Project description:Mesendodermal specification and cardiac differentiation are key issues for developmental biology and heart regeneration medicine. Previously, we demonstrated that FAM122A, a highly conserved housekeeping gene, is an endogenous inhibitor of protein phosphatase 2A (PP2A) and participates in multifaceted physiological and pathological processes. However, the in vivo function of FAM122A is largely unknown. In this study, we observed that Fam122 deletion resulted in embryonic lethality with severe defects of cardiovascular developments and significantly attenuated cardiac functions in conditional cardiac-specific knockout mice. More importantly, Fam122a deficiency impaired mesendodermal specification and cardiac differentiation from mouse embryonic stem cells but showed no influence on pluripotent identity. Mechanical investigation revealed that the impaired differentiation potential was caused by the dysregulation of histone modification and Wnt and Hippo signaling pathways through modulation of PP2A activity. These findings suggest that FAM122A is a novel and critical regulator in mesendodermal specification and cardiac differentiation. This research not only significantly extends our understanding of the regulatory network of mesendodermal/cardiac differentiation but also proposes the potential significance of FAM122A in cardiac regeneration.
Project description:The electron transport chain promotes aspartate synthesis, which is required for cancer cell proliferation. However, it is unclear whether aspartate is limiting in normal stem cells. We found that mouse hematopoietic stem cells (HSCs) depend entirely on cell-autonomous aspartate synthesis, which increases upon HSC activation. Overexpression of the glutamate/aspartate transporter, Glast, or deletion of glutamic-oxaloacetic transaminase 1 (Got1) each increased aspartate levels in HSCs/progenitor cells and increased the function of HSCs but not colony-forming progenitors. Conversely, deletion of Got2 reduced aspartate levels and the function of HSCs but not colony-forming progenitors. Deletion of Got1 and Got2 eliminated HSCs. Isotope tracing showed aspartate was used to synthesize asparagine and purines. Both contributed to increased HSC function as deletion of asparagine synthetase or treatment with 6-mercaptopurine attenuated the increased function of GLAST-overexpressing HSCs. HSC function is thus limited by aspartate, purine, and asparagine availability during hematopoietic regeneration.
Project description:A tightly controlled balance between hematopoietic stem and progenitor cell compartments is required to maintain normal blood cell homeostasis throughout life, and this balance is regulated by intrinsic and extrinsic cellular factors. Cav-1 is a 22-kDa protein that is located in plasma membrane invaginations and is implicated in regulating neural stem cell and embryonic stem cell proliferation. However, the role of Cav-1 in hematopoietic stem cell (HSC) function is largely unknown. In this study, we used Cav-1(-/-) mice to investigate the role of Cav-1 in HSCs function during aging. The results showed that Cav-1(-/-) mice displayed a decreased percentage of B cells and an increased percentage of M cells in the bone marrow and peripheral blood, and these changes were due to an increased number of HSCs. FACS analysis showed that the numbers of Lin(-)Sca1(+)c-kit(+) cells (LSKs), long-term HSCs (LT-HSCs), short-term HSCs and multipotent progenitors were increased in Cav-1(-/-) mice compared with Cav-1(+/+) mice, and this increase became more pronounced with aging. An in vitro clonogenic assay showed that LT-HSCs from Cav-1(-/-) mice had reduced ability to self-renew. Consistently, an in vivo competitive transplantation assay showed that Cav-1(-/-) mice failed to reconstitute hematopoiesis. Moreover, a Cav-1 deletion disrupted the quiescence of LSKs and promoted cell cycle progression through G2/M phase. In addition, we found that Cav-1 deletion impaired the ability of HSCs to differentiate into mature blood cells. Taken together, these data suggest that Cav-1-deficient cells impaired HSCs quiescence and induced environmental alterations, which limited HSCs self-renewal and function.
Project description:Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure, variable congenital malformations and a predisposition to malignancies. FANCB (also known as FAAP95), is the only X-linked FA gene discovered thus far. In the present study, we investigated hematopoiesis in adult Fancb deficient (Fancb(-/y)) mice and found that Fancb(-/y) mice have decreased hematopoietic stem cell (HSC) quiescence accompanied by reduced progenitor activity in vitro and reduced repopulating capacity in vivo. Like other FA mouse models previously reported, the hematopoietic system of Fancb(-/y) mice is hypersensitive to DNA cross-linking agent mitomycin C (MMC), which induces bone marrow failure in Fancb(-/y) mice. Furthermore, Fancb(-/y) BM exhibits slower recovery kinetics and less tolerance to myelotoxic stress induced by 5-fluorouracil than wild-type littermates. RNA-seq analysis reveals altered expression of genes involved in HSC function and cell cycle regulation in Fancb(-/y) HSC and progenitor cells. Thus, this Fancb(-/y) mouse model provides a novel approach for studying the critical role of the FA pathway not only in germ cell development but also in the maintenance of HSC function.
Project description:CCCTC binding factor (CTCF) is an important factor in the maintenance of chromatin-chromatin interactions, yet the mechanism regulating its binding to chromatin is unknown. We demonstrate that zinc finger protein 143 (ZNF143) is a key regulator for CTCF-bound promoter-enhancer loops. In the murine genome, a large percentage of CTCF and ZNF143 DNA binding motifs are distributed 37 bp apart in the convergent orientation. Furthermore, deletion of ZNF143 leads to loss of CTCF binding on promoter and enhancer regions associated with gene expression changes. CTCF-bound promoter-enhancer loops are also disrupted after excision of ZNF143. ZNF143-CTCF-bound promoter-enhancer loops regulate gene expression patterns essential for maintenance of murine hematopoietic stem and progenitor cell integrity. Our data suggest a common feature of gene regulation is that ZNF143 is a critical factor for CTCF-bound promoter-enhancer loops.
Project description:U2AF1 is involved in the recognition of the 3' splice site during pre-mRNA splicing. Mutations in U2AF1 are frequently observed in myelodysplastic syndromes. However, the role of wild-type U2AF1 in normal hematopoiesis has remained elusive. Using a novel conditional U2af1 knockout allele, we have found that deletion of U2af1 results in profound defects in hematopoiesis characterized by pancytopenia, ablation of hematopoietic stem/progenitor cells (HSPC) leading to bone marrow failure and early lethality in mice. U2af1 deletion impairs HSPC function and repopulation capacity. U2af1 deletion also causes increased DNA damage and reduced survival in hematopoietic progenitors. RNA sequencing analysis reveals significant alterations in the expression of genes related to HSC maintenance, cell proliferation, and DNA damage response-related pathways in U2af1-deficient HSPC. U2af1 deficiency also induces splicing alterations in genes important for HSPC function. This includes altered splicing and perturbed expression of Nfya and Pbx1 transcription factors in U2af1-deficient HSPC. Collectively, these results suggest an important role for U2af1 in the maintenance and function of HSPC in normal hematopoiesis. A better understanding of the normal function of U2AF1 in hematopoiesis is important for development of appropriate therapeutic approaches for U2AF1 mutant induced hematologic malignancies.
Project description:SH2-domain-containing inositol 5'-phosphatase-1 (SHIP) deficiency significantly increases the number of hematopoietic stem cells (HSCs) present in the bone marrow (BM). However, the reconstitution capacity of these HSCs is severely impaired, suggesting that SHIP expression might be an intrinsic requirement for HSC function. To further examine this question, we developed a model in which SHIP expression is ablated in HSCs while they are resident in a SHIP-competent milieu. In this setting, we find that long-term repopulation by SHIP-deficient HSCs is not compromised. Moreover, SHIP-deficient HSCs from this model repopulate at levels comparable with wild-type HSCs upon serial transfer. However, when HSCs from mice with systemic ablation of SHIP are transplanted, they are functionally compromised for repopulation. These findings demonstrate that SHIP is not an intrinsic requirement for HSC function, but rather that SHIP is required for the BM milieu to support functionally competent HSCs. Consistent with these findings, cells that comprise the BM niche express SHIP and SHIP deficiency profoundly alters their function.
Project description:Hematopoietic stem cells (HSCs) have the potential to replenish the blood system for the lifetime of the organism. Their 2 defining properties, self-renewal and differentiation, are tightly regulated by the epigenetic machineries. Using conditional gene-knockout models, we demonstrated a critical requirement of lysine acetyltransferase 5 (Kat5, also known as Tip60) for murine HSC maintenance in both the embryonic and adult stages, which depends on its acetyltransferase activity. Genome-wide chromatin and transcriptome profiling in murine hematopoietic stem and progenitor cells revealed that Tip60 colocalizes with c-Myc and that Tip60 deletion suppress the expression of Myc target genes, which are associated with critical biological processes for HSC maintenance, cell cycling, and DNA repair. Notably, acetylated H2A.Z (acH2A.Z) was enriched at the Tip60-bound active chromatin, and Tip60 deletion induced a robust reduction in the acH2A.Z/H2A.Z ratio. These results uncover a critical epigenetic regulatory layer for HSC maintenance, at least in part through Tip60-dependent H2A.Z acetylation to activate Myc target genes.
Project description:Age-related changes in the hematopoietic compartment are primarily attributed to cell-intrinsic alterations in hematopoietic stem cells (HSCs); however, the contribution of the aged microenvironment has not been adequately evaluated. Understanding the role of the bone marrow (BM) microenvironment in supporting HSC function may prove to be beneficial in treating age-related functional hematopoietic decline. Here, we determined that aging of endothelial cells (ECs), a critical component of the BM microenvironment, was sufficient to drive hematopoietic aging phenotypes in young HSCs. We used an ex vivo hematopoietic stem and progenitor cell/EC (HSPC/EC) coculture system as well as in vivo EC infusions following myelosuppressive injury in mice to demonstrate that aged ECs impair the repopulating activity of young HSCs and impart a myeloid bias. Conversely, young ECs restored the repopulating capacity of aged HSCs but were unable to reverse the intrinsic myeloid bias. Infusion of young, HSC-supportive BM ECs enhanced hematopoietic recovery following myelosuppressive injury and restored endogenous HSC function in aged mice. Coinfusion of young ECs augmented aged HSC engraftment and enhanced overall survival in lethally irradiated mice by mitigating damage to the BM vascular microenvironment. These data lay the groundwork for the exploration of EC therapies that can serve as adjuvant modalities to enhance HSC engraftment and accelerate hematopoietic recovery in the elderly population following myelosuppressive regimens.
Project description:Hematopoietic stem cell (HSC) maintenance depends on extrinsic cues. Currently, only local signals arising from the bone marrow niche have been shown to maintain HSCs. However, it is not known whether systemic factors also sustain HSCs. We assessed the physiological source of thrombopoietin (TPO), a key cytokine required for maintaining HSCs. Using TpoDsRed-CreER knock-in mice, we showed that TPO is expressed by hepatocytes but not by bone marrow cells. Deletion of Tpo from hematopoietic cells, osteoblasts, or bone marrow mesenchymal stromal cells does not affect HSC number or function. However, when Tpo is deleted from hepatocytes, bone marrow HSCs are depleted. Thus, a cross-organ factor, circulating TPO made in the liver by hepatocytes, is required for bone marrow HSC maintenance. Our results demonstrate that systemic factors, in addition to the local niche, are a critical extrinsic component for HSC maintenance.