scholarly journals The RNA Splicing Factor U2AF1 Controls Hematopoietic Stem Cell Survival and Function

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 9-9
Author(s):  
Avik Dutta ◽  
Yue Yang ◽  
Bao Le ◽  
Golam Mohi
Keyword(s):  
Dna Damage ◽  
Rna Splicing ◽  
Marked Decrease ◽  
Wild Type ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem ◽  
Damage Response ◽  
And Function ◽  
Deficient Mice

Somatic mutations in U2AF1 have been identified in ~11% cases of MDS. U2AF1 is involved in the recognition of the 3' splice site required for the recruitment of the U2 snRNP during pre-mRNA splicing. Most U2AF1 mutations are found in two hotspots (S34 and Q157) within the first and second zinc finger domains. Transgenic and knock-in mice expressing U2AF1 S34F mutant exhibit impaired hematopoiesis. However, the role of wild-type U2AF1 in regulating hematopoietic stem cell (HSC) function and normal hematopoiesis has remained unknown. To determine the role of U2AF1 in normal hematopoiesis, we generated a new conditional U2af1 knockout (floxed) mouse. We crossed U2af1 floxed mouse with Mx1-Cre mouse and the expression of Cre recombinase was induced with polyinosine-polycytosine (pI-pC) injection at 5 to 6 weeks after birth. We observed that deletion of U2af1 significantly reduced white blood cell, neutrophil, red blood cell and platelet counts in their peripheral blood compared with control animals within 10-14 days after pI-pC injection. Histopathologic analysis of the BM sections from U2af1-deficient mice showed severe BM aplasia. Flow cytometric analyses revealed a marked decrease in myeloid, erythroid and megakaryocytic precursors in the BM of U2af1-deficient mice compared with control animals. We also observed a marked decrease in Lin-Sca-1+c-kit+(LSK) and long-term hematopoietic stem cells (LT-HSC), short-term HSC (ST-HSC), and multipotential progenitors (MPP) as well as common myeloid progenitors (CMP), granulocyte-macrophage progenitors (GMP), and megakaryocyte-erythroid progenitors (MEP) in the BM of U2af1-deleted mice. Hematopoietic progenitor colony assays showed a significant decrease in myeloid (CFU-GM), erythroid (BFU-E), and megakaryocytic (CFU-Mk) colonies in the BM of U2af1-deficient mice.Together, these data suggest that loss of U2af1 causes severe defects in hematopoiesis. We performed both non-competitive and competitive BM transplantation assays using U2af1-deficient BM to determine the role of U2af1 in HSC function. There was marked reduction of HSC, progenitors and all types of blood and BM cell precursors upon U2af1 deletion (by pI-pC administration) in the transplanted animals. Also, U2af1-deficient HSCs were unable to compete with WT HSCs and there was rapid loss of hematopoietic progenitors/precursors derived from the U2af1-deficient HSCs. Since U2af1 deletion resulted in rapid decrease of hematopoietic progenitors in the BM, we asked whether deletion of U2af1 insulted the genome and induced apoptosis to hematopoietic cells in the BM. We observed significantly increased apoptosis in the total BM as wells as in c-kit+, Gr1+, Ter119+and CD41+cells suggesting that hematopoietic progenitors and precursors of multiple cell lineages underwent apoptosis upon U2af1 deletion. We also performed gamma-H2AX assay using imaging flow cytometry to evaluate DNA damage in total BM, Gr1+(myeloid) and CD71+(erythroid) cells in control and U2af1-deleted mice. We observed markedly elevated gamma-H2AX in total BM, Gr1+and CD71+cells from U2af1-deficient mice compared with control mice.In addition, we observed increased Chk1 phosphorylation (ser345), a hallmark for activation of the ATR pathway, and increased histone H2A K119 ubiquitination (H2AK119Ub), a marker for DNA damage response, in the BM of U2af1-deficient mice. Thus, depletion of U2af1 causes insult to the genome and induces DNA damage and increased cell death. To gain insights into severe hematopoietic defects observed in U2af1-deficient mice, we performed transcriptome profiling of sorted LSK cells from U2af1 wild type (control) and U2af1-deleted mice. GSEA analysis of RNA sequencing data revealed significant downregulation of genes related to HSC maintenance in U2af1-deficient LSK. GSEA also revealed enrichment for cell cycle and DNA damage response-related genes, consistent with decreased proliferation and increased DNA damage and apoptosis observed in U2af1-deficient hematopoietic progenitors. We also determined the effects of U2af1 deletion on RNA splicing. Interestingly, we observed significant changes in gene expression as well as splicing alterations in several genes important for HSC survival and function. In conclusion, our results suggest a crucial role for U2af1 in the survival and function of HSC. Disclosures Mohi: Tolero Pharmaceuticals Inc.: Research Funding.

scholarly journals Critical Requirement of U2AF1 in the Maintenance and Function of Hematopoietic Stem Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1195-1195
Author(s):  
Avik Dutta ◽  
Yue Yang ◽  
Bao Le ◽  
Golam Mohi
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Rna Sequencing ◽  
Marked Decrease ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Hematopoietic Development ◽  
And Function ◽  
Deficient Mice

Mutations in the RNA spliceosome genes have been frequently found in myelodysplastic syndromes (MDS). U2AF1 is involved in the recognition of the 3' splice site required for the recruitment of the U2 snRNP during pre-mRNA splicing. U2AF1 mutations have been identified in ~11% cases of MDS and are associated with poor prognosis. However, the role of wild type U2AF1 in normal hematopoiesis has remained unknown. To determine the role of U2AF1 in hematopoietic stem/progenitor cell (HSPC) function and normal hematopoiesis, we have generated a conditional U2AF1 knockout (floxed) mouse. We crossed the U2AF1 floxed mouse with Mx1Cre mouse and the expression of Cre recombinase was induced with pI-pC injection at 4 weeks after birth. All induced Mx1Cre;U2AF1fl/fl (U2AF1-deleted) mice became moribund or died between 11-12 days after pI-pC induction. U2AF1-deleted mice exhibited marked decrease in bone marrow (BM) cellularity and significantly reduced numbers of WBC, neutrophil, RBC and platelet counts in their peripheral blood compared with control animals. Flow cytometric analyses revealed a dramatic decrease in myeloid, erythroid and megakaryocytic precursor cells in U2AF1-deficient mice compared with control animals. Hematopoietic progenitor colony assays showed a marked decrease in myeloid (CFU-GM), erythroid (BFU-E), and megakaryocytic (CFU-Mk) colonies in the BM of U2AF1-deficient mice. Histopathologic analysis revealed severe BM aplasia in U2AF1-deficient mice. Together, these data suggest that deletion of U2AF1 results in profound defects in hematopoietic development. The fatal BM failure in U2AF1-deficient mice prompted us to examine the HSPC compartments in the BM of these animals. We observed a marked decrease in Lin-Sca-1+c-kit+(LSK) and long-term hematopoietic stem cells (LT-HSC), short-term HSC (ST-HSC), and multipotential progenitors (MPP) as well as early progenitors including common myeloid progenitors (CMP), granulocyte-macrophage progenitors (GMP), and megakaryocyte-erythroid progenitors (MEP) in the BM of U2AF1-deficient mice, indicating a defect at the earliest stage of adult hematopoietic development. To determine whether the loss of HSCs in U2AF1-deficient animals is cell autonomous, BM cells from uninduced control (U2AF1fl/fl; no cre) and Mx1Cre;U2AF1fl/fl mice were transplanted into lethally irradiated WT C57BL/6 mice. Six weeks after transplantation, recipients were injected with pI-pC to induce the deletion of U2AF1. All the recipients of U2AF1-deficient BM became moribund within 14 days after pI-pC induction. Deletion of U2AF1 in the recipient animals resulted in pancytopenia and marked decrease in HSC/progenitors, myeloid, erythroid and megakaryocytic cells similar to that observed in the primary U2AF1-deficient mice, suggesting that the hematopoietic defects in U2AF1-deficient HSCs is cell intrinsic. We performed competitive repopulation assays to further evaluate the function of U2AF1-deficient HSCs. BM cells from uninduced control (U2AF1fl/fl; no cre) and Mx1Cre;U2AF1fl/fl mice (CD45.2+) were mixed with CD45.1+competitor BM cells at a ratio of 1:1 and then transplanted into lethally irradiated congenic recipient animals (CD45.1+). Chimerism analysis in the transplanted animals revealed that U2AF1-deficient mice BM cells were completely unable to compete with WT BM cells. The percentages of U2AF1-deficient CD45.2+(donor-derived) LSK, myeloid, B and T cells were markedly reduced in the recipient animals compared with wild type U2AF1 BM donor at 16 weeks after transplantation, indicating that U2AF1-deficiency impairs the repopulation capacity of the HSCs. To gain insights into the mechanism by which U2AF1controls HSC maintenance and function,we performed RNA-sequencing on purified LSK cells from control and U2AF1-deleted mice. Analysis of RNA-sequencing data revealed significant down regulation of genes related to HSC maintenance, cell cycle and JAK-STAT pathway in U2AF1-deficient LSK cells compared with control LSK. RNA sequencing also identified significantly altered splicing events in several important genes in U2AF1-deficient LSK cells. The most commonly altered splicing events were exon skipping/inclusion. We also observed increased phospho-H2AX and DNA damage in U2AF1-deficient BM cells. Overall, our results suggest an essential role for U2AF1 in the maintenance and function of hematopoietic stem cells. Disclosures No relevant conflicts of interest to declare.

Loss of Gfi1 Impedes Development of T-Cell Lymphoma upon Exposure to N-ethyl-N-nitrosourea but Predisposes to Severe Myleodysplastic Changes.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2221-2221
Author(s):  
Cyrus Khandanpour ◽  
Ulrich Duehrsen ◽  
Tarik Möröy
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Dna Damage ◽  
T Cell ◽  
Bone Marrow Cells ◽  
Cell Lymphoma ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Deficient Mice

Abstract Exogenous toxic substances often cause the initiation and development of leukemia and lymphoma by acting as mutagens. N-ethyl-N-nitrosourea (ENU) is a paradigmatic example for such a substance, which introduces point mutations in the genome through DNA damage and repair pathways. ENU is widely used to experimentally induce T-cell lymphomas in mice. We have used ENU to investigate whether the hematopoietic transcription factor Gfi1 is required for lymphomagenesis. The Gfi1 gene was originally discovered as a proviral target gene and a series of experiments with transgenic mice had suggested a role of Gfi1 as a dominant oncogene with the ability to cooperate with Myc and Pim genes in the generation of T-cell lymphoma. In addition, Gfi1 deficient mice showed a defect in T-cell maturation but also aberration in myeloid differentiation and an accumulation of myelomonocytic cells. ENU was administered i.p. once a week for three weeks with a total dose of 300mg/kg to wild type (wt) and Gfi1 null mice. Wild type mice (12/12) predominantly developed T-cell tumors and rarely acute myeloid leukemia, as expected. However, only 2/8 Gfi1 −/− mice succumbed to lymphoid neoplasia; they rather showed a severe dysplasia of the bone marrow that was more pronounced than in wt controls. These changes in Gfi1 null mice were accompanied by a dramatic decrease of the LSK (Lin-, Sca1- and c-Kit+) bone marrow fraction that contains hematopoietic stem cells and by a higher percentage (18%) of bone marrow cells, not expressing any lineage markers (CD4, CD 8, Ter 119, Mac1, Gr1, B220, CD3). In particular, we found that the LSK subpopulation of Gfi1 deficient mice showed a noticeable increase in cells undergoing apoptosis suggesting a role of Gfi1 in hematopoietic stem cell survival. In addition, Gfi1−/− bone marrow cells and thymic T-cells were more sensitive to DNA damage such as radiation and exposure to ENU than their wt counterparts pointing to a role of Gfi1 in DNA damage response. Our results indicate that Gfi1 is required for development of T-cell tumors and that a loss of Gfi1 may sensitize hematopoietic cells and possibly hematopoietic stem cells for programmed cell death. Further studies have to show whether interfering with Gfi1 expression or function might represent a tool in the therapy of leukemia.

Deficiency of Bone Marrow Stromal Cx43 Expression Induces Hematopoietic Progenitor Homing Deficiency and Cell-Cycle Arrest of Hematopoietic Stem Cells and Progenitors.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 349-349
Author(s):  
Lina Li ◽  
Cynthia A. Presley ◽  
Bryan Kastl ◽  
Jose A. Cancelas
Keyword(s):  
Cell Cycle ◽  
S Phase ◽  
Chimeric Mice ◽  
Wild Type ◽  
Hematopoietic Progenitor ◽  
Cx43 Expression ◽  
Hematopoietic Stem ◽  
M Phase ◽  
Deficient Mice

Abstract Contact between bone marrow (BM) hematopoietic stem cells (HSC) and osteoblast/stromal (OS) cells has been shown to be critical in the regulation of hematopoiesis. However, very little is known about the regulatory mechanisms of direct cell-to-cell communication in the hematopoietic microenvironment. BM cells are directly connected through gap junctions (GJs) which consist of narrow channels between contacting cells and are composed by connexins. Connexin 43 (Cx43) is expressed by BM OS cells. Multiple osteogenic defects have been reported in human Cx43 mutations and Cx43 has been shown to be essential in controlling osteoblast functions. Due to the perinatal death of Cx43 germline null mice, an interferon-inducible, conditional genetic approach (Mx1-Cre), expressed by both hematopoietic and stromal BM cells, was used to study the role of Cx43 in stem cell function. We have previously reported that Cx43 is critical for the interaction between stroma and HSC in CAFC assays (Cancelas J.A. et al., Blood 2000) and in adult hematopoiesis after 5-fluorouracil (5-FU) administration (Presley C, et al., Cell Comm. Adh., 2005). Here, we observed that after 5-FU administration, Cx43 expression is predominantly located in the endosteum. To study the role of stroma-dependent Cx43 in hematopoiesis, we developed hematopoietic chimeras by BM transplantation of wild-type Cx43 HSC into stromal Cx43-deficient mice. Stromal Cx43 deficiency induced a severe impairment of blood cell formation during the recovery phase after 5-FU administration compared to stromal Mx1-Cre-Tg wild-type controls (Table 1), as well as a significant decrease in BM cellularity (~60% reduction) and progenitor cell content (~83% reduction). Cell cycle analysis of 5-FU-treated BM progenitors from stromal Cx43-deficient mice showed an S-phase arrest (S phase: 63.5%; G2/M phase: <1%) compared to wild-type chimeric mice (S phase: 38.6%, G2/M phase: 7.8%, p=0.01) suggesting a cell division blockade. Unlike Cx43-deficient primary mice, a differentiation arrest at the HSC compartment was observed in 5-FU-treated, stromal Cx43-deficient mice, since the content of competitive repopulating units (CRU) at 1 month, of 14-day post-5-FU BM of stromal Cx43-deficient mice was increased (27.7 ± 0.67) compared to recipients of HSC from stromal wild-type counterparts (26.5 ± 0.92 CRU, p < 0.01). Interestingly, wild-type hematopoietic progenitor homing in stromal Cx43-deficient BM was severely impaired with respect to wild-type BM (5.1% vs10.4 %, respectively, p < 0.01), while hematopoietic Cx43-deficient BM progenitors normally homed into the BM, suggesting a differential role for Cx43 in stromal and HSC. In conclusion, expression of Cx43 in osteoblasts and stromal cells appears to play a crucial role in the regulation of HSC homing in BM and hematopoietic regeneration after chemotherapy. Peripheral blood counts of WT and stromal Cx43-deficient chimeric mice after 5-FU administration (150 mg/Kg) Neutrophil counts (×10e9/L) Reticulocyte count (%) Day post-5-FU WT Cx43-deficient WT Cx43-deficient * p < 0.05 Day +8 2.89 ± 0.06 0.81 ± 0.02* 2.0 ± 0.6 3.0 ± 0.9 Day +11 9.11 ± 2.5 3.13 ± 0.8* 6.1 ± 0.6 2.7 ± 0.3* Day +14 6.22 ± 5.7 7.58 ± 8.2 7.5 ± 0.5 2.5 ± 0.5*

Viral Infection Sentinel Rig-I Maintains Hematopoietic Stem Cell Function Through Regulating DNA-Damage Response

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1166-1166
Author(s):  
Wu Zhang ◽  
Meng-Lei Ding ◽  
Xian-Yang Li ◽  
He-Zhou Guo ◽  
Hong-Xin Zhang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Dna Lesions ◽  
Type I ◽  
Hematopoietic Stem ◽  
Damage Response ◽  
Underlying Mechanisms ◽  
And Function

Abstract Throughout life hematopoietic stem cells (HSCs) have to cope with various kinds of insults from inflammation to DNA damage constantly to maintain the integrity of stemness. It is possible that certain core factors are commonly implicated in the maintenance of HSC pool and function under discrete physiological and pathological conditions. However, the underlying mechanisms remain largely unexplored. Previous works have demonstrated that retinoic acid inducible gene I (Rig-I) plays an essential role in recognizing viral RNA and activating type I IFN transcription, but whether Rig-I is involved in the core program governing HSCs’ behaviors is unclear. Here, we report that in the steady status Rig-I deficiency significantly increased HSC number by dysregulating the cell-cycling status of HSCs in mice. However, HSCs in Rig-I-/- mice were actually more sensitive to genotoxic treatments such as irradiation as compared to wild type HSCs, causing more Rig-I-/- mice to die of hematopoietic exhaustion. In accordance, HSC transplantation assays showed a significant impact of Rig-I loss on the hematopoietic regeneration capacity. Mechanistically, we found that Rig-I represented a pivotal component of the molecular pathways that mediate DNA-damage response and the repair of DNA lesions. Taken together, these data indicate a crucial role of innate immunity-regulatory factor Rig-I in the maintenance of HSCs. Disclosures: No relevant conflicts of interest to declare.

scholarly journals U2AF1(S34F) Mutant Hematopoietic Cells Require Expression of Wild-Type U2af1 for Survival

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 941-941
Author(s):  
Brian Wadugu ◽  
Amanda Heard ◽  
Joseph Bradley ◽  
Matthew Ndonwi ◽  
Jin J Shao ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Rna Splicing ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Poly I:C ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Mutant Cells ◽  
Marrow Cells

Abstract Somatic mutations in U2AF1, a spliceosome gene involved in pre-mRNA splicing, occur in up to 11% of MDS patients. While we reported that mice expressing mutant U2AF1(S34F) have altered hematopoiesis and RNA splicing, similar to mutant MDS patients, the role of wild-type U2AF1 in normal hematopoiesis has not been studied. U2AF1mutations are always heterozygous and the wild-type allele is expressed, suggesting that mutant cells require the residual wild-type (WT) allele for survival. A complete understanding of the role of wild-type U2AF1 on hematopoiesis and RNA splicing will enhance our understanding of how mutant U2AF1 contributes to abnormal hematopoiesis and splicing in MDS. In order to understand the role of wild-type U2af1 in normal hematopoiesis, we created a conditional U2af1 knock-out (KO) mouse (U2af1flox/flox). Homozygous embryonic deletion of U2af1using Vav1-Cre was embryonic lethal and led to reduction in fetal liver hematopoietic stem and progenitor cells (KLS and KLS-SLAM, p ≤ 0.05) at embryonic day 15, suggesting that U2af1 is essential for hematopoiesis during embryonic development. To study the hematopoietic cell-intrinsic effects of U2af1 deletion in adult mice, we performed a non-competitive bone marrow transplant of bone marrow cells from Mx1-Cre/U2af1flox/flox, Mx1-Cre/U2af1flox/wtor Mx1-Cre/U2af1wt/wtmice into lethally irradiated congenic recipient mice. Following poly I:C-induced U2af1deletion, homozygous U2af1 KOmice, but not other genotypes (including heterozygous KO mice), became moribund. Analysis of peripheral blood up to 11 days post poly I:C treatment revealed anemia (hemoglobin decrease >1.7 fold) and multilineage cytopenias in homozygous U2af1 KOmice compared to all other genotypes(p ≤ 0.001, n=5 each).Deletion of U2af1 alsoled to rapid bone marrow failure and a reduction in the absolute number of bone marrow neutrophils (p ≤ 0.001), monocytes (p ≤ 0.001), and B-cells (p ≤ 0.05), as well as a depletion of hematopoietic progenitor cells (KL, and KLS cells, p ≤ 0.001, n=5 each). Next, we created mixed bone marrow chimeras (i.e., we mixed equal numbers of homozygous KO and wild-type congenic competitor bone marrow cells and transplanted them into lethally irradiated congenic recipient mice) to study the effects of U2af1 deletion on hematopoietic stem cell (HSC) function. As early as 10 days following Mx1-Cre-induction, we observed a complete loss of peripheral blood neutrophil and monocyte chimerism of the U2af1 KOcells, but not U2af1 heterozygous KO cells, and at 10 months there was a complete loss of homozygous U2af1 KObone marrow hematopoietic stem cells (SLAM, ST-HSCs, and LT-HSCs), neutrophils, and monocytes, as well as a severe reduction in B-cells and T-cells (p ≤ 0.001, n=3-4 for HSCs. p ≤ 0.001, n=9-10 for all other comparisons). The data indicate that normal hematopoiesis is dependent on wild-type U2af1expression, and that U2af1 heterozygous KO cells that retain one U2af1 allele are normal. Next, we tested whether mutant U2AF1(S34F) hematopoietic cells require expression of wild-type U2AF1 for survival. To test this, we used doxycycline-inducible U2AF1(S34F) or U2AF1(WT) transgenic mice. We generated ERT2-Cre/U2af1flox/flox/TgU2AF1-S34F/rtTA(S34F/KO), and ERT2-Cre/U2af1flox/flox/TgU2AF1-WT/rtTA,(WT/KO) mice, as well as all other single genotype control mice. We then created 1:1 mixed bone marrow chimeras with S34F/KO or WT/KO test bone marrow cells and wild-type competitor congenic bone marrow cells and transplanted them into lethally irradiated congenic recipient mice. Following stable engraftment, we induced U2AF1(S34F) (or WT) transgene expression with doxycycline followed by deletion of endogenous mouse U2af1 using tamoxifen. As early as 2 weeks post-deletion of U2af1, S34F/KO neutrophil chimerism dropped to 5.4% indicating loss of mutant cells, while WT/KO neutrophil chimerism remained elevated at 31.6% (p = 0.01, n=6-8). The data suggest that mutant U2AF1(S34F) hematopoietic cells are dependent on expression of wild-type U2af1 for survival. Since U2AF1mutant cells are vulnerable to loss of the residual wild-type U2AF1allele, and heterozygous U2af1KO cells are viable, selectively targeting the wild-type U2AF1allele in heterozygous mutant cells could be a novel therapeutic strategy. Disclosures No relevant conflicts of interest to declare.

MiR-223 is dispensable for platelet production and function in mice

2013 ◽  
Vol 110 (12) ◽  
pp. 1207-1214 ◽  
Author(s):  
Xavier Loyer ◽  
Simon Leierseder ◽  
Tobias Petzold ◽  
Lin Zhang ◽  
Steffen Massberg ◽  
...  
Keyword(s):  
Platelet Adhesion ◽  
Cell Types ◽  
Surface Expression ◽  
Wild Type ◽  
Platelet Production ◽  
Multiple Cell ◽  
And Function ◽  
Deficient Mice ◽  
Platelet Depletion

SummaryMicroRNAs (miRNAs) are key physiological regulators in multiple cell types. Here, we assessed platelet production and function in mice deficient in miR-223, one of the most abundantly expressed miRNAs in platelets and megakaryocytes. We found platelet number, size, lifespan as well as surface expression of platelet adhesion receptors to be unchanged in miR-223-deficient mice. Likewise, loss of miR-223 did not affect platelet activation, adhesion and aggregation and also had no effect on bleeding times. Moreover, miR-223 null megakaryocytes developed normally and were capable to form pro-platelets. However, we detected a transient delay in the recovery of platelet numbers following antibody-induced platelet depletion in miR-223-deficient animals. This delay was not observed after transplantation of bone marrow from miR-223-deficient animals into wild-type recipients, indicating a non-cell-autonomous role of miR-223 for thrombopoiesis. Overall, our data indicate a surprisingly modest role of miR-223 in platelet production, while the function of platelets does not seem to depend on miR-223.

Phosphorylation of FANCA on S1449 Is Important for Fanconi Anemia (FA) Pathway Function.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 186-186
Author(s):  
Natalie B. Collins ◽  
Andrei Tomashevski ◽  
Gary M. Kupfer
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Dna Damage Response ◽  
Fanconi Anemia ◽  
Phosphorylation Site ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Damage Response ◽  
And Function ◽  
Mutant Cells

Abstract Previous work in our lab and others has shown that the Fanconi anemia proteins, FANCG and FANCA, are phosphoproteins. FANCG is phosphorylated at mitosis, and these phosphorylations are required for proper exit from chromatin at mitosis. FANCG is also phosphorylated after DNA damage, with the phosphorylation site required for wild-type sensitivity to DNA damaging agents. FANCA is also phosphorylated after DNA damage and localized to chromatin, but the site and significance of this phosphorylation were previously unknown. Mass spectrometry of FANCA revealed one phosphopeptide with phosphorylation on serine 1449. Site-directed mutagenesis of this residue to alanine (S1449A) abolished a slower mobility form of FANCA seen after MMC treatment. Furthermore, the S1449A mutant failed to completely correct the MMC hypersensitivity of FA-A mutant cells. S1449A mutant cells displayed lower than wild-type levels of FANCD2 monoubiquitination following DNA damage, and an increased number of gross chromosomal aberrations were seen in metaphase spreads from S1449A mutant cells when compared to wild type cells. Using a GFP reporter substrate to measure homologous recombination, cells expressing the S1449A FANCA failed to completely correct the homologous recombination defect seen in FA cells. Taken together, cells expressing FANCA S1449A display a variety of FA-associated phenotypes, suggesting that the phosphorylation of S1449 is a functionally significant event. The DNA damage response in human cells is, in large part, coordinated by phosphorylation events initiated by apical kinases ATM and ATR. S1449 is found in a consensus ATM site, therefore studies are underway to determine if ATM or ATR is the kinase responsible for FANCA phosphorylation at S1449. Phosphorylation is a crucial process in transducing the DNA damage response, and phosphorylation of FA proteins appears critical to both localization and function of the proteins. Understanding how phosphorylation marks are placed on FANCA will give insight into the role of FANCA in the DNA damage response.

Angiopoietin-Like Protein 3 Supports the Activity of Mouse Hematopoietic Stem Cells in the Bone Marrow Niche.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 249-249
Author(s):  
Junke Zheng ◽  
HoangDinh Huynh ◽  
Chengcheng Zhang
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Dna Damage ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Marrow Stromal Cells ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Hsc Niche

Abstract Abstract 249 The physiological role of Angiopoietin-like proteins (Angptls) in hematopoietic system is unknown. Here we showed that Angptl3 is expressed by both hematopoietic stem cells (HSCs) and bone marrow stromal cells. In particular, the expression of Angptl3 in the bone marrow stromal cells is significantly increased upon transplantation, suggesting that this protein may play an important role in the bone marrow under stress. We asked whether Angptl3 expression had a functional role in HSCs by utilizing mice ablated for Angptl3. Using Hoechst/pyronin Y staining and Brdu incorporation analysis, we found that HSCs in Angptl3-null mice exhibited significantly decreased quiescence compared to those in wild-type mice. To test the role of Angptl3 in the stress response of hematopoietic cells, we treated mice with 5-fluorouracil (5-FU), which is toxic to cycling cells and accelerates the entry of HSCs into the cell cycle. The survival of Angptl3-null mice was significantly lower than that of wild-type mice. To further identify the role of Angptl3 in stress response of HSCs, we examined whether Angptl3 affected DNA damage in HSCs upon transplantation. To this end, we transplanted WT bone marrow cells into lethally irradiated Angptl3 null recipients or WT mice. We found that HSCs in the Angptl3 null recipient mice had significantly increased gamma-H2AX foci compared to WT recipients, suggesting that Angptl3 protects HSCs from DNA damage. We further used the competitive reconstitution analysis to determine the roles of Angptl3 on HSC activity. Importantly, both Angptl3-null HSCs transplanted to wild-type recipients and wild-type HSCs transplanted to Angptl3-null recipients showed impaired repopulation. These results conclude that Angptl3 has both cell-autonomous and environmental effects that support the in vivo activity of HSCs. To identify the intracellular target of Angptl3 in HSCs, we performed DNA microarray and real-time RT-PCR analyses to compare the gene expression in HSCs isolated from WT and Angptl3 null mice. We found that Angptl3-null HSCs had increased expression of transcription factor Ikaros. Consistently, extrinsic treatment of HSCs by Angptl3 also suppressed the expression of Ikaros. Ikaros is a zinc finger transcription factor important for differentiation of lymphoid, myeloid, and erythroid cells, and its expression is low in multi-potent HSCs, but high in progenitors with lymphoid-myeloid potential. Since Angptl3 downregulates the expression of Ikaros in HSCs, we examined the effect of forced expression of Ikaros on HSC activities. Indeed, overexpression of Ikaros enhanced HSC cycling and DNA damage, and diminished their repopulation activity, indicating the downregulation of Ikaros by extrinsic Angptl3 maintains the stemness of HSCs. We studied the spatial relationship of Angptl3-expressing cells and the bone-marrow HSCs using immunohistochemical tools. We showed that 58.6% of Angptl3-producing cells were in contact with sinusoidal endothelial cells in bone marrow and that 60.8% of HSCs are adjacent to Angptl3-producing cells in the bone marrow. To directly test whether Angptl3-producing bone marrow stromal cells support HSC expansion, we co-cultured HSCs and CD45-SSEA4+ cells and used competitive reconstitution analysis to measure HSC activity. HSCs co-cultured with WT CD45-SSEA4+ cells had significantly increased repopulation relative to those co-cultured with Angptl3 null CD45-SSEA4+ cells (36% vs. 17%). This result demonstrated that bone marrow CD45-SSEA4+ cells support expansion of HSCs, and provided the functional evidence that Angptl3-producing stromal cells are a part of HSC niche in the bone marrow. Thus, Angptl3-producing cells are an important component of the HSC niche. Our experiments demonstrate a unique example of the extrinsic control of stemness by cell-autonomous effects from stem cells per se and by environmental effects from the niche cells. Disclosures: No relevant conflicts of interest to declare.

Bone Marrow Failure in RPS19-Deficient Mice Is Partly Caused by p53 Activation and Responds to L-Leucine Treatment

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 727-727
Author(s):  
Pekka Jaako ◽  
Shubhranshu Debnath ◽  
Karin Olsson ◽  
Johan Flygare ◽  
Stefan Karlsson
Keyword(s):  
Bone Marrow ◽  
Preliminary Data ◽  
Bone Marrow Failure ◽  
Mtor Pathway ◽  
Wild Type ◽  
Hematopoietic Progenitors ◽  
P53 Activation ◽  
Deficient Mice

Abstract Abstract 727 Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia associated with physical malformations and predisposition to cancer. Of the many different DBA disease genes known, all encode for ribosomal proteins, suggesting that DBA is a disorder relating to ribosomal biogenesis or function. Among these genes, ribosomal protein S19 (RPS19) is the most frequently mutated (25 % of the patients). The generation of animal models for DBA is pivotal in order to understand the disease mechanisms and to evaluate novel therapies. We have generated two mouse models for RPS19-deficient DBA by taking advantage of RNA interference (Jaako et al. Blood. 2010;116:193. ASH meeting abstract). These models contain RPS19-targeting shRNAs expressed by a doxycycline-responsive promoter downstream of the collagen A1 locus allowing an inducible and dose-dependent regulation of shRNA. As we have previously reported, the induction of RPS19 deficiency results in a reduction in the number of erythrocytes, platelets and white blood cells that with time leads to the exhaustion of hematopoietic stem cells and bone marrow failure. In the current study we have analyzed the role of p53 in RPS19-deficient hematopoiesis by crossing the transgenic mice into Trp53 null background. To isolate the hematopoietic phenotype we transplanted bone marrow cells from these mice into lethally irradiated wild-type recipients. We have previously shown that the severity of the hematopoietic phenotype in transplanted recipients is highly dependent on the level of RPS19 downregulation, and the recipients with low RPS19 expression die 2–3 weeks after induction because of bone marrow failure. Remarkably, the inactivation of Trp53 rescued the early mortality in these recipients. However, although the inactivation of Trp53 completely reversed the erythrocyte and leukocyte numbers in the recipients with intermediate RPS19 downregulation, the recipients with low RPS19 expression still developed a mild anemia and macrocytosis. p53 activation is known to inhibit the AKT/mTOR pathway, a central regulator of cell growth and survival. Although the role of this pathway in DBA pathogenesis remains poorly defined, some patients positively respond to treatment with amino acid L-leucine, a nutrient signal that stimulates mTOR activity. Currently we are studying the role of L-leucine in RPS19-deficient hematopoiesis both in vitro and in vivo. Our preliminary data confirm that L-leucine modestly enhances the proliferation of RPS19-deficient c-Kit -enriched hematopoietic progenitors in vitro (1.2 fold in 8 days), while there is no effect on wild-type cultures. Interestingly, the proliferative response in RPS19-deficient cultures appears more pronounced when cells are cultured in low cytokine concentration (1.6 fold in 8 days). Since primary cells from DBA patients are highly responsive to stem cell factor (SCF), which also mediates its effect partly via PI3K/AKT/mTOR pathway, we are studying whether L-leucine has a synergistic role with SCF enhancing the proliferation of hematopoietic progenitors. Finally, a 15% L-leucine supplement in drinking water partly rescues the erythrocyte and leukocyte number in RPS19-deficient mice. In summary, our results demonstrate a key role of p53 activation in RPS19-deficient DBA, although they also suggest that p53-independent pathways may contribute towards phenotype upon severe RPS19 deficiency. Furthermore, our preliminary data supports the role of L-leucine as a therapeutic agent in the treatment of DBA. Disclosures: No relevant conflicts of interest to declare.

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