Nfix is a novel regulator of murine hematopoietic stem and progenitor cell survival
ABSTRACT: Hematopoietic stem cells are both necessary and sufficient to sustain the complete blood system of vertebrates. Here we show that Nfix, a member of the nuclear factor I (Nfi) family of transcription factors, is highly expressed by hematopoietic stem and progenitor cells (HSPC) of murine adult bone marrow. Although shRNA mediated knockdown of Nfix expression in Lineage-Sca-1+c-Kit+ HSPC had no effect on in vitro cell growth or viability, Nfix-depleted HSPC displayed a significant loss of colony forming potential, as well as short- and long-term in vivo hematopoietic repopulating activity. Analysis of recipient mice 4-20 days post-transplant revealed that Nfix-depleted HSPC establish in the bone marrow but fail to persist due to increased apoptotic cell death. Gene expression profiling of Nfix-depleted HSPC reveals that loss of Nfix expression in HSPC is concomitant with a decrease in the expression of multiple genes known to be important for HSPC survival, such as Erg, Mecom, Mpl and Prdm16. These data reveal that Nfix is a novel regulator of HSPC survival post-transplantation and establish, for the first time, a role for Nfi genes in the regulation of this cellular compartment. 3 NFIX depleted samples are compared to 3 wt samples
Project description:Although Hematopoietic Stem Cell Transplantation (HSCT) routinely treats hematologic disease, many patients experience adverse outcomes. Understanding the molecular regulation of HSC engraftment is paramount to improving HSCT regimens. Here, we executed a large-scale transplant-based functional screen for novel regulators of HSC repopulation.. Of >50 gene candidates tested, 18 were required for in vivo hematopoietic repopulation and two were detrimental to repopulation, as their loss enhanced this activity. Each Hit was validated in a second screen. Eleven Hits have never before been implicated in HSC biology. We further show that one novel Hit, Foxa3, is required for optimal engraftment as Foxa3-/- bone marrow is defective in both primary and secondary hematopoietic reconstitution. We also present evidence that Foxa3 is a novel pioneer factor in HSC. Each gene identified in our screen is a window into the cellular mechanisms that control hematopoietic reconstitution. Thus, this work represents a resource to the community to better understand these processes 3 FOXA3 KO samples are compared to 3 wt samples
Project description:Hematopoietic stem cells are both necessary and sufficient to sustain the complete blood system of vertebrates. Here we show that Nfix, a member of the nuclear factor I (Nfi) family of transcription factors, is highly expressed by hematopoietic stem and progenitor cells (HSPCs) of murine adult bone marrow. Although short hairpin RNA-mediated knockdown of Nfix expression in Lineage(-)Sca-1(+)c-Kit(+) HSPCs had no effect on in vitro cell growth or viability, Nfix-depleted HSPCs displayed a significant loss of colony-forming potential, as well as short- and long-term in vivo hematopoietic repopulating activity. Analysis of recipient mice at 4 to 20 days posttransplant revealed that Nfix-depleted HSPCs are established in the bone marrow, but fail to persist due to increased apoptotic cell death. Gene expression profiling of Nfix-depleted HSPCs reveals that loss of Nfix expression in HSPCs is concomitant with a decrease in the expression of multiple genes known to be important for HSPCs survival, such as Erg, Mecom, and Mpl. These data reveal that Nfix is a novel regulator of HSPCs survival posttransplantation and establish a role for Nfi genes in the regulation of this cellular compartment.
Project description:TRAF-interacting protein with forkhead-associated domain B (TIFAB) is a haploinsufficient gene in del(5q) Myelodysplastic syndrome (MDS). Hematopoietic-specific deletion of Tifab results in progressive bone marrow (BM) and blood defects, including skewed hematopoietic stem/progenitor cells (HSPC) proportions, altered myeloid differentiation, and progressive cytopenia. A subset of mice transplanted with Tifab knockout (KO) hematopoietic cells develop a bone marrow failure (BMF)-like disease with neutrophil dysplasia and cytopenia. In competitive transplants, Tifab KO HSPC are out-competed by wild-type (WT) cells, suggesting a cell-intrinsic HSPC defect. Gene expression analysis of Tifab KO HSPC identified dysregulation of immune-related signatures, and hypersensitivity to Toll-like receptor 4 (TLR4) stimulation. TIFAB also forms a complex with TRAF6, a mediator of immune signaling, and reduces TRAF6 protein stability by a lysosome-dependent mechanism. In contrast, TIFAB loss increases TRAF6 protein and the dynamic range of TLR4 signaling in HSPC, contributing to ineffective hematopoiesis. Moreover, combined deletion of TIFAB and miR-146a, two genes associated with del(5q) MDS/AML, results in a cooperative increase in TRAF6 expression and hematopoietic dysfunction in vivo. Re-expression of TIFAB in human del(5q) leukemic cells results in attenuated TLR4 signaling and reduced cell viability. These findings underscore the importance of efficient regulation of innate immune/TRAF6 signaling within HSPC by TIFAB, and its cooperation with miR-146a as it relates to the pathogenesis of hematopoietic malignancies, such as del(5q) MDS/AML. We performed an expression analysis on sorted lineage-Sca1+cKit+ (LSK) isolated from bone marrow (BM) of 3 month old mice transplanted with Tifab+/+ (WT) or Tifab-/- (KO) BM cells (n = 3 mice/group). We selected this time point to capture the gene expression profile of Tifab-/- LSK after engraftment but prior to overt hematopoietic failure. Total RNA was extracted, purified, reverse transcribed, labeled, and hybridized onto the GeneChip MoGene 2.0 ST Array (Affymetrix). Comparison comprises mRNA expression profile of Tifab+/+ LSK vs. Tifab-/- LSK.
Project description:The commitment of stem and progenitor cells toward specific hematopoietic lineages is tightly controlled by a number of transcription factors that regulate differentiation programs via the expression of lineage restricting genes. Nuclear factor one (NFI) transcription factors are important in regulating hematopoiesis and here we report an important physiological role of NFIX in B- and myeloid lineage commitment and differentiation. We demonstrate that NFIX acts as a regulator of lineage specification in the haematopoietic system and the expression of Nfix was transcriptionally downregulated as B cells commit and differentiate, whilst maintained in myeloid progenitor cells. Ectopic Nfix expression in vivo blocked early B cell development stage, coincident with the stage of its downregulation. Furthermore, loss of Nfix resulted in the perturbation of myeloid and lymphoid cell differentiation, and a skewing of gene expression involved in lineage fate determination. Nfix was able to promote myeloid differentiation of total bone marrow cells under B cell specific culture conditions but not when expressed in the hematopoietic stem cell (HSPC), consistent with its role in HSPC survival. The lineage choice determined by Nfix correlated with transcriptional changes in a number of genes, such as E2A, C/EBP, and Id genes. These data highlight a novel and critical role for NFIX transcription factor in hematopoiesis and in lineage specification.
Project description:The contribution of osteoclasts to hematopoietic stem/progenitor cell (HSPC) retention in the bone marrow is controversial. Studies of HSPC trafficking in osteoclast-deficient mice are limited by osteopetrosis. Here, we employed two non-osteopetrotic mouse models to assess the contribution of osteoclasts to basal and granulocyte colony-stimulating factor (G-CSF)-induced HSPC mobilization. We generated Rank(-/-) fetal liver chimeras using Csf3r(-/-) recipients to produce mice lacking G-CSF receptor expression in osteoclasts. Basal and G-CSF-induced HSPC mobilization was normal in these chimeras. We next acutely depleted osteoclasts in wild-type mice using the RANK ligand inhibitor osteoprotegerin. Marked suppression of osteoclasts was observed after a single injection of osteoprotegerin-Fc. Basal and G-CSF-induced HSPC mobilization in osteoprotegerin-Fc-treated mice was comparable to that in control mice. Together, these data indicate that osteoclasts are not required for the efficient retention of HSPCs in the bone marrow and are dispensable for HSPC mobilization by G-CSF.
Project description:Inflammatory signals have been shown to play a critical role in controlling the maintenance and functions of hematopoietic stem cells (HSCs). While the significance of inflammation in hematopoiesis has begun to unfold, molecular mechanisms and players that govern this mode of HSC regulation remain largely unknown. The E3 ubiquitin ligase A20 has been considered as a central gatekeeper of inflammation. Here, we have specifically depleted A20 in multi-potent progenitors (MPPs) and studied its impact on hematopoiesis. Our data suggest that lack of A20 in Flt3+ progenitors causes modest alterations in hematopoietic differentiation. Analysis of hematopoietic stem and progenitor cell (HSPC) pool revealed alterations in HSPC subsets including, HSCs, MPP1, MPP2, MPP3 and MPP4. Interestingly, A20 deficiency in MPPs caused loss of HSC quiescence and compromised long-term hematopoietic reconstitution. Mechanistic studies identified that A20 deficiency caused elevated levels of Interferon-? signaling and downregulation of p57 in HSCs. In essence, these studies identified A20 as a key regulator of HSC quiescence and cell fate decisions.
Project description:Targeted mouse mutants with inactivated Mixed-Lineage-Leukemia-5 (Mll5, MGI:1924825) alleles exhibit numerical, cell cycle and functional abnormalities in their hematopoietic stem and progenitor cell (HSPC) compartments, including hyper-proliferation of otherwise quiescent hematopoietic stem cells, lack of long-term reconstitution potential and profound radiation sensitivity. Most of the HSPC defects are secondary to increased levels of DNA damage and intracellular accumulation of reactive oxygen species (ROS). To obtain first insights into underlying molecular mechanisms, we performed Affymetrix gene chip analysis using total RNA isolated from FACS-sorted Lin-Sca1+Kit+ (LSK) cells of Mll5+/+ and Mll5-/- mice, both with and without prior long-term treatment with the ROS quencher N-Acetyl-L-Cysteine (NAC). As key finding, microarray data revealed elevated hybridization signals for several transcripts of known or putative IFN-1 target genes in LSK cells from Mll5-/- mice irrespective of NAC-treatment. In fact, comprehensive gene set enrichment analysis identified a number of gene sets closely associated with interferon responses that were significantly affected in Mll5-/- LSK cells. RNA was isolated from FACS-sorted Lin-Sca1+Kit+ (LSK) cells of Mll5+/+ and Mll5-/- mice, both with and without prior long-term treatment with the ROS quencher N-Acetyl-L-Cysteine (NAC)
Project description:The small ubiquitin-related modifier (SUMO) participates in various cellular processes, including maintenance of genome integrity, nuclear transport, transcription and signal transduction. However, the biological function of sumoylation in hematopoiesis has not been fully explored. We show here that definitive hematopoietic stem/progenitor cells (HSPCs) are depleted in SUMO-deficient zebrafish embryos. Impairment of sumoylation attenuates HSPC generation and proliferation. The hyposumoylation triggered HSPC defects are CCAAT/enhancer-binding protein ? (C/ebp?) dependent. Critically, a SUMO-C/ebp? fusion rescues the defective hematopoiesis in SUMO-deficient embryos, at least in part through restored runx1 expression. While C/ebp?-dependent transcription is involved in myeloid differentiation, our studies here reveal that C/ebp? sumoylation is essential for HSPC development during definitive hematopoiesis.
Project description:Chronic infections affect a third of the world's population and can cause bone marrow suppression, a severe condition that increases mortality from infection. To uncover the basis for infection-associated bone marrow suppression, we conducted repeated infection of WT mice with Mycobacterium avium. After 4-6 months, mice became pancytopenic. Their hematopoietic stem and progenitor cells (HSPCs) were severely depleted and displayed interferon gamma (IFN-?) signaling-dependent defects in self-renewal. There was no evidence of increased HSPC mobilization or apoptosis. However, consistent with known effects of IFN-?, transcriptome analysis pointed toward increased myeloid differentiation of HSPCs and revealed the transcription factor Batf2 as a potential mediator of IFN-?-induced HSPC differentiation. Gain- and loss-of-function studies uncovered a role for Batf2 in myeloid differentiation in both murine and human systems. We thus demonstrate that chronic infection can deplete HSPCs and identify BATF2 as a mediator of infection-induced HSPC terminal differentiation.
Project description:This SuperSeries is composed of the following subset Series: GSE30444: Retroviral Sox17 over-expression adult hematopoietic stem/progenitor cells microarray GSE30445: Sox17-transgenic hematopoietic stem cell microarray Refer to individual Series