ARID3a is a DNA-binding protein important for normal hematopoiesis in mice and for in vitro lymphocyte development in human cultures. ARID3a knockout mice die in utero with defects in both early hematopoietic stem cell populations and erythropoiesis. Recent transcriptome analyses in human erythropoietic systems revealed increases in ARID3a transcripts implicating potential roles for ARID3a in human erythrocyte development. However, ARID3a transcript levels do not faithfully reflect protein levels in many cells, and the functions and requirements for ARID3a protein in those systems have not been explored. We used the erythroleukemic cell line K562 as a model to elucidate functions of ARID3a protein in early human erythropoiesis. ARID3a knockdown of hemin-stimulated K562 cells resulted in lack of fetal globin production and modifications in gene expression. Temporal RNA sequencing data link ARID3a expression with the important erythroid regulators Gata1, Gata2, and Klf1. Ablation of ARID3a using CRISPR-Cas9 further demonstrated it is required to maintain chromatin structures associated with erythropoietic differentiation potential. These data demonstrate that the ARID3a protein is required for early erythropoietic events and provide evidence for the requirement of ARID3a functions for proper maintenance of appropriate chromatin structures. ImmunoHorizons, 2021, 5: 802-817.
Our previous studies demonstrated that AT-rich interacting domain 3a (ARID3a), a DNA-binding protein associated with increased immunoglobulin heavy chain transcription and chromatin accessibility, is required for normal hematopoiesis in mice and in human cells. ARID3a knockout mice exhibited low numbers of erythrocytes and died due to failed erythropoiesis between days 9 and 12 of gestation. The role of ARID3a in human erythropoiesis has not been studied. Stimulation of the human K562 cell line with hemin induces fetal globin synthesis and erythrocyte differentiation. K562 cells constitutively express ARID3a, so we used this cell line to determine if ARID3a is important for human erythrocyte differentiation. Knockdown of ARID3a with shRNA in hemin-stimulated K562 cells resulted in >75% inhibition of globin. To identify genes affected by ARID3a loss, we performed RNA-seq experiments at various times after hemin stimulation with and without ARID3a. ARID3a shRNA-inhibited cells confirmed inhibition of globin gene products, including HBA1, HBA2, and HBZ. Erythroid specific transcription factors were also inhibited in samples treated with ARID3a shRNA while myeloid surface markers were induced. Several critical upstream regulators of ARID3a were also identified in these analyses. These data suggest that ARID3a is important for cell fate decisions in erythropoiesis in human cells and identifies potential important targets of ARID3a in this process.
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