Baobing Zhao

Nuclear Condensation during Mouse Erythropoiesis Requires Caspase-3-Mediated Nuclear Opening.

Mammalian erythropoiesis involves chromatin condensation that is initiated in the early stage of terminal differentiation. The mechanisms of chromatin condensation during erythropoiesis are unclear. Here, we show that the mouse erythroblast forms large, transient, and recurrent nuclear openings that coincide with the condensation process. The opening lacks nuclear lamina, nuclear pore complexes, and nuclear membrane, but it is distinct from nuclear envelope changes that occur during apoptosis and mitosis. A fraction of the major histones are released from the nuclear opening and degraded in the cytoplasm. We demonstrate that caspase-3 is required for the nuclear opening formation throughout terminal erythropoiesis. Loss of caspase-3 or ectopic expression of a caspase-3 non-cleavable lamin B mutant blocks nuclear opening formation, histone release, chromatin condensation, and terminal erythroid differentiation. We conclude that caspase-3-mediated nuclear opening formation accompanied by histone release from the opening is a critical step toward chromatin condensation during erythropoiesis in mice.

Aberrant overexpression of CD14 on granulocytes sensitizes the innate immune response in mDia1 heterozygous del(5q) MDS

The myelodysplastic syndromes (MDSs) include a spectrum of stem cell malignancies characterized by an increased risk of developing acute myeloid leukemia. Heterozygous loss of chromosome 5q (del[5q]) is the most common cytogenetic abnormality in MDS. DIAPH1 is localized to 5q31 and encodes one of the formin proteins, mDia1, which is involved in linear actin polymerization. Mice with mDia1 deficiency develop hematologic features with age mimicking human myeloid neoplasm, but its role in the pathogenesis of MDS is unclear. Here we report that mDia1 heterozygous and knockout mice develop MDS phenotypes with age. In these mice, CD14 was aberrantly overexpressed on granulocytes in a cell-autonomous manner, leading to a hypersensitive innate immune response to lipopolysaccharide (LPS) stimuli through CD14/Toll-like receptor 4 signaling. Chronic stimulation with LPS accelerated the development of MDS in mDia1 heterozygous and knockout mice that can be rescued by lenalidomide. Similar findings of CD14 overexpression were observed on the bone marrow granulocytes of del(5q) MDS patients. Mechanistically, mDia1 deficiency led to a downregulation of membrane-associated genes and a specific upregulation of CD14 messenger RNA in granulocytes, but not in other lineages. These results underscore the significance of mDia1 heterozygosity in deregulated innate immune responses in del(5q) MDS.

Targeted shRNA screening identified critical roles of pleckstrin-2 in erythropoiesis

Differentiation of erythroblasts to mature red blood cells involves dynamic changes of the membrane and cytoskeleton networks that are not fully characterized. Using a mouse fetal liver erythroblast culture system and a targeted shRNA functional screening strategy, we identified a critical role of pleckstrin-2 in actin dynamics and protection of early stage terminal erythroblasts from oxidative damage. Knockdown of pleckstrin-2 in the early stage of terminal erythropoiesis disrupted the actin cytoskeleton and led to differentiation inhibition and apoptosis. This pro-survival and differentiation function of pleckstrin-2 was mediated through its interaction with cofilin, by preventing cofilin’s mitochondrial entry when the intracellular level of reactive oxygen species was higher in the early stage of terminal erythropoiesis. Treatment of the cells with a scavenger of reactive oxygen species rescued cofilin’s mitochondrial entry and differentiation inhibition induced by pleckstrin-2 knockdown. In contrast, pleckstrin-2 knockdown in late stage terminal erythroblasts had no effect on survival or differentiation but blocked enucleation due to disorganized actin cytoskeleton. Thus, our study identified a dual function of pleckstrin-2 in the early and late stages of terminal erythropoiesis through its regulations of actin dynamics and cofilin’s mitochondrial localization, which reflects intracellular level of reactive oxygen species in different developmental stages.

Ineffective erythropoiesis caused by binucleated late-stage erythroblasts in mDia2 hematopoietic specific knockout mice.

The generation of mature red blood cells is initiated from the commitment of hematopoietic stem cells to erythroid progenitors, which is followed by their differentiation to a series of morphologically recognizable erythroblasts.1 At the end of terminal erythropoiesis, the highly condensed nucleus migrates to one side of the cytoplasm of orthochromatic erythroblast, which is followed by the unique enucleation process producing reticulocytes and mature red blood cells.2–4 Our previous study demonstrated that mDia2, which belongs to the mDia formin protein family,5 is a downstream effector protein of Rac GTPases regulating late-stage terminal erythropoiesis, especially enucleation.6 Herein we generated conditional mDia2 knockout mouse models to reveal the roles of mDia2 in adult erythropoiesis. The conditional mDia2 knockout mouse models utilized an mDia2 targeting allele with exons 10 and 11 floxed. The LacZ and neomycin cassettes were flanked by FRT to be removed by FLP recombinase (Online Supplementary Figure S1A). We first crossed mDia2fl/+ mice with E2A-Cre transgenic mice to generate whole body mDia2 knockout mice. The depletion of mDia2 mRNA and protein were confirmed by real-time PCR and Western blot assays (Online Supplementary Figure S1B and S1C, respectively). As previously reported,7 mDia2fl/fl E2A-Cre mice were never generated alive (Online Supplementary Figure S1D). We found that the mDia2 knockout embryos die in uterus at approximately embryonic day 12.5 (E12.5) (Online Supplementary Figure S1E). This demonstrates that mDia2 is essential for embryonic development, which compromises the study of the roles of mDia2 in vivo in adults.

Age-related inflammatory bone marrow microenvironment induces ineffective erythropoiesis mimicking del(5q) MDS

Anemia is characteristic of myelodysplastic syndromes (MDS). The mechanisms of anemia in MDS are unclear. Using a mouse genetic approach, here we show that dual deficiency of mDia1 and miR-146a, encoded on chromosome 5q and commonly deleted in MDS (del(5q) MDS), causes an age-related anemia and ineffective erythropoiesis mimicking human MDS. We demonstrate that the ageing bone marrow microenvironment is important for the development of ineffective erythropoiesis in these mice. Damage-associated molecular pattern molecules (DAMPs), whose levels increase in ageing bone marrow, induced TNFα and IL-6 upregulation in myeloid-derived suppressor cells (MDSCs) in mDia1/miR-146a double knockout mice. Mechanistically, we reveal that pathologic levels of TNFα and IL-6 inhibit erythroid colony formation and differentially affect terminal erythropoiesis through reactive oxygen species-induced caspase-3 activation and apoptosis. Treatment of the mDia1/miR-146a double knockout mice with all-trans retinoic acid, which promoted the differentiation of MDSCs and ameliorated the inflammatory bone marrow microenvironment, significantly rescued anemia and ineffective erythropoiesis. Our study underscores the dual roles of the ageing microenvironment and genetic abnormalities in the pathogenesis of ineffective erythropoiesis in del(5q) MDS.

H2AX deficiency is associated with erythroid dysplasia and compromised haematopoietic stem cell function.

Myelodysplastic syndromes (MDS) are clonal disorders of haematopoiesis characterised by dysplastic changes of major myeloid cell lines. However, the mechanisms underlying these dysplastic changes are poorly understood. Here, we used a genetically modified mouse model and human patient data to examine the physiological roles of H2AX in haematopoiesis and how the loss of H2AX contributes to dyserythropoiesis in MDS. H2AX knockout mice showed cell-autonomous anaemia and erythroid dysplasia, mimicking dyserythropoiesis in MDS. Also, dyserythropoiesis was increased in MDS patients with the deletion of chromosome 11q23, where H2AX is located. Although loss of H2AX did not affect the early stage of terminal erythropoiesis, enucleation was decreased. H2AX deficiency also led to the loss of quiescence of hematopoietic stem and progenitor cells, which dramatically compromised their bone marrow engraftment. These results reveal important roles of H2AX in late-stage terminal erythropoiesis and hematopoietic stem cell function.

Chromatin condensation during terminal erythropoiesis.

ABSTRACT Mammalian terminal erythropoiesis involves gradual but dramatic chromatin condensation steps that are essential for cell differentiation. Chromatin and nuclear condensation is followed by a unique enucleation process, which is believed to liberate more spaces for hemoglobin enrichment and enable the generation of a physically flexible mature red blood cell. Although these processes have been known for decades, the mechanisms are still unclear. Our recent study reveals an unexpected nuclear opening formation during mouse terminal erythropoiesis that requires caspase-3 activity. Major histones, except H2AZ, are partially released from the opening, which is important for chromatin condensation. Block of the nuclear opening through caspase inhibitor or knockdown of caspase-3 inhibits chromatin condensation and enucleation. We also demonstrate that nuclear opening and histone release are cell cycle regulated. These studies reveal a novel mechanism for chromatin condensation in mammalia terminal erythropoiesis.

Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony forming units (CFU-Es). After CFU-Es, cells are morphologically recognizable and generally termed terminal erythroblasts. One of the challenges for the study of terminal erythropoiesis is the lack of experimental approaches to dissect gene functions in a chronological manner. In this protocol, we describe a unique strategy to determine gene functions in the early and late stages of terminal erythropoiesis. In this system, mouse fetal liver TER119 (mature erythroid cell marker) negative erythroblasts were purified and transduced with exogenous expression of cDNAs or small hairpin RNAs (shRNAs) for the genes of interest. The cells were subsequently cultured in medium containing growth factors other than erythropoietin (Epo) to maintain their progenitor stage for 12 hr while allowing the exogenous cDNAs or shRNAs to express. The cells were changed to Epo medium after 12 hr to induce cell differentiation and proliferation while the exogenous genetic materials were already expressed. This protocol facilitates analysis of gene functions in the early stage of terminal erythropoiesis. To study late stage terminal erythropoiesis, cells were immediately cultured in Epo medium after transduction. In this way, the cells were already differentiated to the late stage of terminal erythropoiesis when the transduced genetic materials were expressed. We recommend a general application of this strategy that would help understand detailed gene functions in different stages of terminal erythropoiesis.

Loss of pleckstrin-2 reverts lethality and vascular occlusions in JAK2V617F-positive myeloproliferative neoplasms

V617F driver mutation of JAK2 is the leading cause of the Philadelphia-chromosome-negative myeloproliferative neoplasms (MPNs). Although thrombosis is a leading cause of mortality and morbidity in MPNs, the mechanisms underlying their pathogenesis are unclear. Here, we identified pleckstrin-2 (Plek2) as a downstream target of the JAK2/STAT5 pathway in erythroid and myeloid cells, and showed that it is upregulated in a JAK2V617F-positive MPN mouse model and in patients with MPNs. Loss of Plek2 ameliorated JAK2V617F-induced myeloproliferative phenotypes including erythrocytosis, neutrophilia, thrombocytosis, and splenomegaly, thereby reverting the widespread vascular occlusions and lethality in JAK2V617F-knockin mice. Additionally, we demonstrated that a reduction in red blood cell mass was the main contributing factor in the reversion of vascular occlusions. Thus, our study identifies Plek2 as an effector of the JAK2/STAT5 pathway and a key factor in the pathogenesis of JAK2V617F-induced MPNs, pointing to Plek2 as a viable target for the treatment of MPNs.

Loss of mDia1 causes neutropenia via attenuated CD11b endocytosis and increased neutrophil adhesion to the endothelium

Formin protein mDia1 is involved in actin polymerization and plays important roles in the migration and adhesion of hematopoietic cells. The mDia1 encoding gene is located on chromosome 5q, which is commonly deleted in patients with del(5q) myelodysplastic syndromes (MDSs). We previously reported that mice with mDia1 deficiency developed neutropenia that closely mimics MDS. However, the mechanism of neutropenia in these mice and patients with del(5q) MDS remains incompletely defined. Here, we reveal that mDia1 knockout mice show cell-autonomously increased CD11b expression on neutrophils in the peripheral blood and bone marrow. The level of CD11b was also higher in patients with del(5q) MDS compared with normal individuals. Mechanistically, loss of mDia1 significantly attenuated the endocytosis of CD11b on neutrophils, which led to an increased number of neutrophils adhering to the blood vessels in mDia1 knockout mice. Administration of CD11b antibody to mDia1 knockout mice reduced the adhesion of neutrophils to the vessels and rescued neutropenia. Our study reveals the role of mDia1 deficiency in the upregulation of CD11b on neutrophils, which leads to their increased binding to the blood vessels. These results may provide important clues for the pathogenesis of neutropenia in patients with del(5q) MDS.