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Background Condensation of chromatin prior to enucleation is an essential component of terminal erythroid maturation, and defects in this process are associated with inefficient erythropoiesis and anemia. However, the mechanisms involved in this phenomenon are not well understood. Here, we describe a potential role for the histone variant H2A.X in erythropoiesis. Results We find in multiple model systems that this histone is essential for normal maturation, and that the loss of H2A.X in erythroid cells results in dysregulation in expression of erythroid-specific genes as well as a nuclear condensation defect. In addition, we demonstrate that erythroid maturation is characterized by phosphorylation at both S139 and Y142 on the C-terminal tail of H2A.X during late-stage erythropoiesis. Knockout of the kinase BAZ1B/WSTF results in loss of Y142 phosphorylation and a defect in nuclear condensation, but does not replicate extensive transcriptional changes to erythroid-specific genes observed in the absence of H2A.X. Conclusions We relate these findings to Caspase-Initiated Chromatin Condensation (CICC) in terminal erythroid maturation, where aspects of the apoptotic pathway are invoked while apoptosis is specifically suppressed.

Opinion statement Acquired aplastic anemia (AA) is a rare, life-threatening bone marrow failure (BMF) disorder that affects patients of all ages and is caused by lymphocyte destruction of early hematopoietic cells. Diagnosis of AA requires a comprehensive approach with prompt evaluation for inherited and secondary causes of bone marrow aplasia, while providing aggressive supportive care. The choice of frontline therapy is determined by a number of factors including AA severity, age of the patient, donor availability, and access to optimal therapies. For newly diagnosed severe aplastic anemia, bone marrow transplant should be pursued in all pediatric patients and in younger adult patients when a matched sibling donor is available. Frontline therapy in older adult patients and in all patients lacking a matched sibling donor involves immunosuppressive therapy (IST) with horse antithymocyte globulin and cyclosporine A. Recent improvements in upfront therapy include encouraging results with closely matched unrelated donor transplants in younger patients and the emerging benefits of eltrombopag combined with initial IST, with randomized studies underway. In the refractory setting, several therapeutic options exist, with improving outcomes of matched unrelated donor and haploidentical bone marrow transplantation as well as the addition of eltrombopag to the non-transplant AA armamentarium. With the recent appreciation of frequent clonal hematopoiesis in AA patients and with the growing use of next-generation sequencing in the clinic, utmost caution should be exercised in interpreting the significance of somatic mutations in AA. Future longitudinal studies of large numbers of patients are needed to determine the prognostic significance of somatic mutations and to guide optimal surveillance and treatment approaches to prevent long-term clonal complications.

The fetal-to-adult switch in hemoglobin production is a model of developmental gene control with relevance to the treatment of hemoglobinopathies. The expression of transcription factor BCL11A, which represses fetal β-type globin ( HBG ) genes in adult erythroid cells, is predominantly controlled at the transcriptional level but the underlying mechanism is unclear. We identify HIC2 as a repressor of BCL11A transcription. HIC2 and BCL11A are reciprocally expressed during development. Forced expression of HIC2 in adult erythroid cells inhibits BCL11A transcription and induces HBG expression. HIC2 binds to erythroid BCL11A enhancers to reduce chromatin accessibility and binding of transcription factor GATA1, diminishing enhancer activity and enhancer–promoter contacts. DNA-binding and crystallography studies reveal direct steric hindrance as one mechanism by which HIC2 inhibits GATA1 binding at a critical BCL11A enhancer. Conversely, loss of HIC2 in fetal erythroblasts increases enhancer accessibility, GATA1 binding and BCL11A transcription. HIC2 emerges as an evolutionarily conserved regulator of hemoglobin switching via developmental control of BCL11A . HIC2 regulates the fetal-to-adult hemoglobin switch. It inactivates an enhancer of the BCL11A gene, a fetal globin repressor, by reducing chromatin accessibility and displacing the transcription factor GATA1.

The mechanisms by which the fetal-type β-globin-like genes HBG1 and HBG2 are silenced in adult erythroid precursor cells remain a fundamental question in human biology and have therapeutic relevance to sickle cell disease and β-thalassemia. Here, we identify via a CRISPR–Cas9 genetic screen two members of the NFI transcription factor family—NFIA and NFIX—as HBG1/2 repressors. NFIA and NFIX are expressed at elevated levels in adult erythroid cells compared with fetal cells, and function cooperatively to repress HBG1/2 in cultured cells and in human-to-mouse xenotransplants. Genomic profiling, genome editing and DNA binding assays demonstrate that the potent concerted activity of NFIA and NFIX is explained in part by their ability to stimulate the expression of BCL11A, a known silencer of the HBG1/2 genes, and in part by directly repressing the HBG1/2 genes. Thus, NFI factors emerge as versatile regulators of the fetal-to-adult switch in β-globin production. NFIA and NFIX directly repress the expression of fetal-type β-globin-like genes HBG1 and HBG2 in adult erythroid cells, and also do it indirectly through the upregulation of BCL11A .

Peroxisome proliferator-activated receptor gamma (PPARγ) is a multifunctional transcription factor that regulates adipogenesis, immunity and inflammation. Our laboratory previously demonstrated that PPARγ ligands induce apoptosis in malignant B cells. While malignant B lineage cells such as B cell lymphoma express PPARγ, its physiological function remains unknown. Herein, we demonstrate that silencing PPARγ expression by RNAi in human Burkitt's type B lymphoma cells increased basal and mitogen-induced proliferation and survival, which was accompanied by enhanced NF-κB activity and increased expression of Bcl-2. These cells also had increased survival upon exposure to PPARγ ligands and exhibited a less differentiated phenotype. In contrast, PPARγ overexpression in B lymphoma cells inhibited cell growth and decreased their proliferative response to mitogenic stimuli. These cells were also more sensitive to PPARγ-ligand induced growth arrest and displayed a more differentiated phenotype. Collectively, these findings support a regulatory role for PPARγ in the proliferation, survival and differentiation of malignant B cells. These findings further suggest the potential of PPARγ as a therapeutic target for B cell malignancy.

Hematopoietic stem cell (HSC) transplantation using umbilical cord blood (UCB) is a potentially life-saving treatment for leukemia and bone marrow failure but is limited by the low number of HSCs in UCB. The loss of HSCs after ex vivo manipulation is also a major obstacle to gene editing for inherited blood disorders. HSCs require a low rate of translation to maintain their capacity for self-renewal, but hematopoietic cytokines used to expand HSCs stimulate protein synthesis and impair long-term self-renewal. We previously described cytokine-free conditions that maintain but do not expand human and mouse HSCs ex vivo. Here we performed a high throughput screen and identified translation inhibitors that allow ex vivo expansion of human HSCs while minimizing cytokine exposure. Transplantation assays show a ~5-fold expansion of long-term HSCs from UCB after one week of culture in low cytokine conditions. Single cell transcriptomic analysis demonstrates maintenance of HSCs expressing mediators of the unfolded protein stress response, further supporting the importance of regulated proteostasis in HSC maintenance and expansion. This expansion method maintains and expands human HSCs after CRISPR/Cas9 editing of the BCL11A+58 enhancer, overcoming a major obstacle to ex vivo gene correction for human hemoglobinopathies.Competing Interest StatementSAP is a consultant for bluebirdbio and Agios Pharmaceutics. SAP and PSK have independent sponsored research agreements with Blueprint Medicines.Footnotes* https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE248311

Erythropoiesis is a robust process of cellular expansion and maturation that is required to maintain the massive steady-state red blood cell mass. However, anemia is common following clastogenic injury such as total body irradiation (TBI), suggesting that erythroid progenitors and precursors may be highly sensitive targets of radiation. Here, we explore the endogenous injury and recovery processes of the erythron following 4 Gy TBI of C57BL/6 mice. Functional colony assays were used to analyze erythroid progenitors, including day 7 burst-forming units (d7 BFU-E) and more mature d3 BFU-E and colony-forming units (CFU-E), and imaging flow cytometry was utilized to quantify erythroblast precursors, consisting of immature proerythroblasts and progressively more mature basophilic, polychromatophilic, and orthochromatic erythroblasts. We find that essentially all bone marrow and splenic erythroid progenitors and precursors are lost within two days following 4 Gy TBI. Phenotypic CFU-E and proerythroblasts in the bone marrow exhibit preferential apoptotic loss immediately following sublethal irradiation, revealing a functional transition at the proerythroblast to basophilic erythroblast maturational stages characterized by a shift from a pro-apoptotic to an anti-apoptotic phenotype. Following this initial loss, erythroid recovery is characterized by specific expansion of late-stage erythroid progenitors (d3 BFU-E and CFU-E) in the bone marrow that is dependent on endogenous erythropoietin (EPO) induction. This robust progenitor expansion is followed by a wave of maturing erythroid precursors in the bone marrow and their transient emergence in the bloodstream before re-initiation of extramedullary erythropoiesis in the spleen. Furthermore, we find that bone marrow macrophages, which constitute the erythroid precursor microenvironmental niche, are relatively radioresistant and form robust erythroblast islands post-4 Gy TBI during erythroid precursor recovery. We conclude that sublethal radiation serves as a model of endogenous stress erythropoiesis that is characterized by specific injury to the extravascular erythron, initial expansion and maturation of EPO-responsive late-stage progenitors exclusively in the bone marrow, and subsequent reseeding of extramedullary sites. This model will facilitate the study of mechanisms regulating erythropoiesis in bone marrow and extramedullary sites as well as the functional evaluation of erythroid lineage-directed therapeutics to mitigate cellular injury.