scholarly journals Deficiency of Antioxidative Paraoxonase 2 (Pon2) Leads to Increased Number of Phenotypic LT-HSCs and Disturbed Erythropoiesis

2021 ◽  
Vol 2021 ◽  
pp. 1-18
Author(s):  
Lisa Spiecker ◽  
Ines Witte ◽  
Julia Mehlig ◽  
Viral Shah ◽  
Markus Meyerhöfer ◽  
...  

Background. Long-term hematopoietic stem cells (LT-HSCs) reside in bone marrow niches with tightly controlled reactive oxygen species (ROS) levels. ROS increase results into LT-HSC differentiation and stem cell exhaustion. Paraoxonase 2 (PON2) has been shown to be important for ROS control. Objectives. We investigate the effects of inactivation of the PON2 gene on hematopoietic cell differentiation and activity. Methods and Results. In young mice with inactivated Pon2 gene (Pon2-/-, <3 months), we observed an increase of LT-HSCs and a reduced frequency of progenitor cells. In competitive transplantations, young Pon2-/- BM outcompeted WT BM at early time points. ROS levels were significantly increased in Pon2-/- whole BM, but not in Pon2-/- LT-HSCs. In more differentiated stages of hematopoiesis, Pon2 deficiency led to a misbalanced erythropoiesis both in physiologic and stress conditions. In older mice (>9 months), Pon2 depletion caused an increase in LT-HSCs as well as increased levels of granulocyte/macrophage progenitors (GMPs) and myeloid skewing, indicating a premature aging phenotype. No significant changes in ROS levels in old Pon2-/- LT- and short-term (ST-) HSCs were observed, but a significant reduction of spontaneous apoptotic cell death was measured. RNA-seq analysis in Pon2-/- LT-HSCs identified overrepresentation of genes involved in the C-X-C chemokine receptor type 4 (Cxcr4) signaling, suggesting compensatory mechanisms to overcome ROS-mediated accelerated aging in hematopoietic progenitor cells. Conclusions. In summary, our current data indicate that PON2 is involved in the regulation of HSC functions.

Blood ◽  
1998 ◽  
Vol 92 (12) ◽  
pp. 4612-4621 ◽  
Author(s):  
M.A. Dao ◽  
K. Hashino ◽  
I. Kato ◽  
J.A. Nolta

Abstract Recent reports have indicated that there is poor engraftment from hematopoietic stem cells (HSC) that have traversed cell cycle ex vivo. However, inducing cells to cycle in culture is critical to the fields of ex vivo stem cell expansion and retroviral-mediated gene therapy. Through the use of a xenograft model, the current data shows that human hematopoietic stem and progenitor cells can traverse M phase ex vivo, integrate retroviral vectors, engraft, and sustain long-term hematopoiesis only if they have had the opportunity to engage their integrin receptors to fibronectin during the culture period. If cultured in suspension under the same conditions, transduction is undetectable and the long-term multilineage regenerative capacity of the primitive cells is severely diminished.


Blood ◽  
2017 ◽  
Vol 129 (18) ◽  
pp. 2479-2492 ◽  
Author(s):  
Wei Shi ◽  
Therese Vu ◽  
Didier Boucher ◽  
Anna Biernacka ◽  
Jules Nde ◽  
...  

Key Points Combined loss of Ssb1/Ssb2 induces rapid lethality due to replication stress–associated loss of hematopoietic stem and progenitor cells. Functionally, loss of Ssb1/Ssb2 activates p53 and IFN pathways, causing enforced cell cycling in quiescent HSPCs and apoptotic cell loss.


Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 509-509
Author(s):  
Jonathan Kenyon ◽  
Emily Thomas ◽  
Karen Lingas ◽  
Stanton L. Gerson

Abstract The etiology of hematologic pathologies such as leukemia, myelodysplasia, anemia, bone marrow failure, altered immune function, and how they are associated with aging, remains unclear. Our hypothesis is that these diseases are caused or aggravated by a subset of hematopoietic stem/progenitor cells (HSC) lacking effective mismatch repair (MMR) and therefore exhibiting a hypermutator phenotype. Microsatellite instability (MSI) is a marker of MMR deficiency. We used cord blood, bone marrow, and bone core samples to isolate and then clonally expand HSC for MSI analysis. Five microsatellite loci previously used in the diagnosis of the MMR defective disease HNPCC (BAT 25, BAT 26, D2S123, D5S346, and D17S250) were analyzed for insertions and deletions. We have analyzed 38 patient samples between the ages of 0 and 86 years, including 8 cancer patients. These data show an age-dependent increase in the frequency of high grade microsatellite instability (MSI-H), i.e. those CFU with microsatellite instability at >20% of loci tested. Additionally, samples obtained from individuals older than 50 years were 6 times more likely to have a > 10% frequency of MSI-H CFU than samples obtained from younger individuals, suggesting an inflection point for the onset of hematopoietic diseases. In all instances this instability is seen only within a subset of human HSC clones. To further characterize the origin of this deficiency, a method to select for MMR deficient hematopoietic cells was developed that first selected for survival of MMR deficient HSC, and then allowed for the examination of expression status of key MMR pathway genes hMLH1 and hMSH2 and their protein products. First, CD34+ HSC were isolated from various aged patient samples. To avoid possible effects of other repair pathways, the cells were treated with O6-Benzylguanine (BG) to remove O6-methylguanine DNA-methyltransferase (MGMT) activity and prevent removal of O6-methylguanine lesions. Next, temozolomide (TMZ) at concentrations of 50–125 μM was used to induce O6-methylguanine (O6-mG) lesions that persist in the presence of BG. These O6-mG lesions mispair with cytosine and are recognized as DNA mismatches by the mismatch repair (MMR) pathway inducing apoptotic cell death. TMZ selected cells that fail to recognize the mispair due to a lack of MMR survive this selection. In these TMZ resistant clones, RT-PCR amplification of hMLH1 transcripts from total RNA isolated reveal a defect in hMLH1 but not hMSH2 expression. In the one AML sample obtained thus far HSC treated with 200 uM TMZ we have observed 0 to 30% of hMLH1 expression within TMZ resistant CFU was observed when compared to untreated controls. Together this data links MSI to MMR defects of a subpopulation of hematopoietic precursors in older individuals. This is the first examination of MMR gene expression in clones of HSC that has shown specific MMR functional deficiencies. Our study suggests that a MMR pathway deficiency in a subset of stem cells could contribute to age related hematopoietic disease processes including stem cell failure and malignant transformation.


2017 ◽  
Vol 114 (33) ◽  
pp. E6875-E6883 ◽  
Author(s):  
Bas Pilzecker ◽  
Olimpia Alessandra Buoninfante ◽  
Paul van den Berk ◽  
Cesare Lancini ◽  
Ji-Ying Song ◽  
...  

DNA damage tolerance (DDT) enables bypassing of DNA lesions during replication, thereby preventing fork stalling, replication stress, and secondary DNA damage related to fork stalling. Three modes of DDT have been documented: translesion synthesis (TLS), template switching (TS), and repriming. TLS and TS depend on site-specific PCNA K164 monoubiquitination and polyubiquitination, respectively. To investigate the role of DDT in maintaining hematopoietic stem cells (HSCs) and progenitors, we used PcnaK164R/K164R mice as a unique DDT-defective mouse model. Analysis of the composition of HSCs and HSC-derived multipotent progenitors (MPPs) revealed a significantly reduced number of HSCs, likely owing to increased differentiation of HSCs toward myeloid/erythroid-associated MPP2s. This skewing came at the expense of the number of lymphoid-primed MPP4s, which appeared to be compensated for by increased MPP4 proliferation. Furthermore, defective DDT decreased the numbers of MPP-derived common lymphoid progenitor (CLP), common myeloid progenitor (CMP), megakaryocyte-erythroid progenitor (MEP), and granulocyte-macrophage progenitor (GMP) cells, accompanied by increased cell cycle arrest in CMPs. The HSC and MPP phenotypes are reminiscent of premature aging and stressed hematopoiesis, and indeed progressed with age and were exacerbated on cisplatin exposure. Bone marrow transplantations revealed a strong cell intrinsic defect of DDT-deficient HSCs in reconstituting lethally irradiated mice and a strong competitive disadvantage when cotransplanted with wild-type HSCs. These findings indicate a critical role of DDT in maintaining HSCs and progenitor cells, and in preventing premature aging.


2021 ◽  
Vol 220 (11) ◽  
Author(s):  
Michael L. Dustin

Hematopoietic stem and progenitor cells (HSPCs) use specialized adhesive structures referred to as magnupodium to stay in hematopoietic niches. Bessey et al. (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202005085) define new characteristics of the magnupodium, including centriole polarization and the necessary and sufficient role of CXCR4 signaling.


Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 941-941
Author(s):  
Pratibha Singh ◽  
Jennifer Speth ◽  
Peirong Hu ◽  
Louis M. Pelus

Abstract Abstract 941 Hematopoietic stem cells reside in osteoblastic and vascular niches within the bone marrow. The osteoblastic niche is composed of mesenchymal stem cell derived progenitor cells (MPC) and osteoblasts and are the main sources of the CXC chemokine CXCL12/SDF-1 in the bone marrow microenvironment. Several published studies suggest that the interaction between CXCR4 expressed on hematopoietic stem cells with SDF-1 produced in the bone marrow microenvironment is important for their retention in the bone-marrow. However, the role of SDF-CXCR4 signaling in formation and maintenance of osteoblastic niches in the bone marrow is not known. In this study, we examined the role of CXCR4 signaling in MPC proliferation and differentiation and its effects on hematopoietic stem cell (HSC) function. Flow cytometry analysis demonstrated that CXCR4 is expressed on the phenotypically defined MPC. Deletion of CXCR4 in tamoxifen cre inducible CXCR4flox-flox mice (verified by PCR and flow cytometry; 90% gene deletion and surface CXCR4 expression) results in significantly decreased numbers of Lin- CD45- CD31- Sca-1+ ALCAM- MPC (39±4.2%) and Lin- CD45- CD31- Sca-1-CD51+ osteoblasts (25±2.6%) in bone marrow 15 days after tamoxifen treatment. SDF-1 induced proliferation of CXCR4 deficient MPC was decreased by 4-fold compared to control, measured by the colony forming unit-fibroblast (CFU-F) assay. To determine, whether CXCR4 deficiency in bone marrow stromal cells affects SDF-1 induced HSC proliferation, we cultured FACS sorted wild-type SLAM SKL (103 cells) on CXCR4 deficient stroma for 5 days and total SLAM SKL cell numbers were counted by flow-cytometey analysis. CXCR4 deficient stroma failed to support optimal HSC proliferation and 48±5.2% less SLAM KSL cells was observed on CXCR4 deficient stroma compared to wild-type stroma. To investigate the mechanisms through which CXCR4-SDF-1 signaling regulates MPC proliferation, we evaluated the effect of SDF-1 treatment on expression of the anti-apoptotic and cell-cycle regulator protein, Survivin, in MPC. Multivariate intracellular flow cytometry demonstrated that Survivin expression increased by 23±4.2% in wild-type MPC after SDF-1 treatment (50ng/ml), however no significant increased was demonstrated in CXCR4 deficient MPC cells. CFU-F formation was reduced by 2.5 fold when the Survivin gene was conditionally deleted in MPC. Moreover, fewer SLAM SKL cells were detected on Survivin deficient stroma compared to wild-type stroma after SDF-1 treatment for 5 days. In conclusion, our data suggest that CXCR4-SDF-1 signaling mediated Survivin expression in MPC is important for their proliferation and maintenance of the bone-marrow hematopoietic niche. Disclosures: No relevant conflicts of interest to declare.


Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5126-5126
Author(s):  
Atsushi Sato ◽  
Hideyo Hirai ◽  
Akihiro Tamura ◽  
Asumi Yokota ◽  
Yoshihiro Hayashi ◽  
...  

Abstract Our previous findings have revealed the requirement of CCAAT Enhancer Binding Protein (C/EBPb), a leucine zipper transcription factor, in granulopoiesis (Hirai et al. Nat Immunol, 2006). During emergency situations such as infection, C/EBPb is involved in the sufficient supply of granulocytes through amplification of hematopoietic stem and progenitor cells (HSPCs) (Satake et al. J Immunol, 2012). In addition, we have shown that C/EBPb is upregulated by downstream signaling of BCR-ABL and promotes myeloid expansion and exhaustion of leukemic stem cells in chronic phase chronic myeloid leukemia (Hayashi et al. Leukemia, 2013). These observations suggested that C/EBPb plays important roles in regulation of normal and leukemic HSPCs. In this study, we focus on the functions of C/EBPb in normal HSPCs under stressed conditions. At steady state, the frequencies of HSPCs in the bone marrow (BM) of C/EBPb knockout (KO) mice were identical to those in the BM of wild type (WT) mice. It suggests that C/EBPb has little impact on the emergence or maintenance of HSPCs during steady state. To investigate function of C/EBPb in HSPCs, competitive repopulation assay was performed. Total BM cells from either WT or KO mice (CD45.2+) and the equal number of competitor cells from the BM of CD45.1+ WT mice were transplanted into lethally irradiated recipient WT mice (CD45.1+), and the chimerism of CD45.2+ cells in the peripheral blood (PB) of the recipient mice was monitored once a month. Chimerism of KO cells in the recipient mice was significantly lower than that of WT cells at 1 month after transplantation (52.2 ± 10.3% vs 37.8 ± 8.8%, p < 0.0000001, n = 37 vs 36) and the differences were maintained thereafter (Figure 1), suggesting that C/EBPb is required at early time points after transplantation. In order to elucidate the early events which make difference in the chimerism, homing ability was assessed first. Sixteen hours after transplantation of lineage depleted WT or KO BM cells (CD45.2+) together with lineage negative CD45.1+ WT BM cells, the frequencies of CD45.2+ WT and KO donor cells in the c-kit+ Sca1+ lineage- (KSL) fraction were identical. Then we compared the initial expansion of HSPCs. Purified 1000 KSL cells from either WT or KO mice (CD45.2+) were transplanted to lethally irradiated recipient WT mice (CD45.1+ / CD45.2+) together with the equal number of competitor KSL cells from WT mice (CD45.1+). The ratio of CD45.2+ KO cells to CD45.1+ competitors in the KSL fraction of the recipient mice was significantly lower than that of CD45.2+ WT cells at 4 weeks after transplantation (6.76 ± 2.35 vs 2.84 ± 1.16, p = 0.040, n = 4 vs 4). These results suggest that C/EBPb is required for initial expansion of HSPCs rather than for homing after transplantation. Next, we investigated the roles of C/EBPb in maintenance of HSPCs under stressed conditions. By staining of intracellular C/EBPb in combination with multi-color flow cytometric analysis, we found that C/EBPb is upregulated at protein level in KSL cells of WT mice 5 days after intraperitoneal injection of 5-fluorouracil (5-FU). Then the recipient mice were repetitively administered with 5-FU (150mg/kg i.p.) after BM transplantation in a competitive way. As mentioned above, the chimerism of KO cells in PB of recipient mice was significantly lower than those of WT mice at 1 month after transplantation. Interestingly, the chimerism of KO cells gradually increased by repetitive administration of 5-FU and even overtook those of WT cells 5 months after transplantation (Figure 2). In accordance with the changes observed in the PB, the chimerism of KO cells in the KSL fraction in the BM of recipient mice was significantly higher than those of WT cells (70.7 ± 25.3% vs 12.1 ± 9.78%, p = 0.016, n = 5 vs 4) 5 months after transplantation, suggesting that WT HSPCs exhausted earlier than KO HSPCs in response to hematopoietic stress. From these findings, we conclude that C/EBPb is required for initial expansion and exhaustion of HSPCs after hematopoietic stresses. We are currently investigating the molecular targets of C/EBPb and its clinical significance in the pathogenesis of leukemia. Disclosures No relevant conflicts of interest to declare.


Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3392-3392
Author(s):  
Adriana C Drost ◽  
Ute Krauß ◽  
Lothar Kanz ◽  
Robert Möhle

Abstract Abstract 3392 CD34+ hematopoietic stem- and progenitor cells (HPC) express high levels of the G protein-coupled receptors (GPCR) CXCR4 and CysLT1. In contrast to the established role of CXCR4 in stem cell homing, the function of CysLT1 in HPC remains only partially understood. We found that upon stimulation of peripheral blood CD34+ HPC in vitro with the respective ligands (CXCL12/SDF1 for CXCR4 and the cysteinyl-leukotriene LTD4 for CysLT1), both receptors similarly mediate the following functional activities: intracellular calcium fluxes, actin polymerization, adhesion to endothelium in vitro and chemotaxis. By Westernblot analysis, we could demonstrate Pyk2- and MAP-kinase phosphorylation as pivotal elements of CysLT1 signaling. These pathways have previously been identified also in CXCR4 signaling. To further analyze signal transduction pathways of both receptors, CD34+ cells were pretreated with pertussis toxin (PTX) or with a specific PKC inhibitor, which is an isoform-specific inhibitory myristoylated peptide derived from the pseudosubstrate (PS) region of PKCzeta, mimics the substrate, and maintains PKC in its nonactive isoform. Subsequently, we determined actin polymerization by flow cytometry using phalloidin-FITC, and adhesion to IL-1 stimulated endothelial cells (HUVEC). CXCR4 signaling leading to actin polymerization was found to be completely blocked by preincubation with pertussis toxin (PTX) and therefore is mediated exclusively by Gi proteins, while CysLT1 also involves Gq proteins as reflected by only partial inhibition by PTX. For both receptors, the Pyk2 signaling pathway leading to actin polymerization and adhesion was completely suppressed by preincubation with PSzeta and therefore dependent on atypical PKCzeta, which is calcium and DAG independent. We further examined whether these two similarly functioning receptors maintain any crosstalk, as has been reported for GPCR. Their possible interaction was explored using actin polymerization as a functional read-out. CXCR4- and CysLT1-mediated actin polymerization in response to their respective ligand was induced within 10 sec and returned to basal levels after 4 min. A second challenge after 4 min with the same ligand resulted in a complete suppression, demonstrating self-desensitization of both receptors. Interestingly, restimulating CXCL12-induced cells with LTD4 after 4 min resulted in complete suppression of actin polymerization, whereas restimulating LTD4-induced cells with CXCL12 lead to F-actin levels comparable to those achieved with the first challenge. These data show for the first time that CXCR4 can cross-desensitize CysLT1 while there is no crosstalk from CysLT1 to CXCR4. In conclusion, CXCR4 and CysLT1 share major signaling pathways. However, the ability to desensitize other GPCR is strikingly different. The finding that CXCR4 cross-desensitizes CysLT1 but not vice versa could explain our observation that a CysLT1 antagonist (montelukast) did not mobilize HPC in vivo, as the presence of CXCL12 in the stem cell niche may result in desensitization of CysLT1. In contrast, CXCR4-dependent bone marrow homing may not be influenced by conditions with high local and systemic cysteinyl-leukotriene concentrations, e.g. during allergy and inflammation. In the absence of CXCR4 activation however, CysLT1 could be important for homing of stem and progenitor cells in areas other than the bone marrow with a high local concentration of cysteinyl leukotrienes, e.g. in inflamed tissues. Disclosures: No relevant conflicts of interest to declare.


Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 890-890
Author(s):  
Amir Schajnovitz ◽  
Alexander Kalinkovich ◽  
Kfir Lapid ◽  
Alexander Berchanski ◽  
Tomer Itkin ◽  
...  

Abstract Background Rapid mobilization of hematopoietic stem and progenitor cells (HSPCs) from the bone marrow (BM) to the peripheral blood by anti-CXCR4 agents such as AMD3100 is a complex process, which requires CXCL12 secretion, activation of proteolytic enzymes and supporting cellular compartments (Dar et. al, Leukemia 2011). Notably, components of innate immune system were also shown to be involved (Ratajczak et. al, Leukemia 2010). Human β-defensin-3 (hBD3) is an antimicrobial peptide possessing also anti-CXCR4 effects on human T cells in vitro (Feng et. al, JI 2006), suggesting its HSPC mobilizing potential. Here, we describe a novel approach for targeting CXCR4 in vivo by utilizing β-defensin-derived peptides, resulting in rapid HSPC mobilization. Results While AMD3100 blocked CXCL12-mediated signaling and migration of enriched BM mononuclear cells (MNCs) in vitro, we unexpectedly detected rapid phosphorylation of AKT, p38 and ERK1/2 in BM stromal cells (BMSCs). Interestingly, single administration of AMD3100 to mice resulted in enhancement of CXCR4 phosphorylation within minutes in both BM residing mesenchymal stem/progenitor cells (MSCs) and HSPCs, thus suggesting a CXCR4 agonistic activity. Aiming to test HSPC mobilizing potential of hBD3 and avoiding potential toxicity of systemic administration, we synthesized short linear peptides, comprising the C-terminal parts of hBD3 and the murine ortholog β-defensin-14 (mBD14), as well as a cyclic peptide of hBD3, comprising the same amino acids as the linear one, to serve as a control. All full-length β-defensins and tested peptides (both linear and cyclic) specifically bound CXCR4 (demonstrated by docking approach and anti-CXCR4 antibody competition assay) and efficiently blocked CXCL12-mediated activity of enriched BM MNCs in vitro including cell migration and CXCR4-dependent HIV infection. Intriguingly, full-length β-defensins and derived linear peptides (but not cyclic) revealed a strong stimulatory effect on BMSCs in vitro: triggering phosphorylation of AKT, p38 and ERK1/2 along with enhancing secretion of functional CXCL12. Administration of linear peptides to mice led to a fast activation of CXCR4 signaling in BMSCs and MSCs as well as in HSPCs accompanied by CXCL12 release to the circulation, increased activity of proteolytic enzymes and consequent rapid mobilization of progenitors as well as long-term repopulating stem cells. In addition, linear peptides augmented AMD3100-induced rapid mobilization. Importantly, the control cyclic peptide, which bound CXCR4 but failed to activate BMSCs in vitro, did not induce HSPC mobilization in vivo. Moreover, it inhibited both steady-state egress and AMD3100-induced mobilization of HSPCs. A series of in vivo inhibitory analyses confirmed dependence of hBD3- and mBD14-derived peptide-induced HSPC mobilization on the activation of CXCL12/CXCR4 axis and revealed involvement of uPA and JNK signaling as well as ROS generation. Conclusions Our study demonstrated for the first time the capability of β-defensin-derived peptides to activate in vivo CXCL12/CXCR4 signaling in both hematopoietic and non-hematopoietic BM cells, leading to rapid HSPC mobilization. We suggest that activation of CXCR4 signaling in non-hematopoietic BM cells is crucial for inducing HSPC mobilization. Accordingly, CXCR4-binding agents capable of triggering CXCR4 signaling in non-hematopoietic BM cells in vitro, would induce rapid HSPC mobilization. The results presented here help to better understand the mechanisms of rapid HSPC mobilization and have the potential of improving existing clinical protocols to increase the yield of HSPC harvest for transplantation. Disclosures: Scadden: Fate Therapeutics: Consultancy, Equity Ownership.


Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2405-2405
Author(s):  
Wei Shi ◽  
Therese Vu ◽  
Glen Boyle ◽  
Fares Al-Ejeh ◽  
Tej Pandita ◽  
...  

Abstract Single-stranded DNA binding (SSB) proteins are essential for a variety of DNA metabolic processes and the maintenance of genomic stability. SSB1 and its homolog SSB2, share greater sequence and domain homology to the archaeal and bacterial SSBs than eukaryotic RPA. They form complexes with two other proteins, C9Orf80 and INTS3, and play roles in mediating transcription and DNA repair. SSB1 (also known as OBFC2B or NABP2) is recurrently mutated in various cancers, however the precise function in normal development is incompletely understood. We have previously shown that Ssb1 is required for skeletogenesis, telomeric homeostasis and genomic stability in vivo while Ssb2 knockout mice are viable and grow normally without any detectable phenotype. Interestingly, we observed pronounced upregulation of Ssb2 in response to Ssb1 deletion and modest up-regulation of Ssb1 in response to Ssb2 deletion, suggesting that Ssb1 and Ssb2 may have some overlapping functions. To investigate the specific roles of both Ssb1 and Ssb2 in adult tissue homeostasis, we generated conditional double-knockout (DKO) mouse models of both genes. DKO in adult mice was achieved by using a tamoxifen-inducible Cre (Ssb1fl/fl Ssb2fl/fl R26-CreERT2), in which Ssb1 and Ssb2 are conditionally deleted by the administration of tamoxifen. Induced DKO mice become moribund within seven days featured with pancytopenia and dramatic loss of hematopoietic stem and progenitor cells (HSPCs), suggesting that Ssb1 and Ssb2 are required for the maintenance of haematopoietic stem and progenitors cells (HSPCs). DKO bone marrow was markedly hypocellular with reduction in all lineages of haematopoietic development. Functionally, HSPCs in DKO mice show decreased quiescence at the early stage followed by decreased proliferation and increased cell loss due to apoptotic cell death at the later stage, suggesting the imbalanced bone marrow homeostasis upon DKO may eventually result in exhaustion of the stem cell pool in DKO mice. Furthermore, bona fide HSPC intrinsic functional deficiency caused by DKO was confirmed by competitive bone marrow transplant, where DKO bone marrows showed abolished differentiation capacity and failed to repopulate the bone marrows of recipient mice after induction of DKO in the established engraftments from the Ssb1fl/fl Ssb2fl/fl R26-CreERT2 donors. Gene expression of DKO HSPCs demonstrated an exacerbated p53/p21 DNA damage response and pronounced interferon response. Validating these findings, stabilization of p53 and increased apoptotic cell death were observed in DKO bone marrows and HSPCs and induction of cell cycle and expression of interferon target genes was confirmed by QPCR. DKO HSPCs have increased expression of IFN induced surface markers such as Sca1. The IFN response was intrinsic to HSPCs. Mechanistically, DKO HSPCs manifest a profile of stalled replication forks on DNA combing analysis, unrepaired double strand breaks (increased gammaH2Ax foci and alkaline comet tail moment) and telomeric loss resulting in widespread chromosomal instability. DKO HSPC showed aberrant cytoplasmic accumulation of single stranded DNAs, with R-loop formation (DNA:RNA hybrid), driving this genetic instability and cell-intrinsic interferon response. Altogether, these data provide strong evidence that Ssb1 and Ssb2 have essential functions in regulating haematopoiesis through repairing replication associated DNA damage as well as resolution of R-loop generated during transcription, to maintain genomic stability during normal HSPC homeostasis. Disclosures No relevant conflicts of interest to declare.


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