CLASP2 safeguards hematopoietic stem cell properties during mouse and fish development.

Hematopoietic stem cells (HSCs) express a large variety of cell surface receptors that are associated with acquisition of self-renewal and multipotent properties. Correct expression of these receptors depends on a delicate balance between cell surface trafficking, recycling, and degradation and is controlled by the microtubule network and Golgi apparatus, whose roles have hardly been explored during embryonic/fetal hematopoiesis. Here we show that, in the absence of CLASP2, a microtubule-associated protein, the overall production of HSCs is reduced, and the produced HSCs fail to self-renew and maintain their stemness throughout mouse and zebrafish development. This phenotype can be attributed to decreased cell surface expression of the hematopoietic receptor c-Kit, which originates from increased lysosomal degradation in combination with a reduction in trafficking to the plasma membrane. A dysfunctional Golgi apparatus in CLASP2-deficient HSCs seems to be the underlying cause of the c-Kit expression and signaling imbalance.

Chemotherapy-induced transposable elements activate MDA5 to enhance haematopoietic regeneration.

Haematopoietic stem cells (HSCs) are normally quiescent, but have evolved mechanisms to respond to stress. Here, we evaluate haematopoietic regeneration induced by chemotherapy. We detect robust chromatin reorganization followed by increased transcription of transposable elements (TEs) during early recovery. TE transcripts bind to and activate the innate immune receptor melanoma differentiation-associated protein 5 (MDA5) that generates an inflammatory response that is necessary for HSCs to exit quiescence. HSCs that lack MDA5 exhibit an impaired inflammatory response after chemotherapy and retain their quiescence, with consequent better long-term repopulation capacity. We show that the overexpression of ERV and LINE superfamily TE copies in wild-type HSCs, but not in Mda5-/- HSCs, results in their cycling. By contrast, after knockdown of LINE1 family copies, HSCs retain their quiescence. Our results show that TE transcripts act as ligands that activate MDA5 during haematopoietic regeneration, thereby enabling HSCs to mount an inflammatory response necessary for their exit from quiescence.

ZFP451-mediated SUMOylation of SATB2 drives embryonic stem cell differentiation.

The establishment of cell fates involves alterations of transcription factor repertoires and repurposing of transcription factors by post-translational modifications. In embryonic stem cells (ESCs), the chromatin organizers SATB2 and SATB1 balance pluripotency and differentiation by activating and repressing pluripotency genes, respectively. Here, we show that conditional Satb2 gene inactivation weakens ESC pluripotency, and we identify SUMO2 modification of SATB2 by the E3 ligase ZFP451 as a potential driver of ESC differentiation. Mutations of two SUMO-acceptor lysines of Satb2 (Satb2K →R ) or knockout of Zfp451 impair the ability of ESCs to silence pluripotency genes and activate differentiation-associated genes in response to retinoic acid (RA) treatment. Notably, the forced expression of a SUMO2-SATB2 fusion protein in either Satb2K →R or Zfp451-/- ESCs rescues, in part, their impaired differentiation potential and enhances the down-regulation of Nanog The differentiation defect of Satb2K →R ESCs correlates with altered higher-order chromatin interactions relative to Satb2wt ESCs. Upon RA treatment of Satb2wt ESCs, SATB2 interacts with ZFP451 and the LSD1/CoREST complex and gains binding at differentiation genes, which is not observed in RA-treated Satb2K →R cells. Thus, SATB2 SUMOylation may contribute to the rewiring of transcriptional networks and the chromatin interactome of ESCs in the transition of pluripotency to differentiation.

Dynamic Cardiolipin Synthesis Is Required for CD8+ T Cell Immunity.

Mitochondria constantly adapt to the metabolic needs of a cell. This mitochondrial plasticity is critical to T cells, which modulate metabolism depending on antigen-driven signals and environment. We show here that de novo synthesis of the mitochondrial membrane-specific lipid cardiolipin maintains CD8+ T cell function. T cells deficient for the cardiolipin-synthesizing enzyme PTPMT1 had reduced cardiolipin and responded poorly to antigen because basal cardiolipin levels were required for activation. However, neither de novo cardiolipin synthesis, nor its Tafazzin-dependent remodeling, was needed for T cell activation. In contrast, PTPMT1-dependent cardiolipin synthesis was vital when mitochondrial fitness was required, most notably during memory T cell differentiation or nutrient stress. We also found CD8+ T cell defects in a small cohort of patients with Barth syndrome, where TAFAZZIN is mutated, and in a Tafazzin-deficient mouse model. Thus, the dynamic regulation of a single mitochondrial lipid is crucial for CD8+ T cell immunity.

Repetitive Elements Trigger RIG-I-like Receptor Signaling that Regulates the Emergence of Hematopoietic Stem and Progenitor Cells.

Inflammatory signaling is required for hematopoietic stem and progenitor cell (HSPC) development. Here, we studied the involvement of RIG-I-like receptors (RLRs) in HSPC formation. Rig-I or Mda5 deficiency impaired, while Lgp2 deficiency enhanced, HSPC emergence in zebrafish embryos. Rig-I or Mda5 deficiency reduced HSPC numbers by inhibiting inflammatory signals that were in turn enhanced in Lgp2 deficient embryos. Simultaneous reduction of Lgp2 and either Rig-I or Mda5 rescued inflammatory signals and HSPC numbers. Modulating the expression of the signaling mediator Traf6 in RLR deficient embryos restored HSPC numbers. Repetitive element transcripts could be detected in hemogenic endothelial cells and HSPCs, suggesting a role as RLR ligands. Indeed, ectopic expression of repetitive elements enhanced HSPC formation in wild-type, but not in Rig-I or Mda5 deficient embryos. Manipulation of RLR expression in mouse fetal liver HSPCs indicated functional conservation among species. Thus, repetitive elements transcribed during development drive RLR-mediated inflammatory signals that regulate HSPC formation.

Lymphocyte-Specific Function of the DNA Polymerase Epsilon Subunit Pole3 Revealed by Neomorphic Alleles.

Immunodeficiencies are typically caused by loss-of-function mutations in lymphocyte-specific genes. Occasionally, mutations in ubiquitous general-purpose factors, including those affecting essential components of the DNA polymerase epsilon (POLE) holoenzyme, have cell-type-specific consequences. POLE3, one of the four components of the POLE holoenzyme, features a histone fold domain and a unique acidic C terminus, making it a particularly attractive candidate mediating cell type-specific activities of POLE. Mice lacking Pole3 survive up to late embryonic stages, indicating that this subunit is dispensable for DNA replication. The phenotypes of viable hypomorphic and neomorphic alleles are surprisingly tissue restricted and reveal a stage-specific function of the histone fold domain of Pole3 during T and B cell development. Gradual introduction of positively charged residues into the acidic C terminus leads to peripheral lymphopenia of increasing severity. Our findings serve as a paradigm to understand the molecular basis of cell-type-specific non-replicative functions of the ubiquitous POLE complex.

Hematopoietic regeneration under the spell of epigenetic-epitranscriptomic factors and transposable elements.

PURPOSE OF REVIEW: Since the discovery of master transcription factors that regulate hematopoietic regeneration following different stressors, many more layers of regulation have been discovered. The purpose of this review is to outline the recent discoveries of epigenetic and epitranscriptomic control of hematopoietic regeneration and highlight the novel involvement of transposable elements in this process. RECENT FINDINGS: Over the past 2 years, we have gained additional knowledge in the role of epigenetic regulators in hematopoietic regeneration. Histone modifiers, like SETD1A, JARID2, KDM6B, and classic DNA methylation regulators, like DNMT3A and TET2, govern hematopoietic regeneration. Concomitantly, the significance of RNA modifications and the expanding functions of transposable elements establish novel layers of regulation of hematopoietic regeneration. Capitalizing on this newly acquired knowledge may provide insights on new therapies or drug targets that will improve or accelerate hematopoietic regeneration. SUMMARY: The spectrum of epigenetic and epitranscriptomic modifications that affect hematopoietic regeneration is continually expanding. Transposable elements are also emerging as potent responders of stress stimuli that affect the self-renewal capacity of hematopoietic stem cells. The future challenge is to understand the hierarchy of these control mechanisms and how they integrate and consolidate information from transcription factors and external stimuli.

A metabolic interplay coordinated by HLX regulates myeloid differentiation and AML through partly overlapping pathways.

The H2.0-like homeobox transcription factor (HLX) regulates hematopoietic differentiation and is overexpressed in Acute Myeloid Leukemia (AML), but the mechanisms underlying these functions remain unclear. We demonstrate here that HLX overexpression leads to a myeloid differentiation block both in zebrafish and human hematopoietic stem and progenitor cells (HSPCs). We show that HLX overexpression leads to downregulation of genes encoding electron transport chain (ETC) components and upregulation of PPARδ gene expression in zebrafish and human HSPCs. HLX overexpression also results in AMPK activation. Pharmacological modulation of PPARδ signaling relieves the HLX-induced myeloid differentiation block and rescues HSPC loss upon HLX knockdown but it has no effect on AML cell lines. In contrast, AMPK inhibition results in reduced viability of AML cell lines, but minimally affects myeloid progenitors. This newly described role of HLX in regulating the metabolic state of hematopoietic cells may have important therapeutic implications.

Stress and Non-Stress Roles of Inflammatory Signals during HSC Emergence and Maintenance.

Hematopoietic stem cells (HSCs) are a rare population that gives rise to almost all cells of the hematopoietic system, including immune cells. Until recently, it was thought that immune cells sense inflammatory signaling and HSCs respond only secondarily to these signals. However, it was later shown that adult HSCs could directly sense and respond to inflammatory signals, resulting in a higher output of immune cells. Recent studies demonstrated that inflammatory signaling is also vital for HSC ontogeny. These signals are thought to arise in the absence of pathogens, are active during development, and indispensable for HSC formation. In contrast, during times of stress and disease, inflammatory responses can be activated and can have devastating effects on HSCs. In this review, we summarize the current knowledge about inflammatory signaling in HSC development and maintenance, as well as the endogenous molecular cues that can trigger inflammatory pathway activation. Finally, we comment of the role of inflammatory signaling in hematopoietic diseases.

GFI1 proteins orchestrate the emergence of haematopoietic stem cells through recruitment of LSD1.

In vertebrates, the first haematopoietic stem cells (HSCs) with multi-lineage and long-term repopulating potential arise in the AGM (aorta-gonad-mesonephros) region. These HSCs are generated from a rare and transient subset of endothelial cells, called haemogenic endothelium (HE), through an endothelial-to-haematopoietic transition (EHT). Here, we establish the absolute requirement of the transcriptional repressors GFI1 and GFI1B (growth factor independence 1 and 1B) in this unique trans-differentiation process. We first demonstrate that Gfi1 expression specifically defines the rare population of HE that generates emerging HSCs. We further establish that in the absence of GFI1 proteins, HSCs and haematopoietic progenitor cells are not produced in the AGM, revealing the critical requirement for GFI1 proteins in intra-embryonic EHT. Finally, we demonstrate that GFI1 proteins recruit the chromatin-modifying protein LSD1, a member of the CoREST repressive complex, to epigenetically silence the endothelial program in HE and allow the emergence of blood cells.

Progressive maturation toward hematopoietic stem cells in the mouse embryo aorta.

Clusters of cells attached to the endothelium of the main embryonic arteries were first observed a century ago. Present in most vertebrate species, such clusters, or intraaortic hematopoietic clusters (IAHCs), derive from specialized hemogenic endothelial cells and contain the first few hematopoietic stem cells (HSCs) generated during embryonic development. However, some discrepancies remained concerning the spatio-temporal appearance and the numbers of IAHCs and HSCs. Therefore, the exact cell composition and function of IAHCs remain unclear to date. We show here that IAHCs contain pre-HSCs (or HSC precursors) that can mature into HSCs in vivo (as shown by the successful long-term multilineage reconstitution of primary neonates and secondary adult recipients). Such IAHC pre-HSCs could contribute to the HSC pool increase observed at midgestation. The novel insights in pre-HSC to HSC transition represent an important step toward generating transplantable HSCs in vitro that are needed for autologous HSC transplantation therapies.

Erythropoietic defect associated with reduced cell proliferation in mice lacking the 26S proteasome shuttling factor Rad23b.

Rad23a and Rad23b proteins are linked to nucleotide excision DNA repair (NER) via association with the DNA damage recognition protein xeroderma pigmentosum group C (XPC) are and known to be implicated in protein turnover by the 26S proteasome. Rad23b-null mice are NER proficient, likely due to the redundant function of the Rad23b paralogue, Rad23a. However, Rad23b-null midgestation embryos are anemic, and most embryos die before birth. Using an unbiased proteomics approach, we found that the majority of Rad23b-interacting partners are associated with the ubiquitin-proteasome system (UPS). We tested the requirement for Rad23b-dependent UPS activity in cellular proliferation and more specifically in the process of erythropoiesis. In cultured fibroblasts derived from embryos lacking Rad23b, proliferation rates were reduced. In fetal livers of Rad23b-null embryos, we observed reduced proliferation, accumulation of early erythroid progenitors, and a block during erythroid maturation. In primary wild-type (WT) erythroid cells, knockdown of Rad23b or chemical inhibition of the proteasome reduced survival and differentiation capability. Finally, the defects linked to Rad23b loss specifically affected fetal definitive erythropoiesis and stress erythropoiesis in adult mice. Together, these data indicate a previously unappreciated requirement for Rad23b and the UPS in regulation of proliferation in different cell types.

Integrin αIIb (CD41) plays a role in the maintenance of hematopoietic stem cell activity in the mouse embryonic aorta.

Integrins are transmembrane receptors that play important roles as modulators of cell behaviour through their adhesion properties and the initiation of signaling cascades. The αIIb integrin subunit (CD41) is one of the first cell surface markers indicative of hematopoietic commitment. αIIb pairs exclusively with ß3 to form the αIIbß3 integrin. ß3 (CD61) also pairs with αv (CD51) to form the αvß3 integrin. The expression and putative role of these integrins during mouse hematopoietic development is as yet unknown. We show here that hematopoietic stem cells (HSCs) differentially express αIIbß3 and αvß3 integrins throughout development. Whereas the first HSCs generated in the aorta at mid-gestation express both integrins, HSCs from the placenta only express αvß3, and most fetal liver HSCs do not express either integrin. By using αIIb deficient embryos, we show that αIIb is not only a reliable HSC marker but it also plays an important and specific function in maintaining the HSC activity in the mouse embryonic aorta.

The microtubule plus-end tracking protein CLASP2 is required for hematopoiesis and hematopoietic stem cell maintenance.

Mammalian CLASPs are microtubule plus-end tracking proteins whose essential function as regulators of microtubule behavior has been studied mainly in cultured cells. We show here that absence of murine CLASP2 in vivo results in thrombocytopenia, progressive anemia, and pancytopenia, due to defects in megakaryopoiesis, in erythropoiesis, and in the maintenance of hematopoietic stem cell activity. Furthermore, microtubule stability and organization are affected upon attachment of Clasp2 knockout hematopoietic stem-cell-enriched populations, and these cells do not home efficiently toward their bone marrow niche. Strikingly, CLASP2-deficient hematopoietic stem cells contain severely reduced mRNA levels of c-Mpl, which encodes the thrombopoietin receptor, an essential factor for megakaryopoiesis and hematopoietic stem cell maintenance. Our data suggest that thrombopoietin signaling is impaired in Clasp2 knockout mice. We propose that the CLASP2-mediated stabilization of microtubules is required for proper attachment, homing, and maintenance of hematopoietic stem cells and that this is necessary to sustain c-Mpl transcription.

Embryonic development of hematopoietic stem cells: implications for clinical use.

Hematopoietic stem cell (HSC) transplantation is an important treatment modality for hematological malignancies or to correct congenital immunodeficiency disorders. Several stem cell sources are currently applied clinically, with a recent increased application of umbilical cord blood. The low number of HSCs available, particularly in umbilical cord blood, is a limiting factor, and different lines of research are ongoing to circumvent this issue. In this review, we will describe the research strategies developed to expand adult HSCs in vitro and to generate new HSCs from pluripotent stem cell lines. We will also discuss the importance of studying the embryonic microenvironment since it allows both generation and extensive expansion of HSCs. Understanding the mechanisms that underlie HSC production, self-renewal and differentiation is necessary for the establishment of optimal in vitro HSC cultures, where a limitless and manipulatable resource of HSCs would be available for both clinical and fundamental research.

Ex vivo time-lapse confocal imaging of the mouse embryo aorta.

Time-lapse confocal microscopy of mouse embryo slices was developed to access and image the living aorta. In this paper, we explain how to label all hematopoietic and endothelial cells inside the intact mouse aorta with fluorescent directly labeled antibodies. Then we describe the technique to cut nonfixed labeled embryos into thick slices that are further imaged by time-lapse confocal imaging. This approach allows direct observation of the dynamic cell behavior in the living aorta, which was previously inaccessible because of its location deep inside the opaque mouse embryo. In particular, this approach is sensitive enough to allow the experimenter to witness the transition from endothelial cells into hematopoietic stem/progenitor cells in the aorta, the first site of hematopoietic stem cell generation during development. The protocol can be applied to observe other embryonic sites throughout mouse development. A complete experiment requires ∼2 d of practical work.