue and gray, middle), and chromatin accessibility (green, right) signals after doxycycline-induced Arid3a expression in miR-125b-expressing Gata1s FLCs. The data were generated using CUT&RUN and ATAC-seq, respectively. Regions ±3 kb of the peak center are shown. Aggregate signals of single- and co-occupied regions are also provided (bottom; ARID3A and SMAD2/3 cobound [black], ARID3A and SMAD2 cobound [blue], ARID3A and SMAD3 cobound [gray], and ARID3A-bound only [green] are indicated). (F) Volcano plot showing the differential expression of ARID3A and SMAD2/3 cobound genes upon Arid3a knockdown in Gata1s FLCs.⁴⁰ Genes involved in apoptosis and cell cycle arrest (green); significantly downregulated (blue) and upregulated (red) genes; non-significantly changed genes (gray). (G) GSEA enrichment plots showing genes bound by ARID3A, SMAD2 and SMAD3, and their response to Arid3a modulation in Gata1s FLCs. (H) Integrative Genomics Viewer snapshots of genes involved in apoptosis and cell cycle arrest, showing occupancy of the ARID3A-SMAD2-SMAD3 protein complex and chromatin accessibility (ATAC). The tracks display coverage (RPKM) (left). Scale and chromosome location are shown (top).