- Email: [email protected]
The fanconi anemia c protein regulates cellular division via the microtubule-associated protein stathmin-1
Poster Presentations/ Experimental Hematology 42 (2014) S23–S68
S47
P1096 - TWIST-1, A NOVEL REGULATOR OF HEMATOPOIETIC STEM CELL SELF-RENEWAL AND MYELOID LINEAGE COMMITMENT Xiao-Yan Liu, Cheng-Ya Dong, Nan Wang, Dan Guo, Yang-Yang Zhao, and Xiao-Tong Ma State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Tianjin, China
P1098 - POLYCOMB-LIKE 2 (PCL2) IS REQUIRED FOR HEMATOPOIETIC DEVELOPMENT Janet Manias-Rothberg1,2, Harinad Maganti1,2, Christopher Porter1, Gareth Palidwor1, Robert Paulson3, Theodore Perkins1, Caryn Ito1, and William Stanford1,2 1 OHRI, Ottawa, Ontario, Canada; 2University of Ottawa, Ottawa, Ontario, Canada; 3 Pennsylvania State University, University Park, Pennsylvania, USA
Transcription factor Twist-1 plays essential roles in specification and differentiation of mesoderm-derived tissues. Growing evidences now link Twist-1 to the acquisition of stem-cell-like properties. However, the role of Twist-1 in hematopoietic stem cell (HSC) remains largely uncharacterized. We report that Twist-1 is highly expressed in murine HSCs and its expression declines with differentiation. Competitive repopulation studies demonstrate that enforced expression of Twist-1 in HSC-enriched Lin c-Kit+Sca-1+ (LKS) cells results in the size of the G0 population, and in their reconstitution ability after the first and a second transplantation. In addition, increased Twist-1 expression causes a shift toward production of myeloid cells. Twist-1 transduction in LKS cells activates myeloid lineage-determining factors PU.1 and GATA-1 and down-regulates lymphoid factor GATA-3 in vitro, suggesting that Twist-1-mediated myeloid skewing occurs in hematopoietic stem and progenitor cells (HSPCs). These findings indicate that Twist-1 is not only involved in the maintenance of HSC dormancy and self-renewal capacity but also implicated in the myeloid lineage fate choice of HSPCs. Exploration of the underlying mechanisms reveals that up-regulated N-cadherin, c-Mpl and Tie2 and down-regulated Runx1 transcription could possibly account for the observed effects caused by Twist-1 overexpression. Our study provides the first evidence supporting a role for Twist-1 in hematopoiesis.
Polycomb genes are epigenetic repressors critical in cell fate decisions. We identified Polycomb-like 2 (PCL2) as a critical regulator of embryonic stem cell (ESC) selfrenewal via its role in pluripotency feed-forward networks (Cell Stem Cell 6:153– 166; Cell Cycle 10: 45-51). Knockdown of Pcl2 in ESCs causes defects in differentiation and increased ESC self-renewal. Pcl2 is expressed highly but is later largely restricted to adult hematopoietic tissues. We have generated Pcl2 knockout mice using gene targeted ES cells to study the role of Pcl2 in vivo. Mutant mice that lack Pcl2 die at e15.5 and exhibit growth defects, hemorrhage and anemia. Pcl2-/- mice have significantly fewer enucleated erythrocytes, suggesting Pcl2 is necessary for definitive erythropoiesis. Moreover, when plated in clonogenic colony forming-unit assays, less BFU-E and more CFU-GEMM colonies are present in the fetal liver of Pcl2-/- mice compared with their wildtype littermates. These data indicate that cells lacking in Pcl2 are in a more primitive/progenitor state. In a Friend Virus erythroleukemia model, Pcl2 represses the development of stress erythroid progenitors. RNA-seq analyses in mouse erythroid progenitors revealed a role for Pcl2 in multiple pathways including regulation of cell cycle. Pcl2 is also required for HSC selfrenewal as Pcl2-/- fetal liver cells fail to reconstitute the hematopoietic system of recipient mice in secondary transplants. Taken together, Pcl2 is required for erythroid development and self-renewal of hematopoietic stem cells.
P1097 - THE FANCONI ANEMIA C PROTEIN REGULATES CELLULAR DIVISION VIA THE MICROTUBULE-ASSOCIATED PROTEIN STATHMIN1 Audrey Magron1,2, Caroline Huard1,2, Sabine Elowe1,2, and Madeleine Carreau1,2 1 Universite Laval, Quebec, Quebec, Canada; 2Department of Pediatrics, CHU de Quebec, CHUL Research Center, Quebec, Quebec, Canada
P1099 - DECLINED PRESENTATION ANALYSIS OF HSC FATE, MAINTENANCE AND FUNCTION DUE TO DYSREGULATION OF THE WNT SIGNALING ANTAGONIST, SCLEROSTIN Jennifer Manilay University of California, Merced, Merced, California, USA
Fanconi Anemia (FA) belongs to the inherited bone marrow failure syndromes characterized by a progressive decline in primitive hematopoietic cells due to accelerated cycling and hypersensitivity to various external cellular cues. These cellular defects favour the development of clonal proliferation and leads to leukemia. Proteins mutated in FA have been implicated in mitotic events assuring the safeguard of chromosome segregation. Consequently, defective FA proteins have been associated with a higher rate of cytokinesis failure, aneuploidy and chromosome missegregation, which are hallmarks of cancer cells. In an attempt to identify FA protein’s function in cellular division events, we identified through Yeasts-2-Hybrid screening approaches, the microtubule-associated protein Stathmin (STMN), as a binding partner of the FA protein C (FANCC). Through different biochemical approaches, we confirmed the FANCC-STMN interaction. FA-disease causing mutations in FANCC prevent its interaction with STMN. We observed that FANCC with Phospho-STMN co-localizes to centrosomes during mitosis. In addition, we found that FANCC modulates STMN phosphorylation status on serine 16 and 38 during cellular division. The phosphorylation status of STMN during the cell cycle requires a functional FANCC. These results suggest that FANCC participates in the regulation of cellular division via the microtubule-associated protein STMN. Previous studies have shown that elevated levels of STMN are found in many cancers including leukemia whereas a dramatic decrease in STMN levels is found in hematopoietic cells upon differentiation along different lineages. Consequently, we propose that misregulation of STMN1 activity combined with the DNA repair defects in FA cells may account for the loss of hematopoietic stem cells and increase in cancer susceptibility seen in patients with FA.
An understanding of how changes in bone health affect hematopoiesis could inform how to retain strong immunity in patients with bone disease and reveal how agerelated changes in bones could affect hematopoiesis during aging. Sclerostin (SOST) is a secreted protein that is critical for normal bone homeostasis, in which bone-building by osteoblast (OB) cells is balanced by bone-breakdown by osteoclast (OC) cells. SOST is produced primarily by mature mineralized bone cells called osteocytes (OCYs) and prevents the maturation of OBs into OCYs by its action as a Wnt signaling antagonist. Although the role of SOST in bone homeostasis has been well-established, we observed that the absence of SOST adversely affected B lymphocyte development in SOST-knockout (KO) mice, due to changes in the bone marrow microenvironment, including reduced expression of stem cell factor (SCF) and CXCL12. These growth factors are also important for hematopoietic stem cell (HSC) maintenance and migration within the bone marrow niche; however, the role of SOST in the regulation of HSC function remains an open question. We hypothesized that HSCs that develop in SOST-deficient bones harbor defects in quiescence, cell cycling and Wnt signaling that lead to HSC depletion and hematopoietic failure. To test this hypothesis, we treated SOST KO mice with 5-fluorouracil in vivo and performed HSC cycle analysis to determine the ratios of cells in quiescence and active cell cycle. The results of these studies demonstrated that sclerostin is not essential for regulation of HSC quiescence and entrance into cell cycle. In contrast, primary hematopoietic progenitor cell (HPC) transplantation assays were performed to ascertain the role of the sclerostin-deficiency on HSC differentiation, and our preliminary results demonstrate that the SOST KO bones affect B lymphocyte development in the bone marrow and might enhance differentiation of granulocytes in the periphery. Secondary HPC transplantation studies, currently ongoing, will test whether these environment-induced changes are temporary or permanent. Taken together, these data suggest that the roles of SOST deficiency on HSC maintenance and HSC differentiation are distinct.
S47
P1096 - TWIST-1, A NOVEL REGULATOR OF HEMATOPOIETIC STEM CELL SELF-RENEWAL AND MYELOID LINEAGE COMMITMENT Xiao-Yan Liu, Cheng-Ya Dong, Nan Wang, Dan Guo, Yang-Yang Zhao, and Xiao-Tong Ma State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Tianjin, China
P1098 - POLYCOMB-LIKE 2 (PCL2) IS REQUIRED FOR HEMATOPOIETIC DEVELOPMENT Janet Manias-Rothberg1,2, Harinad Maganti1,2, Christopher Porter1, Gareth Palidwor1, Robert Paulson3, Theodore Perkins1, Caryn Ito1, and William Stanford1,2 1 OHRI, Ottawa, Ontario, Canada; 2University of Ottawa, Ottawa, Ontario, Canada; 3 Pennsylvania State University, University Park, Pennsylvania, USA
Transcription factor Twist-1 plays essential roles in specification and differentiation of mesoderm-derived tissues. Growing evidences now link Twist-1 to the acquisition of stem-cell-like properties. However, the role of Twist-1 in hematopoietic stem cell (HSC) remains largely uncharacterized. We report that Twist-1 is highly expressed in murine HSCs and its expression declines with differentiation. Competitive repopulation studies demonstrate that enforced expression of Twist-1 in HSC-enriched Lin c-Kit+Sca-1+ (LKS) cells results in the size of the G0 population, and in their reconstitution ability after the first and a second transplantation. In addition, increased Twist-1 expression causes a shift toward production of myeloid cells. Twist-1 transduction in LKS cells activates myeloid lineage-determining factors PU.1 and GATA-1 and down-regulates lymphoid factor GATA-3 in vitro, suggesting that Twist-1-mediated myeloid skewing occurs in hematopoietic stem and progenitor cells (HSPCs). These findings indicate that Twist-1 is not only involved in the maintenance of HSC dormancy and self-renewal capacity but also implicated in the myeloid lineage fate choice of HSPCs. Exploration of the underlying mechanisms reveals that up-regulated N-cadherin, c-Mpl and Tie2 and down-regulated Runx1 transcription could possibly account for the observed effects caused by Twist-1 overexpression. Our study provides the first evidence supporting a role for Twist-1 in hematopoiesis.
Polycomb genes are epigenetic repressors critical in cell fate decisions. We identified Polycomb-like 2 (PCL2) as a critical regulator of embryonic stem cell (ESC) selfrenewal via its role in pluripotency feed-forward networks (Cell Stem Cell 6:153– 166; Cell Cycle 10: 45-51). Knockdown of Pcl2 in ESCs causes defects in differentiation and increased ESC self-renewal. Pcl2 is expressed highly but is later largely restricted to adult hematopoietic tissues. We have generated Pcl2 knockout mice using gene targeted ES cells to study the role of Pcl2 in vivo. Mutant mice that lack Pcl2 die at e15.5 and exhibit growth defects, hemorrhage and anemia. Pcl2-/- mice have significantly fewer enucleated erythrocytes, suggesting Pcl2 is necessary for definitive erythropoiesis. Moreover, when plated in clonogenic colony forming-unit assays, less BFU-E and more CFU-GEMM colonies are present in the fetal liver of Pcl2-/- mice compared with their wildtype littermates. These data indicate that cells lacking in Pcl2 are in a more primitive/progenitor state. In a Friend Virus erythroleukemia model, Pcl2 represses the development of stress erythroid progenitors. RNA-seq analyses in mouse erythroid progenitors revealed a role for Pcl2 in multiple pathways including regulation of cell cycle. Pcl2 is also required for HSC selfrenewal as Pcl2-/- fetal liver cells fail to reconstitute the hematopoietic system of recipient mice in secondary transplants. Taken together, Pcl2 is required for erythroid development and self-renewal of hematopoietic stem cells.
P1097 - THE FANCONI ANEMIA C PROTEIN REGULATES CELLULAR DIVISION VIA THE MICROTUBULE-ASSOCIATED PROTEIN STATHMIN1 Audrey Magron1,2, Caroline Huard1,2, Sabine Elowe1,2, and Madeleine Carreau1,2 1 Universite Laval, Quebec, Quebec, Canada; 2Department of Pediatrics, CHU de Quebec, CHUL Research Center, Quebec, Quebec, Canada
P1099 - DECLINED PRESENTATION ANALYSIS OF HSC FATE, MAINTENANCE AND FUNCTION DUE TO DYSREGULATION OF THE WNT SIGNALING ANTAGONIST, SCLEROSTIN Jennifer Manilay University of California, Merced, Merced, California, USA
Fanconi Anemia (FA) belongs to the inherited bone marrow failure syndromes characterized by a progressive decline in primitive hematopoietic cells due to accelerated cycling and hypersensitivity to various external cellular cues. These cellular defects favour the development of clonal proliferation and leads to leukemia. Proteins mutated in FA have been implicated in mitotic events assuring the safeguard of chromosome segregation. Consequently, defective FA proteins have been associated with a higher rate of cytokinesis failure, aneuploidy and chromosome missegregation, which are hallmarks of cancer cells. In an attempt to identify FA protein’s function in cellular division events, we identified through Yeasts-2-Hybrid screening approaches, the microtubule-associated protein Stathmin (STMN), as a binding partner of the FA protein C (FANCC). Through different biochemical approaches, we confirmed the FANCC-STMN interaction. FA-disease causing mutations in FANCC prevent its interaction with STMN. We observed that FANCC with Phospho-STMN co-localizes to centrosomes during mitosis. In addition, we found that FANCC modulates STMN phosphorylation status on serine 16 and 38 during cellular division. The phosphorylation status of STMN during the cell cycle requires a functional FANCC. These results suggest that FANCC participates in the regulation of cellular division via the microtubule-associated protein STMN. Previous studies have shown that elevated levels of STMN are found in many cancers including leukemia whereas a dramatic decrease in STMN levels is found in hematopoietic cells upon differentiation along different lineages. Consequently, we propose that misregulation of STMN1 activity combined with the DNA repair defects in FA cells may account for the loss of hematopoietic stem cells and increase in cancer susceptibility seen in patients with FA.
An understanding of how changes in bone health affect hematopoiesis could inform how to retain strong immunity in patients with bone disease and reveal how agerelated changes in bones could affect hematopoiesis during aging. Sclerostin (SOST) is a secreted protein that is critical for normal bone homeostasis, in which bone-building by osteoblast (OB) cells is balanced by bone-breakdown by osteoclast (OC) cells. SOST is produced primarily by mature mineralized bone cells called osteocytes (OCYs) and prevents the maturation of OBs into OCYs by its action as a Wnt signaling antagonist. Although the role of SOST in bone homeostasis has been well-established, we observed that the absence of SOST adversely affected B lymphocyte development in SOST-knockout (KO) mice, due to changes in the bone marrow microenvironment, including reduced expression of stem cell factor (SCF) and CXCL12. These growth factors are also important for hematopoietic stem cell (HSC) maintenance and migration within the bone marrow niche; however, the role of SOST in the regulation of HSC function remains an open question. We hypothesized that HSCs that develop in SOST-deficient bones harbor defects in quiescence, cell cycling and Wnt signaling that lead to HSC depletion and hematopoietic failure. To test this hypothesis, we treated SOST KO mice with 5-fluorouracil in vivo and performed HSC cycle analysis to determine the ratios of cells in quiescence and active cell cycle. The results of these studies demonstrated that sclerostin is not essential for regulation of HSC quiescence and entrance into cell cycle. In contrast, primary hematopoietic progenitor cell (HPC) transplantation assays were performed to ascertain the role of the sclerostin-deficiency on HSC differentiation, and our preliminary results demonstrate that the SOST KO bones affect B lymphocyte development in the bone marrow and might enhance differentiation of granulocytes in the periphery. Secondary HPC transplantation studies, currently ongoing, will test whether these environment-induced changes are temporary or permanent. Taken together, these data suggest that the roles of SOST deficiency on HSC maintenance and HSC differentiation are distinct.