Deficient Hematopoietic Stem Cells Research Articles

IκBα controls dormancy in hematopoietic stem cells via retinoic acid during embryonic development

Recent findings suggest that Hematopoietic Stem Cells (HSC) and progenitors arise simultaneously and independently of each other already in the embryonic aorta-gonad mesonephros region, but it is still unknown how their different features are established. Here, we uncover IκBα (Nfkbia, the inhibitor of NF-κB) as a critical regulator of HSC proliferation throughout development. IκBα balances retinoic acid signaling levels together with the epigenetic silencer, PRC2, specifically in HSCs. Loss of IκBα decreases proliferation of HSC and induces a dormancy related gene expression signature instead. Also, IκBα deficient HSCs respond with superior activation to in vitro culture and in serial transplantation. At the molecular level, chromatin regions harboring binding motifs for retinoic acid signaling are hypo-methylated for the PRC2 dependent H3K27me3 mark in IκBα deficient HSCs. Overall, we show that the proliferation index in the developing HSCs is regulated by a IκBα-PRC2 axis, which controls retinoic acid signaling.

Lnk/Sh2b3 Deficiency Increases DNA Damage Tolerance to Promote Hematopoietic Stem Cell Fitness

DNA repair deficiency depicted by the syndrome Fanconi Anemia (FA), results in progressive bone marrow failure (BMF), increased leukemia incidence and cancer susceptibility. Out of the 23 causative genes for FA, FANCD2 is crucial for its central role in the regulation of DNA repair and replication. The FA pathway plays an important role in maintaining hematopoietic stem and progenitor cell (HSPC) homeostasis and genome integrity. Our previous studies revealed that loss of the adaptor protein LNK (SH2B3), a negative regulator of cytokine signaling, restores hematopoietic stem cell (HSC) function and genome integrity in Fancd2 deficient FA mouse models and primary human FA progenitors. Notably, mice deficient in Lnk harbor a remarkable 14-fold expansion of HSCs with superior self-renewal capability. However, the protective mechanisms that LNK controls to prevent stem cell attrition and increase stem cell fitness are not established. Here, we show that Lnk deficiency reduced DNA damage in Fancd2-/- HSCs at the steady-state in vivo, as well as in forced proliferation upon transplantation. Lnk deficiency reduced HSPC DNA damage in replication stress upon cytokine-induced proliferation in ex vivo culture, accompanied by reduced p53 activation. One injection of pIpC induces HSPC into the cell cycle within 24hr, thus we used this as a model to study replication stress in vivo. We found that Lnk deficiency reduced ATR but not ATM pathway activation in Fancd2-/- HSCs in pIpC-induced proliferation in vivo. ATR is activated by single-strand DNA breaks (SSBs) during replication stress; thus we directly examined the amount of single-strand DNA (ssDNA) and the level of ssDNA binding protein RPA (Replication Protein A) in HSCs during the cell cycle. Of note, Lnk deficiency reduced ssDNA as well as chromatin-bound RPA2 in Fancd2-/- HSCs in replication in vivo. Lnk deficiency also reduced RPA2 phosphorylation in HSPCs under Camptothecin (CPT) and hydroxyurea (HU) induced replication stress conditions. Mechanistically, loss of Lnk protected stalled replication fork from degradation, suppressed ssDNA gaps, and promoted replication fork restart in Fancd2-/- HSPCs upon HU-induced replication stress. In light of our data suggesting a new role for LNK in replication stress-induced DNA damage, we examined the DNA damage tolerance (DDT) mechanism that enables DNA replication to circumvent the lesions, thereby allowing the completion of DNA replication, increasing the survival of HSPCs and preventing genome instability. One of the major DDT pathways is translesion synthesis (TLS) in which the replicative DNA polymerase is temporarily replaced by special TLS polymerases pol ζ or η that can replicate across DNA lesions. Indeed, we found that Lnk deficient HSCs have increased chromatin-bound TLS polymerase eta (Pol ƞ) and were resistant to shRNA-mediated knockdown of PolH (the gene encodes polymerase eta) compared to wildtype HSCs in transplant assays. Strikingly, Lnk deficient HSCs were more resistant to the inhibitor of Rev1 that disrupts Rev1/ pol ζ interaction than wildtype HSCs in transplanting assays. These findings highlight a previously-unappreciated role of LNK in limiting DNA damage tolerance mechanisms in both normal and diseased replicative states. The novel link between LNK-controlled cytokine signaling and TLS sheds light on our understanding of replication stress mitigation to promote HSC fitness that can be explored for future regeneration medicine.

The miR-221/222 cluster regulates hematopoietic stem cell quiescence and multipotency by suppressing both Fos/AP-1/IEG pathway activation and stress-like differentiation to granulocytes.

Throughout life, hematopoietic stem cells (HSCs), residing in bone marrow (BM), continuously regenerate erythroid/megakaryocytic, myeloid, and lymphoid cell lineages. This steady-state hematopoiesis from HSC and multipotent progenitors (MPPs) in BM can be perturbed by stress. The molecular controls of how stress can impact hematopoietic output remain poorly understood. MicroRNAs (miRNAs) as posttranscriptional regulators of gene expression have been found to control various functions in hematopoiesis. We find that the miR-221/222 cluster, which is expressed in HSC and in MPPs differentiating from them, perturbs steady-state hematopoiesis in ways comparable to stress. We compare pool sizes and single-cell transcriptomes of HSC and MPPs in unperturbed or stress-perturbed, miR-221/222-proficient or miR-221/222-deficient states. MiR-221/222 deficiency in hematopoietic cells was induced in C57BL/6J mice by conditional vav-cre-mediated deletion of the floxed miR-221/222 gene cluster. Social stress as well as miR-221/222 deficiency, alone or in combination, reduced HSC pools 3-fold and increased MPPs 1.5-fold. It also enhanced granulopoisis in the spleen. Furthermore, combined stress and miR-221/222 deficiency increased the erythroid/myeloid/granulocytic precursor pools in BM. Differential expression analyses of single-cell RNAseq transcriptomes of unperturbed and stressed, proficient HSC and MPPs detected more than 80 genes, selectively up-regulated in stressed cells, among them immediate early genes (IEGs). The same differential single-cell transcriptome analyses of unperturbed, miR-221/222-proficient with deficient HSC and MPPs identified Fos, Jun, JunB, Klf6, Nr4a1, Ier2, Zfp36-all IEGs-as well as CD74 and Ly6a as potential miRNA targets. Three of them, Klf6, Nr4a1, and Zfp36, have previously been found to influence myelogranulopoiesis. Together with increased levels of Jun, Fos forms increased amounts of the heterodimeric activator protein-1 (AP-1), which is known to control the expression of the selectively up-regulated expression of the IEGs. The comparisons of single-cell mRNA-deep sequencing analyses of socially stressed with miR-221/222-deficient HSC identify 5 of the 7 Fos/AP-1-controlled IEGs, Ier2, Jun, Junb, Klf6, and Zfp36, as common activators of HSC from quiescence. Combined with stress, miR-221/222 deficiency enhanced the Fos/AP-1/IEG pathway, extended it to MPPs, and increased the number of granulocyte precursors in BM, inducing selective up-regulation of genes encoding heat shock proteins Hspa5 and Hspa8, tubulin-cytoskeleton-organizing proteins Tuba1b, Tubb 4b and 5, and chromatin remodeling proteins H3f3b, H2afx, H2afz, and Hmgb2. Up-regulated in HSC, MPP1, and/or MPP2, they appear as potential regulators of stress-induced, miR-221/222-dependent increased granulocyte differentiation. Finally, stress by serial transplantations of miR-221/222-deficient HSC selectively exhausted their lymphoid differentiation capacities, while retaining their ability to home to BM and to differentiate to granulocytes. Thus, miR-221/222 maintains HSC quiescence and multipotency by suppressing Fos/AP-1/IEG-mediated activation and by suppressing enhanced stress-like differentiation to granulocytes. Since miR-221/222 is also expressed in human HSC, controlled induction of miR-221/222 in HSC should improve BM transplantations.

Dose-dependent effect of GFI1 expression in the reconstitution and the differentiation capacity of HSCs.

GFI1 is a transcriptional repressor and plays a pivotal role in regulating the differentiation of hematopoietic stem cells (HSCs) towards myeloid and lymphoid cells. Serial transplantation of Gfi1 deficient HSCs repopulated whole hematopoietic system but in a competitive setting involving wild-type HSCs, they lose this ability. The underlying mechanisms to this end are poorly understood. To better understand this, we used different mouse strains that express either loss of both Gfi1 alleles (Gfi1-KO), with reduced expression of GFI1 (GFI1-KD) or wild-type Gfi1/GFI1 (Gfi1-/GFI1-WT; corresponding to the mouse and human alleles). We observed that loss of Gfi1 or reduced expression of GFI1 led to a two to four fold lower number of HSCs (defined as Lin-Sca1+c-Kit+CD150+CD48-) compared to GFI1-WT mice. To study the functional influence of different levels of GFI1 expression on HSCs function, HSCs from Gfi1-WT (expressing CD45.1 + surface antigens) and HSCs from GFI1-KD or -KO (expressing CD45.2 + surface antigens) mice were sorted and co-transplanted into lethally irradiated host mice. Every 4weeks, CD45.1+ and CD45.2 + on different lineage mature cells were analyzed by flow cytometry. At least 16weeks later, mice were sacrificed, and the percentage of HSCs and progenitors including GMPs, CMPs and MEPs in the total bone marrow cells was calculated as well as their CD45.1 and CD45.2 expression. In the case of co-transplantation of GFI1-KD with Gfi1-WT HSCs, the majority of HSCs (81% ± 6%) as well as the majority of mature cells (88% ± 10%) originated from CD45.2 + GFI1-KD HSCs. In the case of co-transplantation of Gfi1-KO HSCs with Gfi1-WT HSCs, the majority of HSCs originated from CD45.2+ and therefore from Gfi1-KO (61% ± 20%); however, only a small fraction of progenitors and mature cells originated from Gfi1-KO HSCs (<1%). We therefore in summary propose that GFI1 has a dose-dependent role in the self-renewal and differentiation of HSCs.

Cellular and Molecular Mechanisms Involved in Hematopoietic Stem Cell Aging as a Clinical Prospect.

There is a hot topic in stem cell research to investigate the process of hematopoietic stem cell (HSC) aging characterized by decreased self-renewal ability, myeloid-biased differentiation, impaired homing, and other abnormalities related to hematopoietic repair function. It is of crucial importance that HSCs preserve self-renewal and differentiation ability to maintain hematopoiesis under homeostatic states over time. Although HSC numbers increase with age in both mice and humans, this cannot compensate for functional defects of aged HSCs. The underlying mechanisms regarding HSC aging have been studied from various perspectives, but the exact molecular events remain unclear. Several cell-intrinsic and cell-extrinsic factors contribute to HSC aging including DNA damage responses, reactive oxygen species (ROS), altered epigenetic profiling, polarity, metabolic alterations, impaired autophagy, Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway, nuclear factor- (NF-) κB pathway, mTOR pathway, transforming growth factor-beta (TGF-β) pathway, and wingless-related integration site (Wnt) pathway. To determine how deficient HSCs develop during aging, we provide an overview of different hallmarks, age-related signaling pathways, and epigenetic modifications in young and aged HSCs. Knowing how such changes occur and progress will help researchers to develop medications and promote the quality of life for the elderly and possibly alleviate age-associated hematopoietic disorders. The present review is aimed at discussing the latest advancements of HSC aging and the role of HSC-intrinsic factors and related events of a bone marrow niche during HSC aging.

Scribble Is a Negative Regulator of Interferon-I Dependent Notch1 Activation in Adult Hematopoietic Stem Cells and Progenitors

Hematopoietic stem cells (HSC) are highly quiescent cells with the ability to rapidly enter cell cycle and differentiate through changes in their polarity and the disposition of intracellular molecular fate determinants in response to microenvironment (ME) cues. Interferons type 1 (IFN-I) are ME cytokines produced during the physiological response mounted to combat a viral infection. In bone marrow hematopoiesis, IFN-I induces activation and proliferation of HSC. Clinically, patients treated with IFN-I, as well as individuals suffering from IFN-I associated chronic disease, often exhibit sustained hematological cytopenias and HSC failure. The precise molecular mechanisms that govern HSC behavior in response to IFN-I are still unclear. Our data highlights that Scribble deficient HSC are less sensitive to IFN-I mediated activation. By using hematopoietic specific deletion of Scribble in murine hematopoiesis (Vav-Cre;Scribble KO); we demonstrated that Scribble deficient LSK CD150 +/CD48 - HSC are less responsive to polyinositide-polycytidine (pI:C) induced IFN-I mediated activation and retain cellular quiescence (G0:45±5.4% vs 63±2.7% in WT and Scribble KO, respectively, p<0.05). IFN-I induced upregulation of Sca-1 expression was also significantly hampered in Scribble deficient HSC. Functionally, serial transplantation experiments demonstrated that in response to poly I:C, Scribble deficient HSC display increased competitive repopulating potential (26±1.3% vs 38±1.2% BM chimerism for WT and KO BM in secondary recipients and 38±2.5% vs 48±2.7% BM chimerism in tertiary recipients, p<0.01). The maintenance of cellular quiescence and function for Scribble deficient HSC are independent of canonical IFN-I driven STAT-1 signaling, as we report no differences in STAT-1 activation, nuclear translocation or the expression of STAT-1 canonical target genes in response to pI:C. Unsupervised transcriptomics analysis of Scribble-deficient HSC supported dysregulation of Notch signaling. Furthermore, Scribble deficiency in non-activated LSK HSC and progenitors (HSPC) was associated with constitutive activation and cleavage of Notch1 (Notch1 ICD;~3 fold) at levels comparable to IFN-I mediated activation of WT HSPC. However, Scribble deficient HSPC did not exhibit further Notch1 cleavage activation upon in vivo IFN-I induction. Pharmacological in vivo γ-secretase inhibition (YO-01027) prevented the protective effect of Scribble deficiency on IFN-I dependent loss of HSC quiescence. These data indicate that Notch1 activation, and subsequent cleavage, is indispensable for Scribble deficient HSC quiescence in response to IFN-I. Active Cdc42 is a critical regulator of HSC quiescence and fate, and previous studies have demonstrated that Scribble controls HSC asymmetric division potential and fate through the PDZ mediated scaffolding of cytosolic Yap1 with activated Cdc42 (Cdc42-GTP). Next to determine whether poly I:C mediated Notch1 cleavage linked with Cdc42 activity, we analyzed the protein interactions between cleaved Notch1 and Cdc42-GTP in relation with Scribble. Our findings revealed that Scribble associates with non-cleaved, membrane bound Notch but upon in vivo IFN-I induction Notch1 is cleaved, activated and translocates with Scribble-free, activated Cdc42 to the nucleus of HSC. Deletion of HSC Scribble associated with a reduced (~45%, p<0.001) proximity interaction between cleaved Notch1 and Cdc42-GTP. Collectively our findings identify that Scribble controls IFN-I mediated HSPC activation through induction of Notch1 cleavage and Cdc42 activity, and highlight such interaction as a new potential target to dampen inflammation driven HSC exhaustion. DisclosuresCancelas: Cerus Co: Research Funding; TerumoBCT: Research Funding; Hemanext: Membership on an entity's Board of Directors or advisory committees, Research Funding; Cytosorbents Inc: Research Funding; Fresenius-Kabi LLC: Research Funding; Westat Inc: Research Funding; Vascular Solutions Inc.: Research Funding; Hemerus LLC: Research Funding; University of South Florida/MEQU Inc: Research Funding.

RNA Binding Protein Syncrip Is Required for the Low-Output HSC By Sustaining Proteome Quality

RNA binding proteins (RBPs) have been increasingly recognized as an important class of regulators of normal and malignant hematopoiesis. However, the exact function and underpinning mechanisms of the RBPs that govern hematopoietic stem cells (HSCs) remains poorly characterized. We had previously identified SYNCRIP as a critical RBP that controls leukemia stem cell program in myeloid leukemia. Here, using the novel murine genetic conditional knockout (cKO) model, we delineated the role of SYNCRIP in regulating the low-output HSC. We developed a Syncrip cKO allele and crossed Syncripf/f mice to the interferon (IFN) -a-inducible Mx-1-Cre mice to create Syncripf/f Mx-1-Cre+. We consistently obtained near complete depletion of SYNCRIP 3 weeks after two consecutive Poly(I:C) injections. We observed that SYNCRIP is dispensable for static hematopoiesis and Syncrip KO animals showed equivalent number and frequencies of stem and progenitor cells (Lin-Sca+cKit+ (LSK)- LT-HSC (CD48-CD150+); MPP1 (CD48-CD150-); MPP2 (CD48+CD150+); MPP4 (CD48-CD150-)). However, KO SyncripD/D deficient cells were outcompeted by WT Syncripf/f cells in the transplantation setting (bone marrow (BM) chimerism WT (n=9) 38% ± 7.8% vs. KO (n=9) 2.7% ± 0.8%, p<0.001 at 16 weeks post-transplant) and completely lost their ability to repopulate in secondary recipient animals (WT (n=5) 58% ± 7.4% vs. KO (n=5) 7.2 %± 2.9%, p<0.001 at 16 weeks post-transplant). These data strongly indicate that SYNCRIP is critical for maintenance of long-term self-renewal of HSCs. To decipher the effect of Syncrip deletion on the transcriptomic changes in different cell types upon Syncrip loss, we performed single cell RNA sequencing analysis (scRNA-seq) of sorted LK cells (Lin-cKit+ cells) from KO SyncripD/D (n=3) vs. WT Syncrip f/f (n=3) mice. While there is no significant change in frequencies of stem and progenitor compartments, we found defective trajectory from the HSC that is closely identified as low-output HSC based on previously performed barcoding studies. We observed a strong activation of cellular response to stress and unfolded proteins, in particular the HSF1-dependent pathways upon Syncrip depletion specifically within the HSC population. To further investigate the impacts of SYNCRIP loss in the HSC unfolded protein stress response, we evaluated unfolded proteins in cells using tetraphenylethene maleimide (TMI)-based flow cytometry. The abundance of accessible thiols in unfolded proteins, which is bound by TMI serves as a surrogate measurement for the state of the unfolded proteome. We consistently observed almost 2.5-fold increase in TMI signals specifically in LT-HSC, but not ST-HSCs or MPPs upon SYNCRIP deletion indicating that SYNCRIP is required to maintain high protein quality in HSCs. Similar results were obtained with the epichaperome probe PU-FITC, which consists of HSP90 inhibitor PU-H71 conjugated to FITC. PU-H71 selectively binds to the altered epichaperome, which reflects an accumulation of chaperon networks in an aberrant cellular stress condition. Altogether, these data further confirmed that SYNCRIP depletion tips off the proteostatic balance. To understand the molecular mechanisms underpinning the functional requirement of SYNCRIP in HSPCs, we identified 534 direct mRNA targets of SYNCRIP using hyper-TRIBE method. We performed transcriptomic and proteomic analysis of sorted LT- HSCs and LSKs respectively upon SYNCRIP deletion. We integrated these datasets and found a strong enrichment of SYNCRIP targets in control of cytoskeleton and RHO GTPase related pathways. Using immunofluorescence imaging, we confirmed that SYNCRIP deletion in HSCs resulted in 2-fold reduction in RHO GTPase CDC42 expression coupled with reduced tubulin and a loss of cellular polarity (percentage of tubulin polarized cells 56% WT vs. 40% KO). We also observed that Syncrip deficient HSCs demonstrated reduced expression of lysosomal-associated membrane protein 1 (LAMP-1) and less asymmetric distribution of LAMP1 marked lysosomes during cell division (LAMP1 asymmetric division 25% WT vs. 19% KO). Overexpression of CDC42 restored cell polarity and partly rescued ability of KO SyncripD/D to serially replate. Overall, SYNCRIP is required for maintenance of protein homeostasis and cell polarity of the reserve HSCs. Our study uncovers a new regulatory axis that controls stem cell stress responses to preserve HSC self-renewal. DisclosuresNo relevant conflicts of interest to declare.

RUNX3 Promotes the Development of MDS/MPN Overlap Syndrome Via Enhancing Expression of Myc in the Absence of Tet2

The RUNX transcription factors have a critical role in normal and malignant hematopoiesis including myeloproliferative neoplasms (MPN) and myelodysplastic syndromes (MDS). RUNX3 is expressed in various hematopoietic cells, and the reduced expression of RUNX3 due to promoter DNA hyper-methylation is seen in patients with acute lymphoid leukemia, indicating a tumor suppressive role of RUNX3. The loss of Runx3 promotes the development of MPN-like disease in mice. However, loss-of-function mutations and/or a deletion of RUNX3 are rarely observed in patients with myeloid malignancies. In fact, given that enhanced levels of RUNX3 expression are found in a part of patients with AML and MDS, RUNX3 may play an oncogenic role for myeloid malignancies.To determine how RUNX3 over-expression promotes the development of myeloid malignancies, we generated a mouse model of RUNX3 -induced MDS/MPN by utilizing Tet2 -deficient hematopoietic stem cells (HSCs). Since TET2 loss-of-function mutations initiate the transformation of HSCs, we harvested bone marrow CD150+CD34-LSK HSCs from tamoxifen-treated Cre-ERT2 (WT) and Cre-ERT2;Tet2Δ/Δ (Tet2 KO) mice, and transduced expression of 3xFLAG-tagged RUNX3 by using a retrovirus vector in vitro. Then, we transplanted them into lethally-irradiated Ly5.1+ recipient mice together with wild-type competitor cells. We observed that Tet2 KO mice died dominantly due to MPN-like disease, whereas RUNX3 -expressing Tet2 wild-type (RUNX3/WT) HSCs neither competed wild-type bone marrow cells nor developed hematological malignancies in mice by 12 months post transplantation. In contrast, RUNX3 -expressing Tet2 KO (RUNX3/Tet2 KO) HSCs preferentially generated myeloid cells, but not lymphoid cells, and subsequently competed wild-type cells in the PB and the BM. We found that 7 out of 18 RUNX3/Tet2 KO mice developed lethal MDS/MPN diseases despite a longer latency. At the time of sacrifice, RUNX3-Tet2 KO mice showed severe pancytopenia accompanied with dyplastic blood cells including pseudo-Pelger neutrophils and micro megakaryocytes. In addition, these moribund mice significantly enlarged spleen due to the expansion of myeloid cells. FACS analysis demonstrated that the expansion of LSK hematopoietic stem/progenitor cells (HSPCs) in the BM and the spleen, consistent with the features of extramedullary hematopoiesis. Thus, RUNX3/Tet2 KO mice recapitulated the phenotypic features of human MDS/MPN overlap syndrome, indicating that RUNX3 over-expression is prerequisite to the development of MDS/MPN in the absence of Tet2, although it has an adverse effect on the function of Tet2 -present HSCs, reminiscent with a tumor suppressive function of RUNX3 as seen in Runx3 KO mice.To understand the underlying molecular mechanism in the pathogenesis of RUNX3 -induced MDS/MPN, we performed gene expression analysis in HSPCs isolated from WT, RUNX3/WT, Tet2 KO and RUNX3/Tet2 KO mice at pre-disease and MDS/MPN stages. Gene Set Enrichment Analysis (GSEA) demonstrated that RUNX3/Tet2 KO MDS/MPN cells significantly enhanced expression of c-Myc target genes, compared to either WT cells or RUNX3/Tet2 KO cells at the pre-disease stage. While RUNX3/Tet2 KO cells showed negative enrichments of differentiation-related pathway genes, compared to WT cells, we found that those MDS/MPN cells enhanced expression of cell cycle, ribosome, and mitochondria-related biological pathways, in which c-Myc may regulate target genes and contribute to the transformation. In order to determine whether RUNX3 directly activates expression of c-Myc, we performed ChIP-sequencing by using an anti-FLAG antibody in RUNX3/Tet2 KO bone marrow cells, and found that c-Myc 3' distal enhancer regions showed conserved binding sites for Runx transcription factors. In addition, we found that RUNX3 -expressing human AML cells showed higher sensitivity to JQ1, a BRD4 inhibitor, following the significant reduction of both RUNX3 and C-MYC expression. The growth inhibition by JQ1 was rescued by ectopic expression of RUNX3 .In conclusion, RUNX3 and c-Myc collaborated to activate the function of oncogenic enhancers, in which Tet2 deficiency may alter the epigenetic status for allowing the bindings of these transcription factors, to an extent not seen in RUNX3-expressing alone. We demonstrated that RUNX3 functions as either an oncogene or a tumor suppressor for myeloid malignancies in the context dependent manner. DisclosuresHarada:Celgene: Research Funding; NIPPON SHINYAKU CO.: Speakers Bureau; NOVARTIS: Research Funding.

Deficiency of Rbpj Leads to Defective Stress-Induced Hematopoietic Stem Cell Functions and Hif Mediated Activation of Non-canonical Notch Signaling Pathways.

Deregulated notch signaling has been associated with human pathobiology. However, functions of notch pathways in hematopoiesis remain incompletely understood. Here, we ablated canonical notch pathways, through genetic deletion of Rbpj, in hematopoietic stem cells (HSCs). Our data identified that loss of canonical notch results in normal adult HSC pool, at steady state conditions. However, HSC maintenance and functions in response to radiation-, chemotherapy-, and cytokine- induced stress were compromised in the absence of canonical notch. Rbpj deficient HSCs exhibit decreased proliferation rates and elevated expression of p57Kip2. Surprisingly, loss of Rbpj resulted in upregulation of key notch target genes and augmented binding of Hes1 to p57 and Gata2 promoters. Further molecular analyses identified an increase in notch activity, elevated expression and nuclear translocation of Hif proteins, and augmented binding of Hif1α to Hes1 promoter in the absence of Rbpj. These studies, for the first time, identify a previously unknown role for non-canonical notch signaling and establish a functional link between Hif and Notch pathways in hematopoiesis.

Bone Marrow Stromal Antigen 2 Is Critical for IFNy-Dependent Hematopoietic Stem Cell Activation

Bone marrow failure is a significant complication of many chronic infections, which affect a third of the world's population. The inflammatory cytokine interferon-gamma (IFNy) contributes to bone marrow failure syndromes by activating hematopoietic stem cells (HSCs) and impairing their self-renewal. IFNy upregulation during many chronic infections such as tuberculosis, HIV and hepatitis B directly depletes hematopoietic stem cells (HSCs). The mechanisms by which IFNy drives the loss of quiescence and ultimate exhaustion of HSCs remain poorly understood, but may be related to changes in the interaction between HSCs and the bone marrow (BM) microenvironment, or BM niche. Current evidence suggests that quiescent HSCs reside predominantly in the vascular niche, where the production of stem cell factor (SCF) from endothelial cells and CXCL12 from perivascular CXCL12 abundant reticular (CAR) stromal cells are critical for maintaining their quiescence. The goal of our work was to determine whether IFNy signaling alters HSC interactions within the niche. We performed transcriptomic analysis of IFNy-stimulated HSCs and focused on changes in cell-surface expressed genes that may influence HSC-niche interactions. Our analysis revealed Bone Marrow Stromal Antigen 2 (BST2) as the only surface protein upregulated on HSCs upon 24-hour IFNy stimulation. To study the effects of BST2 on HSC-niche interactions, we performed intravital imaging using two reporter mouse models: CXCL12-GFP reporter mice in which CAR cells are labeled with GFP, and CXCL12-GFP Krt18-Cre LSL-tdTomato dual reporter mice in which CAR cells are labelled with GFP and HSCs are labeled with tdTomato. After exogenously labeling and transferring HSCs into CXCL12-GFP mice or endogenously labeling HSCs in the CXCL12-GFP Krt18-Cre LSL-tdTomato mice, we observed that HSCs stimulated with IFNy were significantly distanced from CAR cells compared to pre-treated controls. These findings are consistent with other reports that chemotherapeutic and inflammatory stress disrupts HSC interactions with the niche and promotes HSC migration. Conversely, we observed no change in HSC distancing from CAR cells after IFNy stimulation of IFNy-receptor deficient HSCs, suggesting that the observed HSC displacement was due to a cell autonomous mechanism. These changes were not due to a loss of CXCL12 receptor (CXCR4) expression or disrupted capacity of HSCs to migrate towards CXCL12. Interestingly, Intravital imaging using BST2-deficient HSCs revealed that BST2 KO HSCs do not re-localize from CAR cells during IFNy stimulation. Increased BST2 expression has been linked to the migration, adhesion and metastasis of various cancer cells and we explored whether it could serve a similar role in protein binding in HSCs. Using in vitro plate binding assays, we found that IFNy-treatment promoted increased HSC binding to E-selectin via BST2, as well as increased HSC homing to the bone marrow, a property that is dependent on E-selectin binding. Finally, to determine whether BST2 affects IFNy-dependent HSC activation we performed cell cycle analysis of WT and BST2 KO HSCs. We discovered that the loss of BST2 protects against HSC activation during Mycobacterium avium infection. Furthermore, HSC depletion during chronic infection was mitigated in BST2 KO mice. Our data identifies BST2 as a key protein that influences niche relocalization and activation in response to inflammatory stimulation. This study expands our understanding of factors that contribute to HSC activation and loss of quiescence. These findings could shed light on novel therapeutic interventions for patients who develop pancytopenia or bone marrow failure due to chronic inflammation. Disclosures No relevant conflicts of interest to declare.

Reversing Clonal Hematopoiesis and Associated Atherosclerotic Disease By Targeted Antibody-Drug-Conjugate (ADC) Conditioning and Transplant

Cardiovascular disease (CVD) is the leading cause of death worldwide. Recently, age-related clonal hematopoiesis (CH) has been recognized as a risk factor for CVD of comparable magnitude to smoking, hypertension and hypercholesteremia. While these other risk factors can be mitigated by pharmacological intervention or lifestyle changes, there are no such strategies in place for CH. As CH is initiated by mutations in hematopoietic stem cells (HSCs), a hematopoietic stem cell transplantat (HSCT) could serve as a curative therapy. However, stem cell transplantation is associated with significant toxicity due in part from current conditioning regimens. There is also no evidence that depletion of the disease-driving clones impacts established atherosclerosis. We developed an antibody drug conjugate (ADC) targeting murine CD45. In the context of stem cell transplantation, the CD45-ADC efficiently depletes endogenous HSCs as well as mature leukocytes while enabling rapid engraftment of an infused stem cell graft. In addition, the CD45-ADCs are not based on broad-acting genotoxic agents that lead to long-lasting health risks. We decided to test if CD45-ADC and HSCT could halt atherosclerosis progression through elimination Tet2 knockout HSCs and their disease propagating myeloid progeny. To model CH associated atherosclerosis, LDLR knockout mice were transplanted with 20% CFP labeled wild-type (WT) or Tet2 knockout bone marrow. A single dose of isotype- or CD45-ADC was delivered after 6 weeks of atherosclerosis development and was followed by an infusion of WT CD45.1 bone marrow. As has been reported before, we observed in the isotype-ADC treated animals that Tet2 deficiency leads to a competitive advantage over WT cells. Tet2 knockout cells contributed to peripheral blood chimerism at successively increasing levels and mice harboring the knockout graft showed a significant expansion of their HSC population. Despite their obvious advantage, Tet2 deficient HSC were as efficiently depleted as their WT counterparts upon CD45-ADC and HSCT. Peripheral blood and bone marrow chimerism were similar in WT and Tet2 knockout hosts and the expanded HSC pool was successfully curbed 6 weeks following the intervention. More importantly, CD45-ADC also depleted cells in the atherosclerotic plaques as efficiently as in blood in both WT and Tet2 mutant recipients. This resulted in a significant reduction of myeloid cell infiltration in CD45-ADC conditioned and transplanted knockout hosts and ultimately lead to drastically reduced plaque size in these animals. In conclusion, these data demonstrate that CD45-ADC and HSCT efficiently replaces the disease driving myeloid cells in the atherosclerosis plaques leading to an overall reduction in disease burden. CD45-ADC and transplantation may thus offer a novel therapy for CH and its associated morbidities. Disclosures Palchaudhuri: Magenta Therapeutics: Current Employment. Hyzy:Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company. Proctor:Magenta Therapeutics: Current Employment. Gillard:Magenta Therapeutics: Current Employment. Boitano:Magenta Therapeutics: Ended employment in the past 24 months, Patents &amp; Royalties. Cooke:Magenta Therapeutics: Ended employment in the past 24 months. Scadden:Magenta Therapeutics: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees.

1016 – REGULATION OF RIBOSOME BIOGENESIS IN HEMATOPOIETIC STEM CELLS

Dysregulation of ribosome biogenesis is the underlying etiology in a group of bone marrow failure syndromes called ribosomopathies. As prominently illustrated by one type of ribosomopathies, Shwachman-Diamond Syndrome (SDS), germline biallelic mutations in three assembly factors essential for the maturation of the 60S ribosome subunit result in progressive hematopoietic stem and progenitor cell (HSPC) failure, suggesting that HSPCs are sensitive to perturbations in ribosome assembly. However, how ribosome assembly is regulated in HSPCs remains poorly understood, as are approaches to restore in hematopoietic stem cell (HSC) function in the context of defective ribosome biogenesis. Here we uncover a novel role for the E3 ubiquitin ligase, HectD1, in regulating HSC function via ribosome biogenesis. HSCs from hematopoietic-specific <i>Hectd1</i> knockout mice exhibit a striking defect in transplantation ability and self-renewal. <i>Hectd1</i>-deficient HSCs fail to be maintained ex vivo, concomitant with a reduction in global protein synthesis and growth rate. The mechanism underlying HSC dysfunction upon <i>Hectd1</i> deficiency is directly linked to aberrant ribosome assembly. We demonstrate that HectD1 ubiquitinates and degrades an assembly factor for the polypeptide exit tunnel of the 60S ribosome in the cytoplasm. Depletion of <i>HectD1</i> led to an accumulation of the anti-association factor eIF6 on the 60S and decreased 80S to 60S ratio, consistent with a subunit joining defect associated with SDS-like diseases. Importantly, we found that restoration of ribosome assembly in <i>HectD1</i>-deficient cells promoted protein synthesis and enhanced HSC reconstitution capacity. These findings highlight the importance of ribosome assembly in HSC regeneration and represent a rare <i>in vivo</i> example of genetic suppression of HSC defects associated with dysfunctional ribosome biogenesis.

3142 – GLUCOCORTICOID-INDUCED LEUCINE ZIPPER (GILZ) INTRINSICALLY REGULATES HEMATOPOIETIC STEM CELL FUNCTION

The maintenance of hematopoietic stem cells (HSC) is linked to their quiescent state, while HSC proliferation is associated with differentiation and a loss of long-term stem cell potential. The balance between HSC quiescence and proliferation is tightly regulated by intrinsic and extrinsic cues in the bone marrow. Endogenous glucocorticoid hormones (GC) regulate HSC homing via control of CXCR4 expression. Glucocorticoid-Induced Leucine Zipper (GILZ) is a gene rapidly induced by GC. It mediates many of GC' anti-proliferative and anti-inflammatory effects in several cell types. We found that GILZ is expressed in HSC and addressed its role in hematopoiesis using GILZ knock-out (KO) mice. At steady state, young GILZ-KO mice did not show alteration in HSC number and lineage commitment compared to WT mice. However, competitive transplantation studies revealed a significant decrease in the frequency and number of GILZ-KO compared to WT HSC, suggesting that GILZ-deficient HSC have competitive disadvantage compared to WT cells. RNAseq analysis of gene expression revealed several deregulated cellular pathways implicated in HSC function in GILZ KO compared to WT HSC. Importantly, Gene Set Enrichment Analysis showed a significant depletion of the HSC signature and an enrichment in the mTOR signalling signature in GILZ deficient HSC. Consistently, GILZ deficient HSC showed enhanced proliferation as revealed by flow cytomery analysis of Ki67 expression and DNA content. Mice transplanted with GILZ deficient cells showed significantly earlier mortality compared to WT cells-transplanted mice in 5-FU treatment experiments, demonstrating a cell-intrinsic role of GILZ in HSC function. Overall, these data identify GILZ is a novel regulator of HSCs function.

BMP signaling is required for postnatal murine hematopoietic stem cell self-renewal.

Life-long production of blood from hematopoietic stem cells (HSC) is a process of strict modulation. Intrinsic and extrinsic signals govern fate options like self-renewal – a cardinal feature of HSC. Bone morphogenetic proteins (BMP) have an established role in embryonic hematopoiesis, but less is known about its functions in adulthood. Previously, SMAD-mediated BMP signaling has been proven dispensable for HSC. However, the BMP type-II receptor (BMPR-II) is highly expressed in HSC, leaving the possibility that BMP function via alternative pathways. Here, we establish that BMP signaling is required for selfrenewal of adult HSC. Through conditional knockout we show that BMPR-II deficient HSC have impaired self-renewal and regenerative capacity. BMPR-II deficient cells have reduced p38 activation, implying that non-SMAD pathways operate downstream of BMP in HSC. Indeed, a majority of primitive hematopoietic cells do not engage in SMADmediated responses downstream of BMP in vivo. Furthermore, deficiency of BMPR-II results in increased expression of TJP1, a known regulator of self-renewal in other stem cells, and knockdown of TJP1 in primitive hematopoietic cells partly rescues the BMPR-II null phenotype. This suggests TJP1 may be a universal stem cell regulator. In conclusion, BMP signaling, in part mediated through TJP1, is required endogenously by adult HSC to maintain self-renewal capacity and proper resilience of the hematopoietic system during regeneration.

IFNy Stimulation Induces Hematopoietic Stem Cell Homing and Niche Relocalization

Interferon gamma (IFNy) is a pro-inflammatory cytokine that is upregulated during chronic infections and chronic diseases, such as aplastic anemia, and has been associated with pancytopenia and diminished hematopoiesis. Studies have shown that IFNy negatively regulates hematopoietic stem cell (HSC) homeostasis by decreasing self-renewal and promoting terminal differentiation. The tight regulation of HSC homeostasis is dependent upon the bone marrow (BM) microenvironment, or BM niche. The BM niche is composed of a network of cell types that provide elaborate cell-cell interactions, cellular metabolites, transcriptional regulators, and local and distant humoral and neural signals that allow for hematopoietic homeostasis. In particular, CXCL12-abundant reticular (CAR) cells are vital to HSC maintenance, as depletion of CXCL12, or its receptor, leads to HSC depletion. However, the mechanism which IFNy activates HSCs and influences its interaction with the BM niche is unknown. We hypothesize that IFNy promotes HSC terminal differentiation and loss of quiescence by altering HSC interactions with the BM niche. To assess changes in HSC interactions with the BM niche upon IFNy stimulation, we performed intravital imaging using CXCL12 GFP reporter mice before and after administration of recombinant IFNy. We found that HSCs stimulated with IFNy were significantly distanced from CAR cells compared to pre-treated controls. There was no change in distance with IFNy-receptor deficient HSCs, suggesting that movement away from the CAR cells was due to a cell autonomous IFNy-dependent mechanism. We performed gene expression analysis and transwell migration assays on HSCs from IFNy treated mice, and determined that there was no change in CXCL12 receptor (CXCR4) expression upon IFNy treatment, and IFNy did not alter migration towards CXCL12. These results suggest that HSC re-localization upon IFNy is independent of CXCL12 signaling. To explore the mechanism by which IFNy induces re-localization of HSCs, we first performed microarray analysis on HSCs from IFNy stimulated mice to assess what surface proteins were changed upon IFNy treatment. While there was no change in common HSC receptors thought to influence HSC homeostasis (cKit, Cdh2, Mpl, Itgb1, Itbg2, Itga4, and Itga1), we observed an increase in expression of bone marrow stromal antigen 2 (BST2). To explore the impact of BST2 on HSC homeostasis, quantification and proliferation analysis was performed on HSCs from Bst2-/- mice. Interestingly, Bst2-/- HSCs were significantly less proliferative and more abundant compared to controls. These studies suggest that BST2 may play a role in maintaining HSC homeostasis. The functional role of BST2 in cellular movement and adhesion has been studied in cancer. Increased BST2 expression has been associated with promoting the migration, adhesion and metastasis of various cancer cells. Since migration and adhesion is important for HSC homing, we assessed the effects of IFNy on HSC homing. Hematopoietic progenitors from IFNy-treated mice homed to the bone marrow with greater efficiency than PBS-treated controls, whereas progenitors from IFNy-receptor-deficientmice showed a decrease in homing. Additionally, WBM from IFNyR-/- had reduced engraftment than wildtype, consistent with a role for IFNy signaling in promoting HSC homing. The impact of BST2 on homing is currently being explored. In summary, we show that IFNy induces re-localization of HSCs away from quiescence-promoting CAR cells within the bone marrow niche via a mechanism that is independent of CXCL12 signaling. We further show that IFNy promotes HSC homing. The increased expression of BST2 on IFNy-stimulated HSCs appears to impact HSC proliferation and abundance in the bone marrow. Thus, BST2 may play a role in HSC activation and exit from quiescence. Expanding our understanding of the mechanism that drives HSC activation and terminal differentiation has important implications for patients who develop pancytopenia or bone marrow failure due to chronic inflammation. Disclosures No relevant conflicts of interest to declare.

The Fanconi Anemia-Associated Protein NIPA Is Essential for the Nuclear Abundance of FANCD2

Inherited bone marrow failure syndromes (IBMFS) are a heterogeneous group of disorders characterized by impaired stem cell function resulting in pancytopenia. Diagnosis of IBMFS presents a major challenge due to limited diagnostic tests and overlapping phenotypes. For that reason, novel and clinical relevant biomarkers and possible targets are urgently needed. Our study defines NIPA as an IBMFS gene, which is significantly downregulated in a distinct subset of MDS-type refractory cytopenia of childhood patients. Mechanistically, NIPA binds FANCD2 and regulates its nuclear abundance. The stabilization of both non- and monoubiquitinated FANCD2 identifies NIPA as an essential player in the Fanconi Anemia (FA) pathway. NIPA thereby prevents MMC hypersensitivity visualized by increased numbers of chromosome radials and reduced cell survival after induction of interstrand crosslinks. To provide proof of principle, re-expression of FANCD2 in Nipa deficient cells restores MMC sensitivity. In a knockout mouse model, Nipa deficiency leads to major cell intrinsic long-term repopulation defects of hematopoietic stem cells (HSCs), with impaired self-renewal in serial transplantations and a bias towards myeloid differentiation. Unresolved DNA damage in Nipa deficient HSCs causes increased sensitivity to cell death and leads to progressive, age-related loss of the HSC pool. Induction of replication stress triggers the phenotypic reduction and functional decline of murine HSCs, resulting in complete bone marrow failure and death of the mice thereby mimicing Fanconi Anemia. Taken together, our study adds NIPA to the short list of FA-associated proteins being essential for a functional DNA repair/FA/BRCA axis and thereby emphasizing its impact as potential diagnostic marker and/or possible target in bone marrow failure syndromes. Disclosures Niemeyer: Celgene: Consultancy.

YY1 Controls Hematopoietic Stem Cell Quiescence By Repressing Cohesin Expression

Hematopoietic stem cells (HSCs) are undifferentiated, self-renewing, pluripotent cells that have the capacity to differentiate into all mature lineage-specific cells in adult blood. Adult HSCs can remain in a quiescent state for a prolonged time, and quiescence is a fundamental characteristic of HSCs in adult bone marrow. Thus, the cell cycle must be precisely regulated. Yin Yang 1 (YY1) is a multifunctional transcription factor and Polycomb Group Protein (PcG) that is important for embryonic development, adult hematopoiesis, cell proliferation and maintaining higher-order chromosomal structure. We have generated YY1 conditional knockout mice (Yy1f/f Mx1-Cre) and showed that Yy1 deficient HSCs fail to self-renewal and had disrupted HSCs quiescence. Stem cell factor (SCF)/c-Kit signaling, a critical regulatory pathway in HSC development, is significantly downregulated in Yy1-/- HSCs. Interestingly, YY1 regulation of HSCs self-renewal and quiescence is independent on its PcG domain/function. Instead, YY1 occupied at Smc3 promoter area and repressed Smc3 expression. In Yy1-/-HSCs, Smc3 expression was upregulated. SMC3 is a core component of cohesin protein complex and plays critical roles in HSC self-renewal, myeloid differentiation and leukemogenesis. To further dissect the underlying mechanisms by which YY1 regulates SMC3 expression in HSCs, we have generated conditional knockout mice with YY1 homozygous deletion and SMC3 heterozygous deletion (Yy1f/f Smc3f/+Mx1-Cre). In Yy1-/- Smc3+/- bone marrow cells, SMC3 expression was normalized to the wild-type level. In adult bone marrow cells, YY1 physically interacted with cohesin complex proteins through its zinc finger domain. By analyzing the YY1, SMC1A and SMC3 ChIP-Seq database, our study showed that YY1 and cohesin co-occupied at promoter areas of genes that are critical for cell metabolism. Evidence from previous study showed that impaired metabolism, including increased reactive oxygen species (ROS) and decreased mitochondrial function, can cause defect in stem cell self-renewal and quiescence. In Yy1-/- Smc3+/-HSCs, cell quiescence was restored although HSC self-renewal was still impaired, indicates that YY1 and SMC3 may control HSC cell quiescence via regulating genes critical for cell metabolism. Our study identified YY1 as the first transcription factor that regulates expression of cohesin complex component SMC3. We are currently further dissecting underlying mechanisms and functional significances of metabolic pathways regulated by YY1-SMC3 axis in HSCs. DisclosuresLevine:Imago: Equity Ownership; Isoplexis: Equity Ownership; Janssen: Consultancy, Honoraria; Gilead: Honoraria; Epizyme: Patents & Royalties; Loxo: Consultancy, Equity Ownership; C4 Therapeutics: Equity Ownership; Roche: Consultancy, Research Funding; Prelude: Research Funding; Qiagen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Novartis: Consultancy.

SRC-3 is involved in maintaining hematopoietic stem cell quiescence by regulation of mitochondrial metabolism in mice

AbstractQuiescence maintenance is an important property of hematopoietic stem cells (HSCs), whereas the regulatory factors and underlying mechanisms involved in HSC quiescence maintenance are not fully uncovered. Here, we show that steroid receptor coactivator 3 (SRC-3) is highly expressed in HSCs, and SRC-3–deficient HSCs are less quiescent and more proliferative, resulting in increased sensitivity to chemotherapy and irradiation. Moreover, the long-term reconstituting ability of HSCs is markedly impaired in the absence of SRC-3, and SRC-3 knockout (SRC-3−/−) mice exhibit a significant disruption of hematopoietic stem and progenitor cell homeostasis. Further investigations show that SRC-3 deficiency leads to enhanced mitochondrial metabolism, accompanied by overproduction of reactive oxygen species (ROS) in HSCs. Notably, the downstream target genes of peroxisome proliferator–activated receptor-coactivators 1α (PGC-1α) involved in the regulation of mitochondrial metabolism are significantly upregulated in SRC-3–deficient HSCs. Meanwhile, a significant decrease in the expression of histone acetyltransferase GCN5 accompanied by downregulation of PGC-1α acetylation is observed in SRC-3–null HSCs. Conversely, overexpression of GCN5 can inhibit SRC-3 deficiency-induced mitochondrial metabolism enhancement and ROS overproduction, thereby evidently rescuing the impairment of HSCs in SRC-3−/− mice. Collectively, our findings demonstrate that SRC-3 plays an important role in HSC quiescence maintenance by regulating mitochondrial metabolism.

PDK1 regulates definitive HSCs via the FOXO pathway during murine fetal liver hematopoiesis

PDK1 (phosphoinositide dependent kinase-1) plays an important regulatory role in B cells, T cells and platelets. Less is known about how PDK1 acts in hematopoietic stem cells (HSCs), especially in the fetal liver (FL) during embryonic hematopoiesis, as the FL is the primary fetal hematopoietic organ and the main site of HSC expansion and differentiation. Here, we deleted the PDK1 gene in hematopoietic cells by crossing Vav-Cre transgenic mice with PDK1f/f mice. Using a transplantation assay, we found that HSCs from the E15.5 FL of Vav-Cre;PDK1f/f embryos are severely impaired compared when compared with HSCs from PDK1f/f or PDK1f/+ FLs. Additionally, we found that there were more FL HSCs in an apoptotic state and active cell cycle in PDK1-deficient embryos than in control embryos. By comparing the expression profiles of FL-derived LSKs in Vav-Cre;PDK1f/f embryos to the controls, we found that the BH3-only protein PUMA and the cyclin family proteins were expressed higher in the Vav-Cre;PDK1f/f group, which may account for the increased apoptosis and activated cell cycle in the deficient HSCs. Furthermore, we demonstrated that the expression of FoxO3a was higher in PDK1-deficient LSKs, indicating that the Akt-FoxO3a-PUMA axis may participate in regulating LSKs apoptosis in the E15.5 FL. In contrast, FoxO1 expression was lower in PDK1-deficient LSK cells, suggesting that Akt-FoxO1-CCND may regulate the HSC cell cycle. Taken together, our findings support a critical role for PDK1 in maintaining FL hematopoiesis via regulating apoptosis and cell cycle of definitive hematopoiesis by the Akt-FOXO signaling pathways.

The Chromatin Remodeler BPTF Activates a Stemness Gene-Expression Program Essential for the Maintenance of Adult Hematopoietic Stem Cells.

SummarySelf-renewal and differentiation of adult stem cells are tightly regulated partly through configuration of chromatin structure by chromatin remodelers. Using knockout mice, we here demonstrate that bromodomain PHD finger transcription factor (BPTF), a component of the nucleosome remodeling factor (NURF) chromatin-remodeling complex, is essential for maintaining the population size of hematopoietic stem/progenitor cells (HSPCs), including long-term hematopoietic stem cells (HSCs). Bptf-deficient HSCs are defective in reconstituted hematopoiesis, and hematopoietic-specific knockout of Bptf caused profound defects including bone marrow failure and anemia. Genome-wide transcriptome profiling revealed that BPTF loss caused downregulation of HSC-specific gene-expression programs, which contain several master transcription factors (Meis1, Pbx1, Mn1, and Lmo2) required for HSC maintenance and self-renewal. Furthermore, we show that BPTF potentiates the chromatin accessibility of key HSC “stemness” genes. These results demonstrate an essential requirement of the chromatin remodeler BPTF and NURF for activation of “stemness” gene-expression programs and proper function of adult HSCs.