Bright/Arid3a has been characterized both as an activator of immunoglobulin heavy-chain transcription and as a proto-oncogene. Although Bright expression is highly B lineage stage restricted in adult mice, its expression in the earliest identifiable hematopoietic stem cell (HSC) population suggests that Bright might have additional functions. We showed that >99% of Bright ؊/؊ embryos die at midgestation from failed hematopoiesis. Bright ؊/؊ embryonic day 12.5 (E12.5) fetal livers showed an increase in the expression of immature markers. Colony-forming assays indicated that the hematopoietic potential of Bright ؊/؊ mice is markedly reduced. Rare survivors of lethality, which were not compensated by the closely related paralogue Bright-derived protein (Bdp)/Arid3b, suffered HSC deficits in their bone marrow as well as B lineage-intrinsic developmental and functional deficiencies in their peripheries. These include a reduction in a natural antibody, B-1 responses to phosphocholine, and selective T-dependent impairment of IgG1 class switching. Our results place Bright/Arid3a on a select list of transcriptional regulators required to program both HSC and lineage-specific differentiation.The formation and maintenance of blood throughout fetal and adult life rely on the self-renewal of hematopoietic stem cells (HSCs). Rare HSCs arise in the embryonic yolk sac and aorta-gonad mesonephros AGM, seed the fetal liver, and then circulate in the bone marrow of adult mammals. Fetal and adult HSC progenitors become progressively dedicated to differentiation into erythrocytes, myeloid cells, and lymphocytes. Transcription factors critical for the specification and formation of HSCs cover a wide range of DNA binding protein families. An emerging theme is that many of these same regulators are required later for the differentiation of individual blood lineages, which explains why a number of HSC transcription factors were discovered and originally characterized because of their deregulation in hematopoietic malignancies.Bright/Arid3a/Dril1 is the founder of the AT-rich interaction domain (ARID) superfamily of DNA binding proteins (18,60). Bright, in a complex with Bruton's tyrosine kinase (Btk) and TFII-I, binds to specific AT-rich motifs within the nuclearmatrix attachment regions (MARs) of the immunoglobulin heavy-chain (IgH) intronic enhancer (E) and selected IgH promoters to activate IgH transcription (18,25,30,43,44,55,57,58). B cell-specific, transgenic overexpression of Bright leads to partial blocks at both the late-pre-B and T1 immature stages, skewed marginal-zone (MZ) B cell development, increased natural IgM antibody production, and intrinsic autoimmunity (49). Transgenic dominant negative (DN) inhibition of Bright DNA binding results in reduced levels of IgM in serum and functional perturbation of IgM secretion by B-1 cells (39,48). A small pool of Bright cycles from the nucleus into plasma membrane lipid rafts, where it associates with Btk to dampen antigen receptor signaling (48).While highly B lineage restricted in...
Objective Systemic lupus erythematosus (SLE) is a complex and multifactorial autoimmune disease with striking clinical, immunologic and genetic heterogeneity, despite nearly ubiquitous antinuclear antibody (ANA) production. Multiple gene polymorphisms have been associated with the disease, but individually account for only a very small percentage of overall SLE risk. In earlier studies, constitutive expression of the DNA-binding protein, A+T rich interacting domain 3a (ARID3a) in transgenic mouse B lymphocyte lineage cells led to spontaneous ANA production and preferential development of B cells associated with production of polyreactive antibodies. Therefore, we asked if ARID3a was over-expressed in B lymphocytes of SLE patients and if ARID3a expression was associated with disease severity. Methods A cross section of SLE patients and age and gender-matched controls were analyzed longitudinally for lupus disease activity, numbers of ARID3a+ peripheral blood mononuclear B cells from multiple B cell subsets, immunoglobulin and cytokine levels. Results Fifty of 115 patients (43%) had dramatically increased numbers of ARID3a+ B cells compared to healthy controls. ARID3a is not expressed in naïve B cells of healthy controls, but was abundant in these precursors of antibody-secreting cells in SLE patients. Total numbers of ARID3a+ B cells correlated with increased disease activity as defined by SLE Disease Activity Index scores in individuals assessed at three time points. Conclusion These findings identify B cell anomalies in SLE that allow stratification of patient samples based on ARID3a expression and implicate ARID3a as a potential marker of CD19+ B lymphocytes correlated with disease activity.
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.
Lethal toxin, a key virulence factor produced by Bacillus anthracis, induces cell death, in part by disrupting numerous signaling pathways, in mouse macrophages. However, exposure to sublethal doses of lethal toxin allows some cells to survive. Because these pro-survival signaling events occur within a few hours after exposure to sublethal doses, we hypothesized that acute phase proteins might influence macrophage survival. Our data show that serum amyloid A (SAA) is produced in response to lethal toxin treatment. Moreover, pre-treatment of macrophages with exogenous SAA protected macrophages from lethal toxin-mediated death. Exogenous SAA activated the p38 mitogen activated protein kinase (MAP) kinase pathway, while lethal toxin mutants incapable of p38 activation were incapable of causing cell death. Chemical inhibition of the p38 activation pathway abrogated the protective effects of SAA. These data show that SAA affords protection against lethal toxin in mouse macrophages and link this response to the p38 pathway.
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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