Articles de revues : « Molecular biology, Hematopoiesis, Gene regulation »

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Stellrecht, C. M., G. Fraizer, C. Selvanayagam, L. Y. Chao, A. Lee et G. F. Saunders. « Transcriptional regulation of a hematopoietic proteoglycan core protein gene during hematopoiesis. » Journal of Biological Chemistry 268, n^o 6 (février 1993) : 4078–84. http://dx.doi.org/10.1016/s0021-9258(18)53582-1.

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de Rooij, Laura P. M. H., Derek C. H. Chan, Ava Keyvani Chahi et Kristin J. Hope. « Post-transcriptional regulation in hematopoiesis : RNA binding proteins take control ». Biochemistry and Cell Biology 97, n^o 1 (février 2019) : 10–20. http://dx.doi.org/10.1139/bcb-2017-0310.

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Normal hematopoiesis is sustained through a carefully orchestrated balance between hematopoietic stem cell (HSC) self-renewal and differentiation. The functional importance of this axis is underscored by the severity of disease phenotypes initiated by abnormal HSC function, including myelodysplastic syndromes and hematopoietic malignancies. Major advances in the understanding of transcriptional regulation of primitive hematopoietic cells have been achieved; however, the post-transcriptional regulatory layer that may impinge on their behavior remains underexplored by comparison. Key players at this level include RNA-binding proteins (RBPs), which execute precise and highly coordinated control of gene expression through modulation of RNA properties that include its splicing, polyadenylation, localization, degradation, or translation. With the recent identification of RBPs having essential roles in regulating proliferation and cell fate decisions in other systems, there has been an increasing appreciation of the importance of post-transcriptional control at the stem cell level. Here we discuss our current understanding of RBP-driven post-transcriptional regulation in HSCs, its implications for normal, perturbed, and malignant hematopoiesis, and the most recent technological innovations aimed at RBP–RNA network characterization at the systems level. Emerging evidence highlights RBP-driven control as an underappreciated feature of primitive hematopoiesis, the greater understanding of which has important clinical implications.

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Choi, Kyunghee. « Hemangioblast development and regulation ». Biochemistry and Cell Biology 76, n^o 6 (1 décembre 1998) : 947–56. http://dx.doi.org/10.1139/o99-007.

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Hematopoietic and endothelial cell lineages are the first to mature from mesoderm in the developing embryo. However, little is known about the molecular and (or) cellular events leading to hematopoietic commitment. The recent applications of technology utilizing gene targeted mice and the employment of many available in vitro systems have facilitated our understanding of hematopoietic establishment in the developing embryo. It is becoming clear that embryonic hematopoiesis occurs both in the extra-embryonic yolk sac and within the embryo proper in the mouse. The existence of the long pursued hemangioblast, a common progenitor of hematopoietic and endothelial cells, is now formally demonstrated. Based on this new information, many studies are being conducted to understand hematopoietic commitment events from mesoderm. In this review, we will first discuss the establishment of the hematopoietic system with special emphasis on the most primitive hematopoietic committed cells, the hemangioblast. We will then discuss mesoderm-inducing factors and their possible role in hematopoietic lineage commitment.Key words: hematopoietic commitment, hemangioblast, in vitro embryonic stem cell differentiation.

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Gandillet, Arnaud, Alicia G. Serrano, Stella Pearson, Michael Lie-A-Ling, Georges Lacaud et Valerie Kouskoff. « Sox7-sustained expression alters the balance between proliferation and differentiation of hematopoietic progenitors at the onset of blood specification ». Blood 114, n^o 23 (26 novembre 2009) : 4813–22. http://dx.doi.org/10.1182/blood-2009-06-226290.

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Abstract The molecular mechanisms that regulate the balance between proliferation and differentiation of precursors at the onset of hematopoiesis specification are poorly understood. By using a global gene expression profiling approach during the course of embryonic stem cell differentiation, we identified Sox7 as a potential candidate gene involved in the regulation of blood lineage formation from the mesoderm germ layer. In the present study, we show that Sox7 is transiently expressed in mesodermal precursors as they undergo specification to the hematopoietic program. Sox7 knockdown in vitro significantly decreases the formation of both primitive erythroid and definitive hematopoietic progenitors as well as endothelial progenitors. In contrast, Sox7-sustained expression in the earliest committed hematopoietic precursors promotes the maintenance of their multipotent and self-renewing status. Removal of this differentiation block driven by Sox7-enforced expression leads to the efficient differentiation of hematopoietic progenitors to all erythroid and myeloid lineages. This study identifies Sox7 as a novel and important player in the molecular regulation of the first committed blood precursors. Furthermore, our data demonstrate that the mere sustained expression of Sox7 is sufficient to completely alter the balance between proliferation and differentiation at the onset of hematopoiesis.

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Guo, Fukun, Wei Liu, Kankana Chava, Jose Cancelas, George Thomas, Sara C. Kozma et Yi Zheng. « Role of mTOR in Hematopoiesis and Hematopoietic Stem Cell Regulation. » Blood 114, n^o 22 (20 novembre 2009) : 1490. http://dx.doi.org/10.1182/blood.v114.22.1490.1490.

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Abstract Abstract 1490 Poster Board I-513 The mammalian target of rapamycin (mTOR) integrates nutrients, growth factors, and cellular energy status to control protein synthesis that determines cell growth and metabolism. It is also known that mTOR plays an essential role in cell survival by regulating Akt/PKB signaling. By using the inhibitor rapamycin, mTOR has previously been suggested to regulate proliferation of megakaryocyte progenitors and late stage of megakaryocyte differentiation without a general impact on normal hematopoiesis or hematopoietic stem cell (HSC) function. Due to limitations of rapamycin and the early lethality of conventional mTOR gene targeted mice, the physiological role of mTOR in blood development remains undefined. In this study, we have utilized an inducible conditional mTOR knockout mouse model by crossbreeding mTORflox/flox mice with Mx-Cre mice that allow interferon-induced mTOR deletion in the bone marrow following a transplantation and polyI:C induction protocol, in an effort to determine the genetic role of mTOR in hematopoiesis. Depletion of mTOR drastically affected hematopoiesis in a blood cell autonomous manner in Mx-Cre;mTORflox/flox bone marrow transplant recipients: the mice showed marked reduction in BM cellularity and in the numbers of myeloid and lymphoid lineage cells, erythrocytes, and platelets in peripheral blood, bone marrow, and thymus, leading to bone marrow failure, blood cell exhaustion and lethality. In vitro colony-forming activities by bone marrow or spleen progenitors were completely abolished in the absence of mTOR. Interestingly, the number and frequency of HSCs in bone marrow (Lin−Sca-1+c-Kit+) increased transiently while the number of early progenitors (CMP, GMP, MEP, CLP) detected by cell surface markers remained unchanged or only mildly affected in the mutant mice within 14 days after polyI:C treatment. Concomitantly, mTOR deletion led to a massive egress of HSCs from bone marrow to distal organs including spleen (∼60-fold increase). Transplantation of mTOR−/− bone marrow cells into NOD-SCID mice or competitive transplantation of mTOR−/− bone marrow cells into BoyJ mice further demonstrated that mTOR deficiency caused a complete failure in HSC engraftment and repopulation. Surprisingly, at the cellular level these phenotypes are associated with increased proliferation of HSCs in vivo and in vitro by 60% and 2.5-fold, respectively, as assessed by 5-bromodeoxyuridine incorporation assays whereas the cell survival index appears to be unaffected. Moreover, mTOR−/− HSCs and progenitor cells displayed impaired adhesion to fibronectin CH296 fragment (∼30% decrease) and migration toward SDF-1α gradients (∼30% decrease). At the molecular level, gene chip microarray analysis of mTOR−/− HSCs revealed that the cell cycle regulators myb, wee1, FANCD2, and FANCE were significantly downregulated while Rb and E2F5 were upregulated, the survival/apoptosis regulators MCL1 and BCL2L1 were upregulated, and the actin cytoskeleton and cell extracellular matrix adhesion regulators Arp2/3 complex subunit 5, paxillin, laminin α5, integrin β3, and myosin light chain 6B were upregulated. Further, immunoblotting analysis of isolated Lin− cells showed that SCF-stimulated activation of translational regulators S6K and 4E-BP and survival regulator Akt were abolished upon mTOR deletion. Taken together, these data suggest that mTOR is a critical regulator of HSC quiescence, self-renewal, and engraftment through the regulation of cell cycle, survival and actin cytoskeleton signals, and is essential in multiple stages of hematopoiesis. Disclosures Cancelas: CERUS CO: Research Funding; CARIDIAN BCT: Research Funding; HEMERUS INC: Research Funding.

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Gu, Yi, Michael C. Byrne, Nivanka C. Paranavitana, Bruce Aronow, Jamie E. Siefring, Maria D'Souza, Heidi F. Horton, Lawrence A. Quilliam et David A. Williams. « Rac2, a Hematopoiesis-Specific Rho GTPase, Specifically Regulates Mast Cell Protease Gene Expression in Bone Marrow-Derived Mast Cells ». Molecular and Cellular Biology 22, n^o 21 (1 novembre 2002) : 7645–57. http://dx.doi.org/10.1128/mcb.22.21.7645-7657.2002.

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ABSTRACT Rho family GTPases activate intracellular kinase cascades to modulate transcription of multiple genes. Previous studies have examined the roles of the ubiquitously expressed Rho GTPase, Rac1, in regulation of gene expression in cell lines and implicated NF-κB, serum response factor, and kinase signaling pathways in this regulation. To understand the role of the closely related but hematopoiesis-specific Rho GTPase, Rac2, in regulation of gene transcription, we compared the gene expression profiles between wild-type and Rac2−/− bone marrow-derived mast cells. Our data demonstrate remarkable specificity in the regulation of gene expression by Rac2 versus Rac1. Microarray analysis demonstrated that expression of 38 known genes was significantly altered in Rac2−/− mast cells after cytokine stimulation compared with those in wild-type cells. Of these, the expression of the mouse mast cell protease 7 (MMCP-7) gene in wild-type cells was highly induced at the transcriptional level after stimulation with stem cell factor (SCF). In spite of compensatorily increased expression of Rac1 in Rac2-deficient cells, SCF-induced MMCP-7 transcription did not occur. Surprisingly, the loss of MMCP-7 induction was not due to decreased activation of NF-κB, a transcription factor postulated to lie downstream of Rac1 and known to play a critical role in hematopoietic cell differentiation and proliferation. However, the activities of c-Jun N-terminal kinases (JNKs) were markedly decreased in Rac2−/− mast cells. Our results suggest that cytokine-stimulated activation of MMCP-7 gene transcription is selectively regulated by a Rac2-dependent JNK signaling pathway in primary mast cells and imply a remarkable specificity in the regulation of transcriptional activity by these two highly related Rho GTPases.

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Liao, Eric C., Nikolaus S. Trede, David Ransom, Augustin Zapata, Mark Kieran et Leonard I. Zon. « Non-cell autonomous requirement for thebloodlessgene in primitive hematopoiesis of zebrafish ». Development 129, n^o 3 (1 février 2002) : 649–59. http://dx.doi.org/10.1242/dev.129.3.649.

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Vertebrate hematopoiesis occurs in two distinct phases, primitive (embryonic) and definitive (adult). Genes that are required specifically for the definitive program, or for both phases of hematopoiesis, have been described. However, a specific regulator of primitive hematopoiesis has yet to be reported. The zebrafish bloodless (bls) mutation causes absence of embryonic erythrocytes in a dominant but incompletely penetrant manner. Primitive macrophages appear to develop normally in bls mutants. Although the thymic epithelium forms normally in bls mutants, lymphoid precursors are absent. Nonetheless, the bloodless mutants can progress through embryogenesis, where red cells begin to accumulate after 5 days post-fertilization (dpf). Lymphocytes also begin to populate the thymic organs by 7.5 dpf. Expression analysis of hematopoietic genes suggests that formation of primitive hematopoietic precursors is deficient in bls mutants and those few blood precursors that are specified fail to differentiate and undergo apoptosis. Overexpression of scl, but not bmp4 or gata1, can lead to partial rescue of embryonic blood cells in bls. Cell transplantation experiments show that cells derived from bls mutant donors can differentiate into blood cells in a wild-type host, but wild-type donor cells fail to form blood in the mutant host. These observations demonstrate that the bls gene product is uniquely required in a non-cell autonomous manner for primitive hematopoiesis, potentially acting via regulation of scl.

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Contreras, Jorge R., Thilini Fernando, Tiffany M. Tran, Matteo Zampini, Norma Iris Rodriguez-Malave, Jayanth Kumar Palanichamy, Jasmine Gajeton et al. « Molecular Characterization of Long Non-Coding RNA CASC15 in Leukemogenesis ». Blood 128, n^o 22 (2 décembre 2016) : 5103. http://dx.doi.org/10.1182/blood.v128.22.5103.5103.

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Abstract High throughput transcriptome sequencing has uncovered a previously uncharacterized layer of gene regulation by long non-coding RNAs (lncRNAs). LncRNAs are characterized by capped, polyadenylated, and spliced transcripts that lack an open reading frame. Despite the similarities in their genetic organization, they play variety of roles at the cellular level, including regulation of transcription and translation, leading to alterations in gene expression. One of these functions is the regulation of expression of chromosomally adjacent genes. Here, we examined the function of the lncRNA CASC15 that was originally discovered as being dysregulated in in ETV6-RUNX1-translocated B-acute lymphoblastic leukemia. Enforced expression of CASC15 in hematopoietic stem and progenitor cells led to a myeloid bias in development with an overall decrease in engraftment and colony formation. Conversely, using a CRISPR-based approach, CASC15 deletion skewed hematopoietic cell progenitors towards a B cell fate. CASC15 was also demonstrated to regulate cellular survival, proliferation, and the expression of its chromosomally adjacent gene, SOX4. Differentially regulated genes following CASC15 knockdown in cell lines were enrichment for predicted transcriptional targets of the Yin and Yang-1 (YY1) transcription factor. To further characterize this, we queried a functional relationship between YY1 and CASC15. Interestingly, we found that YY1 interacts with CASC15, and that CASC15 enhanced YY1-mediated transcription at the SOX4 promoter. Together these studies represent some of the first functional characterizations of lncRNAs in leukemia and highlight the importance of non-coding regulatory mechanisms in malignant hematopoiesis. Disclosures No relevant conflicts of interest to declare.

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He, XY, VP Antao, D. Basila, JC Marx et BR Davis. « Isolation and molecular characterization of the human CD34 gene ». Blood 79, n^o 9 (1 mai 1992) : 2296–302. http://dx.doi.org/10.1182/blood.v79.9.2296.2296.

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Abstract The human CD34 surface antigen is selectively expressed on hematopoietic stem/progenitor cells, suggesting that it plays an essential role in early hematopoiesis. Using a 1.5-kb partial human CD34 cDNA sequence, RNA-polymerase chain reaction (PCR), and rapid amplification of cDNA ends (RACE) methods, we cloned and sequenced the full-length (2.65 kb) cDNA. The cDNA encodes a type I transmembrane protein with no obvious homology to other known proteins. The entire CD34 gene of 28 kb was cloned, and the coding sequences mapped to eight exons. Mapping of the 5′ termini of mRNAs by 5′-RACE and RNAase protection analyses has indicated that the human CD34 gene uses multiple transcription initiation sites. Analysis of the upstream regulatory sequences revealed the absence of TATA and CAAT box sequences, and the presence of myb, myc, and ets-like DNA binding motifs. We have identified significant homology between human and mouse CD34 genes in 5′ and 3′ untranslated regions, amino acid coding sequences, and 5′ flanking sequences. This investigation of the CD34 gene should facilitate study of the function and regulation of this stem cell antigen.

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He, XY, VP Antao, D. Basila, JC Marx et BR Davis. « Isolation and molecular characterization of the human CD34 gene ». Blood 79, n^o 9 (1 mai 1992) : 2296–302. http://dx.doi.org/10.1182/blood.v79.9.2296.bloodjournal7992296.

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The human CD34 surface antigen is selectively expressed on hematopoietic stem/progenitor cells, suggesting that it plays an essential role in early hematopoiesis. Using a 1.5-kb partial human CD34 cDNA sequence, RNA-polymerase chain reaction (PCR), and rapid amplification of cDNA ends (RACE) methods, we cloned and sequenced the full-length (2.65 kb) cDNA. The cDNA encodes a type I transmembrane protein with no obvious homology to other known proteins. The entire CD34 gene of 28 kb was cloned, and the coding sequences mapped to eight exons. Mapping of the 5′ termini of mRNAs by 5′-RACE and RNAase protection analyses has indicated that the human CD34 gene uses multiple transcription initiation sites. Analysis of the upstream regulatory sequences revealed the absence of TATA and CAAT box sequences, and the presence of myb, myc, and ets-like DNA binding motifs. We have identified significant homology between human and mouse CD34 genes in 5′ and 3′ untranslated regions, amino acid coding sequences, and 5′ flanking sequences. This investigation of the CD34 gene should facilitate study of the function and regulation of this stem cell antigen.

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SAHA, Nirmalya, James Ropa, Lili Chen, Hsiang-Yu Hu, Maria Mysliwski, Ann Friedman, Ivan Maillard et Andrew G. Muntean. « The PAF1c Subunit Cdc73 Is Essential for Hematopoiesis and Displays Differential Gene Regulation in MLL-AF9 Driven Leukemia ». Blood 132, Supplement 1 (29 novembre 2018) : 1280. http://dx.doi.org/10.1182/blood-2018-99-118703.

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Abstract The Polymerase Associated Factor 1 complex (PAF1c) functions at the interface of epigenetics and gene transcription. The PAF1c is a multi-protein complex composed of Paf1, Cdc73, Leo1, Ctr9, Rtf1 and WDR61, which have all been shown to play a role in disease progression and different types of cancer. Previous reports demonstrated that the PAF1c is required for MLL-fusion driven acute myeloid leukemia. This is due, in part, to a direct interaction between the PAF1c and wild type MLL or MLL fusion proteins. Importantly, targeted disruption of the PAF1c-MLL interaction impairs the growth of MLL-fusion leukemic cells but is tolerated by normal hematopoietic stem cells. These data point to differential functions for the PAF1c in normal and malignant hematopoietic cells that may be exploited for therapeutic purposes. However, a detailed exploration of the PAF1c in normal hematopoiesis is currently lacking. Here, we utilize a mouse genetic model to interrogate the role of the PAF1c subunit, Cdc73, in the development and sustenance of normal hematopoiesis. Using hematopoietic-specific constitutive and conditional drivers to express Cre recombinase, we efficiently excise floxed alleles of Cdc73 in hematopoietic cells. VavCre mediated excision of Cdc73 results in embryonic lethality due to hematopoietic failure. Characterization of the hematopoietic system demonstrated that cKit+ hematopoietic stem and progenitor cells (HSPC) are depleted due to Cdc73 knockout. We next investigated the role of Cdc73 in adult hematopoiesis using Mx1Cre mediated excision. Conditional knockout of Cdc73 in the adult hematopoietic system leads to lethality within 15 days of Cdc73 excision while no phenotype was observed in heterozygous Cdc73fl/wt controls. Pathological examination of bones in these mice showed extensive bone marrow failure. Flow cytometry analysis revealed that cKit+ HSPCs in adult mice are ablated following loss of Cdc73. Bone marrow transplantation assays demonstrated a cell autonomous requirement of Cdc73 for HSC function in vivo. To perform cellular characterization of HSPCs upon Cdc73 KO, we optimized excision conditions to capture cKit+ HSPCs with excised Cdc73 but before their exhaustion. Flow cytometry analysis demonstrated that Cdc73 KO leads to a cell cycle defect. Cdc73 excision leads to a 2.5 fold increase in the accumulation of HSPCs in the G0 phase of cell cycle with a reduction in the proliferative phases. This is accompanied with an increase in cellular death as indicated by Annexin V staining. Together, these data indicate that Cdc73 is required for cell cycle progression and HSPC survival. To understand the molecular function of Cdc73, we performed RNAseq analysis to identify genes regulated by Cdc73 in HSPCs. We observed 390 genes are upregulated and 433 genes are downregulated upon loss of Cdc73. Specifically, Cdc73 excision results in upregulation of cell cycle inhibitor genes such as p21 and p57, consistent with the cell cycle defect observed following Cdc73 excision. Further, when comparing our results to leukemic cells, we uncovered key differences in Cdc73 gene program regulation between ckit+ hematopoietic cells and MLL-AF9 AML cells. Loss of Cdc73 in leukemic cells leads to downregulation of genes associated with early hematopoietic progenitors and upregulation of myeloid differentiation genes consistent with previous studies. Interestingly, we observed a more even distribution of expression changes (non-directional) within these gene programs following Cdc73 inactivation in HSPCs. Most importantly, while loss of Cdc73 in MLL-AF9 AML cells leads to a profound downregulation of the Hoxa9/Meis1 gene program, excision of Cdc73 in HSPCs results in a modest non-directional change in expression of the Hoxa9/Meis1 gene program. This was attributed to no change in Hoxa9 and Meis1 expression in HSPCs following excision of Cdc73, in contrast to MLL-AF9 cells where these pro leukemic targets are significantly downregulated. Together, these data indicate an essential role for the PAF1c subunit Cdc73 in normal hematopoiesis but differential roles and context specific functions in normal and malignant hematopoiesis, which may be of therapeutic value for patients with AMLs expressing Hoxa9/Meis1 gene programs. Disclosures No relevant conflicts of interest to declare.

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Inoue, Hirofumi, Ikuo Nobuhisa, Keisuke Okita, Makiko Takizawa, Marie-Josèphe Pébusque et Tetsuya Taga. « Negative regulation of hematopoiesis by the fused in myeloproliferative disorders gene product ». Biochemical and Biophysical Research Communications 313, n^o 1 (janvier 2004) : 125–28. http://dx.doi.org/10.1016/j.bbrc.2003.11.097.

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Huang, Mengling, Abrar Ahmed, Wei Wang, Xue Wang, Cui Ma, Haowei Jiang, Wei Li et Lili Jing. « Negative Elongation Factor (NELF) Inhibits Premature Granulocytic Development in Zebrafish ». International Journal of Molecular Sciences 23, n^o 7 (30 mars 2022) : 3833. http://dx.doi.org/10.3390/ijms23073833.

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Gene expression is tightly regulated during hematopoiesis. Recent studies have suggested that RNA polymerase II (Pol II) promoter proximal pausing, a temporary stalling downstream of the promoter region after initiation, plays a critical role in regulating the expression of various genes in metazoans. However, the function of proximal pausing in hematopoietic gene regulation remains largely unknown. The negative elongation factor (NELF) complex is a key factor important for this proximal pausing. Previous studies have suggested that NELF regulates granulocytic differentiation in vitro, but its in vivo function during hematopoiesis remains uncharacterized. Here, we generated the zebrafish mutant for one NELF complex subunit Nelfb using the CRISPR-Cas9 technology. We found that the loss of nelfb selectively induced excessive granulocytic development during primitive and definitive hematopoiesis. The loss of nelfb reduced hematopoietic progenitor cell formation and did not affect erythroid development. Moreover, the accelerated granulocytic differentiation and reduced progenitor cell development could be reversed by inhibiting Pol II elongation. Further experiments demonstrated that the other NELF complex subunits (Nelfa and Nelfe) played similar roles in controlling granulocytic development. Together, our studies suggested that NELF is critical in controlling the proper granulocytic development in vivo, and that promoter proximal pausing might help maintain the undifferentiated state of hematopoietic progenitor cells.

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Fuller, John F., Jeanne McAdara, Yifah Yaron, Mark Sakaguchi, John K. Fraser et Judith C. Gasson. « Characterization of HOX Gene Expression During Myelopoiesis : Role of HOX A5 in Lineage Commitment and Maturation ». Blood 93, n^o 10 (15 mai 1999) : 3391–400. http://dx.doi.org/10.1182/blood.v93.10.3391.410k26_3391_3400.

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During the process of normal hematopoiesis, proliferation is tightly linked to maturation. The molecular mechanisms that lead to production of mature effector cells with a variety of phenotypes and functions from a single multipotent progenitor are only beginning to be elucidated. It is important to determine how these maturation events are regulated at the molecular level, because this will provide significant insights into the process of normal hematopoiesis as well as leukemogenesis. Transcription factors containing the highly conserved homeobox motif show considerable promise as potential regulators of hematopoietic maturation events. In this study, we focused on identification and characterization of homeobox genes of the HOX family that are important in regulating normal human myeloid differentiation induced by the hematopoietic growth factor, granulocyte-macrophage colony-stimulating factor (GM-CSF). We have identified three homeobox genes, HOX A5, HOX B6, and HOX B7, which are expressed during early myelopoiesis. Treating bone marrow cells with antisense oligodeoxynucleotides to HOX A5 resulted in inhibition of granulocytic/monocytic hematopoiesis and increased the generation of erythroid progenitors. Also, overexpression of HOX A5 inhibited erythroid differentiation of the K562 cell line. Based on these observations, we propose that HOX A5 functions as an important regulator of hematopoietic lineage determination and maturation.

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Zhaojun, Zhang, Xiong Qian, Wang Shaobin, Wang Hai, Zhang Qian, Qi Heyuan, Li Yanming et al. « Comprehensive Investigation of the Molecular Mechanism of Primitive Hematopoiesis Regulating by KLF3 ». Blood 120, n^o 21 (16 novembre 2012) : 4729. http://dx.doi.org/10.1182/blood.v120.21.4729.4729.

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Abstract Abstract 4729 KLF3 is a member of the Krüppel-like transcription factor family. By recognizing CC/ACACCC motifs in the promoters and enhancers of its regulating genes, KLF3 plays critical roles in cell differentiation and development including B lymphocytes maturation and adipocyte differentiation. Previous studies demonstrated that KLF3-deficient mice displayed myeloproliferative disorders and abnormalities in hematopoiesis. KLF3 prefers to bind to the CACCC box in the yolk sac and fetal liver, indicating that KLF3 probably participates in the primitive hematopoiesis. However, the mechanism that KLF3 regulates primitive hematopoiesis is not fully understood. To characterize the role of KLF3 in primitive hematopoiesis, we firstly detected the expression of KLF3 during erythroid differentiation by RNA-seq in undifferentiated human embryonic stem cells (hESC) as well as three primary erythroid cells at different developmental stages including ES-derived erythroid cells (ESER), fetal- and adult-type erythroid cells (FLER, PBER). The results show that KLF3 is significantly higher expressed in ESER cells than that in other cells, which is an indicating of the role of KLF3 in primitive hematopoiesis. The expression level of KLF3 decreased at later erythroid developmental stages, which was also verified by the decreased KLF3 expression level when K562 cells induced with 50 mM of hemin for up to 72h. Secondly, to further clarify the mechanism that KLF3 regulates primitive hematopoiesis, we depleted KLF3 by shRNA interference in K562 cells, the representative of early development of erythroid cells, and performed microarray analysis to comprehensively characterize the target genes of KLF3 as well as the networks in which the target genes involved. The results indicate that down-regulated KLF3 exhibits remarkable impacts on genes expression profile in K562 cells. Total 655 (p-value<0.01, fold change>1.5) differentially expressed genes were largely disturbed and recognized as potential target genes of KLF3, in which up-regulated genes (372) were more than down-regulated genes (283). Erythroid differentiation markers including HBE, HBA1/A2, HBZ and HBD globin genes are observably up-regulated in KLF3 depleted K562 cells. These results suggest that KLF3 probably exhibits suppressive activities in primitive hematopoiesis. The IPA analysis demonstrates that the potential target genes are specifically enriched in the biofunctions of hematopoiesis and hematological system development. The IPA networks analysis demonstrates that the potential target genes are closely associated with the networks of hematological diseases and hematological system development. IPA analysis also predicted the upstream regulators to drive KLF3 in erythroid cells including GATA1 (p-value<2.83E-12) and EPO (p-value<8.51E-08) which were significantly activated. Thirdly, to clarify whether the erythroid-specific enhancers in the genomic region of KLF3 participate in the KLF3 biology of primitive hematopoiesis, we identified erythroid-specific DNaseI hypersensitive sites (DHSs) in the KLF3 locus from DNase-seq data in four erythroid cells including ESER, FLER, PBER, K562 and seven non-erythroid cells. The enhancer activity of the erythroid DHSs was comprehensively characterized by dual-luciferase reporter assays in K562 cells and non-erythroid Hela and HEK293 cells. No erythroid-specific KLF3 enhancers was finally confirmed, suggesting the regulation of primitive hematopoiesis by KLF3 could depend on the upstream regulators, downstream target genes, as well as the other cis regulatory elements (CREs), but not erythroid-specific enhancers in KLF3 locus. In conclusion, we clarified the expression pattern of KLF3 during erythroid differentiation and confirmed the important functions of KLF3 in primitive hematopoises. Moerover, we ruled out the possibility that erythroid-specific enhancers in KLF3 gene locus participate in primitive hematopoiesis. Next, ChIP and dual luciferase reporter assay will be performed to confirm the regulation of KLF3 on the target genes. The relationship between these upstream regulators and KLF3 potential target genes will be further clarified. Finally, the related observations will be verified in hematopoietic stem cells (HSCs) as well as KLF3 morpholino knockdown zebrafish to fully understand the molecular mechanism of KLF3 in regulating primitive hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

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Kwan, Tommy T., Raymond Liang, Catherine M. Verfaillie, Stephen C. Ekker, Li C. Chan, Shuo Lin et Anskar Y. H. Leung. « Regulation of primitive hematopoiesis in zebrafish embryos by the death receptor gene ». Experimental Hematology 34, n^o 1 (janvier 2006) : 27–34. http://dx.doi.org/10.1016/j.exphem.2005.09.017.

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Grech, Godfrey, et Marieke von Lindern. « The Role of Translation Initiation Regulation in Haematopoiesis ». Comparative and Functional Genomics 2012 (2012) : 1–10. http://dx.doi.org/10.1155/2012/576540.

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Organisation of RNAs into functional subgroups that are translated in response to extrinsic and intrinsic factors underlines a relatively unexplored gene expression modulation that drives cell fate in the same manner as regulation of the transcriptome by transcription factors. Recent studies on the molecular mechanisms of inflammatory responses and haematological disorders indicate clearly that the regulation of mRNA translation at the level of translation initiation, mRNA stability, and protein isoform synthesis is implicated in the tight regulation of gene expression. This paper outlines how these posttranscriptional control mechanisms, including control at the level of translation initiation factors and the role of RNA binding proteins, affect hematopoiesis. The clinical relevance of these mechanisms in haematological disorders indicates clearly the potential therapeutic implications and the need of molecular tools that allow measurement at the level of translational control. Although the importance of miRNAs in translation control is well recognised and studied extensively, this paper will exclude detailed account of this level of control.

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Basu, Sreemanti, Irene Hernandez, Mark Zogg, Karen-Sue B. Carlson et Hartmut Weiler. « Regulation of Hematopoiesis By the Coagulation Receptor Thrombomodulin ». Blood 126, n^o 23 (3 décembre 2015) : 4750. http://dx.doi.org/10.1182/blood.v126.23.4750.4750.

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Abstract BACKGROUND: Pharmacologic supplementation of protein C pathway function by infusion of recombinant Thbd or activated protein C supports recovery of hematopoietic function from lethal radiation injury in mice [Geiger et al., Nature Medicine, 2012]. Partial Thbd deficiency in hematopoietic stem and progenitor cells (HSPC) or bone marrow endothelium results in augmented sensitivity towards radiation injury [Geiger et al., Nature Medicine, 2012]. The underlying cellular and molecular mechanisms of Thbd function in hematopoiesis are not yet characterized. The objective of the current study was to determine the expression pattern and functional role of Thbd in HSPC. RESULTS: Flow cytometric analysis was employed to detect Thbd expression in defined subsets of murine HSPC. Thbd was co-expressed with the endothelial protein C receptor (Procr/EPCR) in the majority of bona fide stem cells with long-term-repopulating capacity (LT-HSC), and was also expressed on EPCR-negative stem cells with short-term repopulating capacity (ST-HSC), multipotent progenitors (MPP), common lymphoid progenitors (CLP), common myeloid progenitors (CMP), and granulocyte-monocyte progenitors (GMP). In contrast, only a subset of megakaryocyte-erythrocyte progenitors (MEP) expressed low levels of Thbd. In the bone marrow, Thbd was also expressed by B cells in early stages of maturation (from progenitor B cell stage till immature stage). In this lineage, the fraction of Thbd-positive cells was inversely correlated with the stage of B cell maturation. In addition, Thbd was detected in three distinct subsets of bone marrow-resident myeloid cells (CD11b+CD115+, CD11b+CD11c+ and CD11b-CD115+). Thbd expression outside the bone marrow was limited to a small fraction of hematopoietic cells (2-5% in the peripheral blood and spleen). These cells included myeloid cells (macrophage/monocyte and dendritic cells). Approximately 1-2% of all B cells in the peripheral blood and the spleen expressed Thbd, possibly reflecting recent bone marrow emigrants. Thbd expression was largely absent from splenic follicular and marginal zone B cells. Adult mice with complete, ubiquitous ablation of Thbd gene function (Meox2Cre-ThbdloxP -mice; "Thbd-null") were generated to analyze the functional role of Thbd in hematopoiesis. Thbd-null mice exhibited low birth weight, but only a mild prothrombotic diathesis, reflected in occasional peripheral vascular occlusion limited to the tail vein. Flow cytometric analyses revealed increased frequency of LT- and ST-HSC, a trend towards reduced CLP frequency, but normal relative abundance of MPP, CMP, GMP, and MEP. Thbd deficiency was also associated with a significant increase in the absolute number of LT-HSC and a reduction in the absolute number of CLP in the bone marrow. No such derangements were observed in mice lacking EPCR. In functional assays, bone marrow from wildtype and mutant mice yielded comparable numbers of CFU-GM. In contrast, the number of CFU-GM was increased in the spleen and peripheral blood of Thbd-null animals. The absolute number and frequency of all B cell precursors, as well as mature B cells in the bone marrow was reduced by ~50%. In the spleen, the absolute number of B cells was increased, whereas other hematopoietic populations in peripheral organs of Thbd-null mice were identical to that of wildtype controls. CONCLUSION: Thbd is expressed in the majority of hematopoietic progenitor cells in the bone marrow, including LT- and ST-HSC, and B cell precursors. Despite abundant Thbd expression in bone marrow resident cells and a modest prothrombotic phenotype, complete Thbd deficiency had only mild effects on steady-state hematopoiesis. Hematopoietic derangements were limited to the B cell compartment, and an Increased presence of CFU-GM in the spleen and peripheral blood of Thbd-null mice, possibly reflecting stimaulation of extramedullary hematopoiesis and/or altered bone marrow retention of precursors. Disclosures No relevant conflicts of interest to declare.

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Sugiyama, Daisuke, Tomoko Inoue-Yokoo, Stuart T. Fraser, Kasem Kulkeaw, Chiyo Mizuochi et Yuka Horio. « Embryonic Regulation of the Mouse Hematopoietic Niche ». Scientific World JOURNAL 11 (2011) : 1770–80. http://dx.doi.org/10.1100/2011/598097.

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Hematopoietic stem cells (HSCs) can differentiate into several types of hematopoietic cells (HCs) (such as erythrocytes, megakaryocytes, lymphocytes, neutrophils, or macrophages) and also undergo self-renewal to sustain hematopoiesis throughout an organism's lifetime. HSCs are currently used clinically as transplantation therapy in regenerative medicine and are typically obtained from healthy donors or cord blood. However, problems remain in HSC transplantation, such as shortage of cells, donor risks, rejection, and graft-versus-host disease (GVHD). Thus, increased understanding of HSC regulation should enable us to improve HSC therapy and develop novel regenerative medicine techniques. HSC regulation is governed by two types of activity: intrinsic regulation, programmed primarily by cell autonomous gene expression, and extrinsic factors, which originate from so-called “niche cells” surrounding HSCs. Here, we focus on the latter and discuss HSC regulation with special emphasis on the role played by niche cells.

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Duan, Zhijun, Richard E. Person, Hu-Hui Lee, Shi Huang, Jean Donadieu, Raffaele Badolato, H. Leighton Grimes, Thalia Papayannopoulou et Marshall S. Horwitz. « Epigenetic Regulation of Protein-Coding and MicroRNA Genes by the Gfi1-Interacting Tumor Suppressor PRDM5 ». Molecular and Cellular Biology 27, n^o 19 (16 juillet 2007) : 6889–902. http://dx.doi.org/10.1128/mcb.00762-07.

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ABSTRACT Gfi1 transcriptionally governs hematopoiesis, and its mutations produce neutropenia. In an effort to identify Gfi1-interacting proteins and also to generate new candidate genes causing neutropenia, we performed a yeast two-hybrid screen with Gfi1. Among other Gfi1-interacting proteins, we identified a previously uncharacterized member of the PR domain-containing family of tumor suppressors, PRDM5. PRDM5 has 16 zinc fingers, and we show that it acts as a sequence-specific, DNA binding transcription factor that targets hematopoiesis-associated protein-coding and microRNA genes, including many that are also targets of Gfi1. PRDM5 epigenetically regulates transcription similarly to Gfi1: it recruits the histone methyltransferase G9a and class I histone deacetylases to its target gene promoters and demonstrates repressor activity on synthetic reporters; on endogenous target genes, however, it functions as an activator, in addition to a repressor. Interestingly, genes that PRDM5 activates, as opposed to those it represses, are also targets of Gfi1, suggesting a competitive mechanism through which two repressors could cooperate in order to become transcriptional activators. In neutropenic patients, we identified PRDM5 protein sequence variants perturbing transcriptional function, suggesting a potentially important role in hematopoiesis.

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Zambidis, Elias T., Bruno Peault, Tea Soon Park, Fred Bunz et Curt I. Civin. « Hematopoietic differentiation of human embryonic stem cells progresses through sequential hematoendothelial, primitive, and definitive stages resembling human yolk sac development ». Blood 106, n^o 3 (1 août 2005) : 860–70. http://dx.doi.org/10.1182/blood-2004-11-4522.

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AbstractWe elucidate the cellular and molecular kinetics of the stepwise differentiation of human embryonic stem cells (hESCs) to primitive and definitive erythromyelopoiesis from human embryoid bodies (hEBs) in serum-free clonogenic assays. Hematopoiesis initiates from CD45 hEB cells with emergence of semiadherent mesodermal-hematoendothelial (MHE) colonies that can generate endothelium and form organized, yolk sac–like structures that secondarily generate multipotent primitive hematopoietic stem progenitor cells (HSPCs), erythroblasts, and CD13+CD45+ macrophages. A first wave of hematopoiesis follows MHE colony emergence and is predominated by primitive erythropoiesis characterized by a brilliant red hemoglobinization, CD71/CD325a (glycophorin A) expression, and exclusively embryonic/fetal hemoglobin expression. A second wave of definitive-type erythroid burst-forming units (BFU-e's), erythroid colony-forming units (CFU-e's), granulocyte-macrophage colony-forming cells (GM-CFCs), and multilineage CFCs follows next from hEB progenitors. These stages of hematopoiesis proceed spontaneously from hEB-derived cells without requirement for supplemental growth factors during hEB differentiation. Gene expression analysis of differentiating hEBs revealed that initiation of hematopoiesis correlated with increased levels of SCL/TAL1, GATA1, GATA2, CD34, CD31, and the homeobox gene-regulating factor CDX4 These data indicate that hematopoietic differentiation of hESCs models the earliest events of embryonic and definitive hematopoiesis in a manner resembling human yolk sac development, thus providing a valuable tool for dissecting the earliest events in human HSPC genesis.

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Sato, Tomohiko, Susumu Goyama et Mineo Kurokawa. « Evi-1 Promotes Para-Aortic Splanchnopleural Hematopoiesis through Up-Regulation of GATA-2 and Repression of TGF-β Signaling. » Blood 110, n^o 11 (16 novembre 2007) : 1236. http://dx.doi.org/10.1182/blood.v110.11.1236.1236.

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Abstract The ecotropic viral integration site-1 (Evi-1) gene was first identified as a common locus of retroviral integration in myeloid tumors in mice. Evi-1 is highly expressed in cases with human acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) as a consequence of chromosomal rearrangements involving 3q26, where Evi-1 is mapped. Mice deficient in Evi-1 die during embryogenesis with widespread hypocellularity, hemorrhaging, and disruption in the development of the heart, somite, and neural crest-derived cells. It was recently reported that Evi-1 is expressed at a high level in the para-aortic splanchnopleural (P-Sp) region, from which definitive hematopoiesis originates. Hematopoietic stem cells (HSCs) in Evi-1-deficient embryos are decreased in number with defective proliferation capacity, and this defective proliferation of Evi-1-deficient P-Sp cells is successfully rescued in vitro by retroviral transfer of GATA-2. However, detailed molecular mechanisms underlying Evi-1-mediated hematopoiesis remain to be elucidated. In the present study, we used a coculture system of cells derived from the P-Sp region with a layer of a stromal cell line OP9, on which hematopoietic cell development is efficiently induced. In this culture system, Evi-1-deficient P-Sp-derived cells showed severely decreased colony forming capacity. This defect was overcome by reactivating Evi-1 retrovirally. Using this assay, we examined a hematopoietic potential of a series of Evi-1 mutants and found that the first zinc finger domain and the acidic domain are required for the hematopoietic rescue of Evi-1-deficient P-Sp cells. These two domains were also related to some extent for GATA-2 up-regulation, suggesting that GATA-2 is one of the key molecules in Evi-1-mediated hematopoiesis. Furthermore, we found that blocking of TGF-β signaling is also able to recover the hematopoietic defect of Evi-1-deficient P-Sp cells. These findings suggest that Evi-1 promotes hematopoietic stem/progenitor expansion during embryogenesis through at least two pathways: up-regulation of GATA-2 and inhibition of TGF-β signaling.

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Okoye-Okafor, Ujunwa Cynthia, Laura Barreyro, Heng Rui Wang, Boris Bartholdy, Britta Will, Tihomira I. Todorova, Swathi-Rao Narayanagari et al. « Molecular and Functional Characterization Of The Novel Protein-Coding Gene Tihl (Translocated in Hodgkin’s Lymphoma) in Hematopoiesis ». Blood 122, n^o 21 (15 novembre 2013) : 3680. http://dx.doi.org/10.1182/blood.v122.21.3680.3680.

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Abstract Cell cycling is a tightly regulated process involving the structured expression modulation of various regulatory genes. This process is crucial for the maintenance of cell survival/proliferation in both normal and malignant hematopoietic cells. We have previously described the highly expressed CIITA-BX648577 gene fusion (Steidl C. et al., Nature 2011), involving the novel gene locus BX648577/FLJ27352 /hypothetical LOC 145788/C15orf65 and the Class II Transactivator (CIITA) in the Hodgkin’s lymphoma cell line KM-H2. While CIITA is well known to be involved in the regulation of immune responses, specifically through regulation of the Major Histocompatibility Complex (MHC)-II, nothing is known about the expression and function of the BX648577 locus. The objective of the current study was to (I) study RNA and protein expression of the putative full length gene encoded by the BX648577 (TIHL) gene locus, and (II) study its biological function in normal and malignant hematopoietic cells, including its effects on cell proliferation, clonogenicity and cell death. We detected robust endogenous TIHL RNA and protein expression in a variety of healthy and malignant hematopoietic cell types using quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis using a TIHL-specific antibody. At the functional level, we found that ectopic expression of the highly conserved full length TIHL protein in human NB4 leukemia cells and murine hematopoietic progenitor HPC-7 cells leads to enhanced clonogenicity and increased proliferative capacity with significant increases in the percentage of cells in S-phase of the cell cycle. Furthermore, we observed more aggressive leukemia and decreased survival of NSG mice following retro-orbital transplantation of TIHL-expressing compared to empty control-expressing NB4 cells. Interestingly, although we did not observe a change in the rate of cell proliferation or colony forming ability following TIHL overexpression in the ATRA-resistant cell line NB4.306, there was a significant alteration in its cell cycle distribution, with an increase in the fraction of cells in S-phase. The increase in S-phase cells was confirmed by 5-ethynyl-2’-deoxyuridine (EdU) incorporation assays and flow cytometry. Similarly, knockdown of TIHL using 2 independent lentiviral shRNAs, led to a significant decrease in the growth of both NB4 and acute myeloid leukemia KG1a cells in both suspension cultures and semi-solid media. Although we observed slightly increased apoptosis upon TIHL downregulation, the changes could be more significantly attributed to a decrease in the percentage of cells in S-phase within 2-3 days after transduction with the lentiviral shRNAs. Finally, in silico analysis of the TIHL promoter identified various predicted transcriptional regulators of TIHL, the majority of which are cell cycle specific transcription factors including Nuclear Receptor Subfamily 5 Group A Member 1 (NR5A1), the ets domain transcription factors ELF5 and ELF1, and Glioma-Associated Oncogene Homolog 1 (GLI-1). Our findings thus far strongly support a novel role for TIHL in cell cycle regulation/modulation in both normal and malignant hematopoiesis. Future directions include gene expression studies to identify downstream targets of TIHL following overexpression and knockdown and co-immunoprecipitation coupled to mass spectrometry analysis will be used to identify direct interacting protein partners of this novel gene. Disclosures: No relevant conflicts of interest to declare.

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Hung, Chun-Hao, Keh-Yang Wang, Yae-Huei Liou, Jing-Ping Wang, Anna Yu-Szu Huang, Tung-Liang Lee, Si-Tse Jiang, Nah-Shih Liao, Yu-Chiau Shyu et Che-Kun James Shen. « Negative Regulation of the Differentiation of Flk2− CD34− LSK Hematopoietic Stem Cells by EKLF/KLF1 ». International Journal of Molecular Sciences 21, n^o 22 (10 novembre 2020) : 8448. http://dx.doi.org/10.3390/ijms21228448.

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Erythroid Krüppel-like factor (EKLF/KLF1) was identified initially as a critical erythroid-specific transcription factor and was later found to be also expressed in other types of hematopoietic cells, including megakaryocytes and several progenitors. In this study, we have examined the regulatory effects of EKLF on hematopoiesis by comparative analysis of E14.5 fetal livers from wild-type and Eklf gene knockout (KO) mouse embryos. Depletion of EKLF expression greatly changes the populations of different types of hematopoietic cells, including, unexpectedly, the long-term hematopoietic stem cells Flk2− CD34− Lin− Sca1+ c-Kit+ (LSK)-HSC. In an interesting correlation, Eklf is expressed at a relatively high level in multipotent progenitor (MPP). Furthermore, EKLF appears to repress the expression of the colony-stimulating factor 2 receptor β subunit (CSF2RB). As a result, Flk2− CD34− LSK-HSC gains increased differentiation capability upon depletion of EKLF, as demonstrated by the methylcellulose colony formation assay and by serial transplantation experiments in vivo. Together, these data demonstrate the regulation of hematopoiesis in vertebrates by EKLF through its negative regulatory effects on the differentiation of the hematopoietic stem and progenitor cells, including Flk2− CD34− LSK-HSCs.

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Nagel, Stefan. « The Role of NKL Homeobox Genes in T-Cell Malignancies ». Biomedicines 9, n^o 11 (12 novembre 2021) : 1676. http://dx.doi.org/10.3390/biomedicines9111676.

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Homeobox genes encode transcription factors controlling basic developmental processes. The homeodomain is encoded by the homeobox and mediates sequence-specific DNA binding and interaction with cofactors, thus operating as a basic regulatory platform. Similarities in their homeobox sequences serve to arrange these genes in classes and subclasses, including NKL homeobox genes. In accordance with their normal functions, deregulated homeobox genes contribute to carcinogenesis along with hematopoietic malignancies. We have recently described the physiological expression of eleven NKL homeobox genes in the course of hematopoiesis and termed this gene expression pattern NKL-code. Due to the developmental impact of NKL homeobox genes these data suggest a key role for their activity in the normal regulation of hematopoietic cell differentiation including T-cells. On the other hand, aberrant overexpression of NKL-code members or ectopical activation of non-code members has been frequently reported in lymphoid and myeloid leukemia/lymphoma, demonstrating their oncogenic impact in the hematopoietic compartment. Here, we provide an overview of the NKL-code in normal hematopoiesis and discuss the oncogenic role of deregulated NKL homeobox genes in T-cell malignancies.

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Nagel, Stefan, Stefan Ehrentraut, Corinna Meyer, Maren Kaufmann, Hans G. Drexler et Roderick AF MacLeod. « NKL Homeobox Gene MSX1 Reactivates An Oncogenic Network In Lymphoid and Myeloid Malignancies ». Blood 122, n^o 21 (15 novembre 2013) : 3765. http://dx.doi.org/10.1182/blood.v122.21.3765.3765.

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Abstract Homeobox genes encode conserved transcription factors (TFs) which regulate fundamental cellular processes during development. Many members of the NKL homeobox gene subfamily are aberrantly expressed in T-cell leukemia and compromise cell differentiation. NKL homeobox gene MSX1 is expressed during embryonic hematopoiesis and its deregulation in Hodgkin lymphoma suggests an oncogenic role of this gene in hematopoietic malignancies. After screening 114 leukemia/lymphoma cell lines by microarray profiling, we detected MSX1 overexpression in three examples each from T-cell acute lymphoblastic leukemia (T-ALL) and mantle cell lymphoma (MCL), and one from acute myeloid leukemia (AML). In silico analysis by R-based statistical tools identified conspicuous expression of MSX1 in 11% of pediatric T-ALL patients, and in 3% each of MCL and AML patients. Thus, we found aberrant MSX1 expression in subsets of both lymphoid and myeloid malignancies. Focusing on MCL and AML we excluded chromosomal rearrangements by classical and molecular cytogenetics at the MSX1 locus underlying overexpression in affected cell lines. However, comparative expression profiling data indicted aberrant histone acetylation involving PHF16 and RTN1, together with TFs FOXC1, HLXB9 and TAL1, as activators of MSX1 transcription. Their involvement was confirmed by siRNA-mediated knockdown and overexpression studies. Reciprocal regulation of MSX1 involved CCND1 and NOTCH signalling. Reporter gene analyses demonstrated that CCND1 and CDKN2D are direct transcriptional targets of MSX1 and its repressive cofactor histone H1C. Fluorescence in situ hybridization showed that t(11;14)(q13;q32) in MCL results in detachment of CCND1 from its corresponding repressive MSX1 binding site. In conclusion, we uncovered a regulatory network around MSX1 in leukemia/lymphoma cells, involving factors and pathways implicated in embryonic hematopoiesis. The reciprocal regulation of MSX1 and the NOTCH pathway in B-cells parallels that of MSX2 in T-cells. These data support the view of a recurrent genetic network involved in hematopoietic development which is reactivated in malignant transformation. Disclosures: No relevant conflicts of interest to declare.

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Yang, Yu-Chung, et Steven C. Clark. « Human interleukin 3 : Analysis of the gene and its role in the regulation of hematopoiesis ». International Journal of Cell Cloning 8, S1 (1990) : 121–29. http://dx.doi.org/10.1002/stem.5530080711.

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Raaijmakers, Marc HGP, Siddhartha Mukherjee, Shangqin Guo, Tatsuya Kobayashi, Jesse Schoonmaker, Zinmar Aung, Benjamin L. Ebert et al. « Niche Induced Myelodysplasia and Secondary Hematopoietic Neoplasia Caused by Deletion of Dicer1 in Osteoprogenitor Cells. » Blood 114, n^o 22 (20 novembre 2009) : 247. http://dx.doi.org/10.1182/blood.v114.22.247.247.

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Abstract Abstract 247 Introduction: Mesenchymal cells are a part of virtually every tissue in metazoans and are thought to participate in organ formation and homeostasis. In the hematopoietic system, mesenchymal cells of the osteoblast lineage have revealed their role as regulators of normal stem cell and hematopoietic physiology. Whether these cells, which have been relegated a relatively non-descript role of ‘stroma', participate in processes that result in disease is relatively understudied. Methods: To explore this, we conditionally deleted Dicer1, the endonuclease essential for miRNA biogenesis, from osteoprogenitor cells by intercrossing transgenic mice expressing a GFP-Cre recombinase under the transcriptional control of the osteoblastic lineage specific osterix promoter to mice containing conditional (floxed) Dicer1 alleles. Results: Deletion of Dicer1 from osteoprogenitor cells resulted in markedly disordered hematopoiesis, affecting multiple lineages and recapitulating key features of human myelodysplastic syndrome (MDS). These features included ineffective hematopoiesis with cytopenia, multilineage dysplasia, increased proliferation and intramedullary apoptosis of primitive hematopoietic cells, decreased B-cell progenitors, increased bone marrow vascularity and the propensity to develop hematopoietic neoplasms (myeloid sarcoma and acute monocytic leukemia-like disease). Comparative genomic hybridization of tumor and germline tissues revealed multiple genetic aberrancies in myeloid sarcomas induced by the Dicer1 deleted environment. The hematopoietic abnormalities were entirely microenvironment dependent with intact Dicer1 in hematopoietic cells. Transplantation of wild-type hematopoietic cells into the mutant environment recapitulated these abnormalities whereas, conversely, transplantation of hematopoietic cells from mutant mice into a wild-type environment resulted in complete normalization of hematopoiesis. In addition, hematopoietic abnormalities were not observed when Dicer1 was deleted from mature osteoblasts indicating a central role of osteoprogenitor cells in the regulation of hematopoiesis. Finally, gene expression profiling and cytokine arrays from osteoprogenitor cells identified candidate molecular effectors of the observed hematopoietic abnormalities. Conclusions: The data demonstrate that a distinctive, differentiation stage specific, stromal subset of osteolineage cells can induce a highly dysfunctional hematopoietic system, recapitulating key characteristics of human myelodysplastic syndrome. Further, they reveal that primary changes in a tissue microenvironment can initiate neoplastic disease. Disclosures: Scadden: Fate Therapeutics: Consultancy.

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Kramarzova, Karolina, Harry Drabkin, Jan Zuna, Zuzana Zemanova, Jan Stary, Jan Trka et Julia Starkova. « Transcription Regulation of HOX Genes in Normal Hematopoiesis and Leukemogenesis in Children ». Blood 120, n^o 21 (16 novembre 2012) : 4614. http://dx.doi.org/10.1182/blood.v120.21.4614.4614.

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Abstract Abstract 4614 Introduction: The homeodomain genes (HOX genes) encode a family of highly conserved transcription factors that play fundamental roles during embryogenesis. HOX genes are also important regulators in hematopoiesis. In leukemogenesis, dysregulated expression of HOX genes has been found. Despite many correlative studies, the mechanism of establishment of leukemia specific HOX gene expression patterns in hematopoietic cells remains to be elucidated. Histone methylases and demethylases (Trithorax (TrxG), JMJD3 and Polycomb-group (PcG) genes) are chromatin modifiers regulating global gene expression through chromatin remodeling in many biological processes. PcG genes can also interact with DNA methyltransferases and alter their activity. Our previously published data showed that HOX gene expression correlated with the level of DNA methylation. These data together with the stabilizing function of PcG genes on HOX expression in embryogenesis suggest the involvement of histone modifiers in the regulation of hematopoietic HOX gene expression. Methods: To investigate the regulation of HOX expression in leukemogenesis, we determined mRNA levels of the representative groups of HOX genes (HOXA, HOXB, CDX1/2), PcG genes (EZH2, BMI1), MLL and demethylases (JMJD3, UTX) in samples of childhood AML (N=41) and healthy controls (N=5). We also studied the dynamics of HOX genes and chromatin modifiers in preleukemic and diagnostic samples of a patient who underwent secondary leukemia. Quantification of gene expression was performed using qPCR assays as previously described. Results: Expression patterns for the majority of HOX genes differed significantly among morphologically defined subgroups of AML with AML M3 having the lowest expression of all HOX genes. Children with AML M5 expressed HOXA cluster at the highest level, while HOXB genes were highly expressed in M5 and M4 subtype. Subgroups defined according to molecular genetics showed similar results. The presence of PML/RARa fusion gene was associated with very low expression of all HOX genes whereas MLL+ and CBFb/MYH11+ patients expressed higher levels of HOXA genes. We also assessed the prognostic significance of particular HOX genes and found that the HOXA cluster was expressed at very low levels in standard risk cases compared to the high risk group (P<0.0001 for most HOXA genes), which is in concordance with previously published results in adult AML (Andreeff et al. 2008). Determination of mRNA levels of histone modifiers showed an overall level of high expression across various AML subgroups. Nevertheless, some were uniformly expressed in AML patients (EZH2, MLL), while others were differentially expressed with the lowest level in the M3 subtype (BMI1, JMJD3). Interestingly, we found a correlation between HOX gene expression and levels of JMJD3, which was mainly evident in CBFb-MYH11+, PML-RARa+ and AML1-ETO+ patients. JMJD3 levels were also correlated with another demethylase, UTX. A positive trend between HOX gene expression and JMJD3 was identified in healthy controls as well. Analysis of the sample from preleukemic period of the patient with secondary leukemia (secALL with MLL translocation) allowed us to study the dynamics of HOX gene expression during leukemogenesis. The diagnostic secALL sample showed an expression pattern of HOX genes typical for MLL+ leukemia. However, the profile of HOX genes in preleukemic sample (16 months before secALL) resembled the pattern found in healthy controls. Nonetheless, 90% of these seemingly normal hematopoietic cells were confirmed by FISH analysis to carry MLL/FOXO3A. Thus, even though MLL is a well known regulator of HOX genes, there must be an additional mechanism, that establishes the expression pattern of HOX genes typical in MLL+ patients. Conclusion: In summary, we identified different expression patterns of HOX genes in particular subtypes of childhood AML that significantly correlated with prognosis. Our results indicate that histone modifiers JMJD3 and UTX might be involved in the regulation of HOX gene expression. Moreover, these data also suggest that histone demethylases could cooperate with specific genetic aberrations implicated in chromatin remodeling on regulation of HOX genes. The analysis of secondary leukemia suggests that additional alterations are required to deregulate HOX expression in at least some MLL+ patients. Disclosures: No relevant conflicts of interest to declare.

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Weiss, Mitchell J. « Role of Long Coding RNAs in Epigenetic Modulation of Hematopoiesis ». Blood 122, n^o 21 (15 novembre 2013) : SCI—28—SCI—28. http://dx.doi.org/10.1182/blood.v122.21.sci-28.sci-28.

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Abstract Long noncoding (Lnc) RNAs are RNA transcripts greater than 200 nucleotides (nt) that regulate gene expression independent of protein coding potential (1-3). It is estimated that thousands of lncRNAs play vital roles in diverse cellular processes. LncRNAs modulate many stages of gene expression by regulating transcription, epigenetics, splicing, translation, and protein localization. We hypothesize that multiple lncRNAs are expressed specifically during erythrocyte and megakaryocyte differentiation, and are likely to have important roles. To identify lncRNAs in erythro-megakaryopoiesis, we performed strand-specific, paired-end deep sequencing (RNA-Seq) to a depth of 200 million reads per sample on two replicates each of murine Ter119+erythroblasts, CD41+ megakaryocytes and bipotential megakaryocyte-erythroid progenitors (MEPs) [lin- Kit+ Sca1- CD16/32- CD34-], and used bioinformatic filtering tools to identify approximately 1,100 candidate lncRNAs. Over 60 percent of these lncRNAs are novel unannotated transcripts with exquisite lineage-specific expression. Using erythroid and megakaryocytic primary cell ChIP-Seq for key transcription factors (TFs) GATA1, TAL1, GATA2,and FLI1, we found that the loci of lncRNAs show similar degree of TF binding as coding genes. We used the erythroid line G1E-ER4 (which expresses estrogen-activated GATA1) to confirm that lncRNAs bound by GATA1 are also directly regulated by it. Furthermore, we used histone methylation ChIP-Seq to show that most lncRNAs arise from classical “promoters” with high H3K4me3 levels and low H3K4me1 levels. Thus, we find that lncRNAs show epigenetic features similar to the promoters of coding genes and are directly regulated by similar TF networks. Comparison of the transcriptomes of mouse fetal liver and human cord blood erythroblasts demonstrated that lncRNAs are expressed in a highly species-specific fashion, i.e., most lncRNAs identifiable in one species are not transcribed in the other, even though the corresponding genomic region is present in both species. Numerous non-conserved but functional lncRNAs are reported in the literature, and the significance of conservation in lncRNA biology is greatly debated. In order to identify functional lncRNAs, we are currently performing RNAi knockdown on numerous candidates to assess how loss of function affects erythroid maturation. We are also performing HITS-CLIP of key chromatin modifying complexes and erythroid transcription factors to identify lncRNAs bound to them. Our studies are beginning to define new layers of gene regulation in normal erythro-megakaryopoiesis, which may be relevant to the pathophysiology of related disorders including various anemias, myeloproliferative and myelodysplastic syndromes and leukemias. 1. Wang K.C., Chang H.Y. Molecular mechanisms of long noncoding RNAs. Molecular Cell. 2011;43(6):904-914. Prepublished on 2011/09/20 as DOI 10.1016/j.molcel.2011.08.018. 2. Hu W., Alvarez-Dominguez J.R., Lodish H.F. Regulation of mammalian cell differentiation by long non-coding RNAs. EMBO reports. 2012;13(11):971-983. Prepublished on 2012/10/17 as DOI 10.1038/embor.2012.145. 3. Paralkar V.R., Weiss M.J. Long noncoding RNAs in biology and hematopoiesis. Blood. 2013;121(24):4842-4846. Prepublished on 2013/05/07 as DOI 10.1182/blood-2013-03-456111. Disclosures: No relevant conflicts of interest to declare.

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Li, Feng X., Jing W. Zhu, Christopher J. Hogan et James DeGregori. « Defective Gene Expression, S Phase Progression, and Maturation during Hematopoiesis in E2F1/E2F2 Mutant Mice ». Molecular and Cellular Biology 23, n^o 10 (15 mai 2003) : 3607–22. http://dx.doi.org/10.1128/mcb.23.10.3607-3622.2003.

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ABSTRACT E2F plays critical roles in cell cycle progression by regulating the expression of genes involved in nucleotide synthesis, DNA replication, and cell cycle control. We show that the combined loss of E2F1 and E2F2 in mice leads to profound cell-autonomous defects in the hematopoietic development of multiple cell lineages. E2F2 mutant mice show erythroid maturation defects that are comparable with those observed in patients with megaloblastic anemia. Importantly, hematopoietic defects observed in E2F1/E2F2 double-knockout (DKO) mice appear to result from impeded S phase progression in hematopoietic progenitor cells. During DKO B-cell maturation, differentiation beyond the large pre-BII-cell stage is defective, presumably due to failed cell cycle exit, and the cells undergo apoptosis. However, apoptosis appears to be the consequence of failed maturation, not the cause. Despite the accumulation of hematopoietic progenitor cells in S phase, the combined loss of E2F1 and E2F2 results in significantly decreased expression and activities of several E2F target genes including cyclin A2. Our results indicate specific roles for E2F1 and E2F2 in the induction of E2F target genes, which contribute to efficient expansion and maturation of hematopoietic progenitor cells. Thus, E2F1 and E2F2 play essential and redundant roles in the proper coordination of cell cycle progression with differentiation which is necessary for efficient hematopoiesis.

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Levantini, Elena, Francesca Bertolotti, Francesco Cerisoli, Anna L. Ferri, Elisa Brescia, Daniele Galvagno, Daniel G. Tenen, Sergio Ottolenghi, Silvia K. Nicolis et Maria C. Magli. « New Role of the Regulatory Gene SOX2 in Hematopoiesis. » Blood 104, n^o 11 (16 novembre 2004) : 4195. http://dx.doi.org/10.1182/blood.v104.11.4195.4195.

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Abstract Several genes encoding transcription factors of different families have been implicated in the development and differentiation of multiple cell systems. The Sry-type high-mobility-group box 2 gene (Sox2) encodes a transcription factor that is expressed in very early cells such as embryonic stem cells and neural stem cells, where it plays important functional roles (Genes and Dev.17:126, 2003; Development131:3805, 2004). To investigate whether Sox2 plays a role also in blood cell production, we first analyzed its expression in murine hematopoietic cells. Results indicate that the gene is transcriptionally active at low levels in primitive progenitors. Furthermore, in order to address the functional implication of Sox2 in hematopoiesis we analyzed mature and precursor cells in mutant mice compound heterozygotes for a null Sox2 allele and for the deletion of a Sox2 5′ enhancer, as the complete inactivation of the gene in homozygosis is embryonic lethal. At the peripheral blood level we did not detect significant variations in the mutants. However analysis of bone marrow precursors in clonogenic assays showed that Sox2 knock-down mice exhibited a significant increase in the number of multipotent precursors, as compared to wild type animals. Moreover, bone marrow cells of wild type and mutant mice were analyzed for the expression of a panel of regulatory genes involved in the control of different somatic stem cells. Preliminary evidence suggests that some of these genes are modulated in the mutant cells. These observations support the view that Sox2 plays a role at early stages of blood cell production, providing further evidence that common molecular mechanisms may be involved in the regulation of several different types of multipotent cells.

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Engel, Michael E., Hong N. Nguyen, Jolene Mariotti, Aubrey Hunt et Scott W. Hiebert. « Myeloid Translocation Gene 16 (MTG16) Interacts with Notch Transcription Complex Components To Integrate Notch Signaling in Hematopoietic Cell Fate Specification ». Molecular and Cellular Biology 30, n^o 7 (1 février 2010) : 1852–63. http://dx.doi.org/10.1128/mcb.01342-09.

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ABSTRACT The Notch signaling pathway regulates gene expression programs to influence the specification of cell fate in diverse tissues. In response to ligand binding, the intracellular domain of the Notch receptor is cleaved by the γ-secretase complex and then translocates to the nucleus. There, it binds the transcriptional repressor CSL, triggering its conversion to an activator of Notch target gene expression. The events that control this conversion are poorly understood. We show that the transcriptional corepressor, MTG16, interacts with both CSL and the intracellular domains of Notch receptors, suggesting a pivotal role in regulation of the Notch transcription complex. The Notch1 intracellular domain disrupts the MTG16-CSL interaction. Ex vivo fate specification in response to Notch signal activation is impaired in Mtg16 −/− hematopoietic progenitors, and restored by MTG16 expression. An MTG16 derivative lacking the binding site for the intracellular domain of Notch1 fails to restore Notch-dependent cell fate. These data suggest that MTG16 interfaces with critical components of the Notch transcription complex to affect Notch-dependent lineage allocation in hematopoiesis.

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Sattler, Martin, Shalini Verma, Christopher H. Byrne, Gautam Shrikhande, Thomas Winkler, Paul A. Algate, Larry R. Rohrschneider et James D. Griffin. « BCR/ABL Directly Inhibits Expression of SHIP, an SH2-Containing Polyinositol-5-Phosphatase Involved in the Regulation of Hematopoiesis ». Molecular and Cellular Biology 19, n^o 11 (1 novembre 1999) : 7473–80. http://dx.doi.org/10.1128/mcb.19.11.7473.

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ABSTRACT The BCR/ABL oncogene causes chronic myelogenous leukemia (CML), a myeloproliferative disorder characterized by clonal expansion of hematopoietic progenitor cells and granulocyte lineage cells. The SH2-containing inositol-5-phosphatase SHIP is a 145-kDa protein which has been shown to regulate hematopoiesis in mice. Targeted disruption of the murine SHIP gene results in a myeloproliferative syndrome characterized by a dramatic increase in numbers of granulocyte-macrophage progenitor cells in the marrow and spleen. Also, hematopoietic progenitor cells from SHIP−/−mice are hyperresponsive to certain hematopoietic growth factors, a phenotype very similar to the effects of BCR/ABL in murine cells. In a series of BCR/ABL-transformed hematopoietic cell lines, Philadelphia chromosome (Ph)-positive cell lines, and primary cells from patients with CML, the expression of SHIP was found to be absent or substantially reduced compared to untransformed cell lines or leukemia cells lacking BCR/ABL. Ba/F3 cells in which expression of BCR/ABL was under the control of a tetracycline-inducible promoter showed rapid loss of p145 SHIP, coincident with induction of BCR/ABL expression. Also, an ABL-specific tyrosine kinase inhibitor, CGP57148B (STI571), rapidly caused reexpression of SHIP, indicating that BCR/ABL directly, but reversibly, regulates the expression of SHIP protein. The estimated half-life of SHIP protein was reduced from 18 h to less than 3 h. However, SHIP mRNA also decreased in response to BCR/ABL, suggesting that SHIP protein levels could be affected by more than one mechanism. Reexpression of SHIP in BCR/ABL-transformed Ba/F3 cells altered the biological behavior of cells in culture. The reduction of SHIP due to BCR/ABL is likely to directly contribute to the pathogenesis of CML.

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Conserva, Maria Rosa, Immacolata Redavid, Luisa Anelli, Antonella Zagaria, Francesco Tarantini, Cosimo Cumbo, Giuseppina Tota et al. « IKAROS in Acute Leukemia : A Positive Influencer or a Mean Hater ? » International Journal of Molecular Sciences 24, n^o 4 (7 février 2023) : 3282. http://dx.doi.org/10.3390/ijms24043282.

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One key process that controls leukemogenesis is the regulation of oncogenic gene expression by transcription factors acting as tumor suppressors. Understanding this intricate mechanism is crucial to elucidating leukemia pathophysiology and discovering new targeted treatments. In this review, we make a brief overview of the physiological role of IKAROS and the molecular pathway that contributes to acute leukemia pathogenesis through IKZF1 gene lesions. IKAROS is a zinc finger transcription factor of the Krüppel family that acts as the main character during hematopoiesis and leukemogenesis. It can activate or repress tumor suppressors or oncogenes, regulating the survival and proliferation of leukemic cells. More than 70% of Ph+ and Ph-like cases of acute lymphoblastic leukemia exhibit IKZF1 gene variants, which are linked to worse treatment outcomes in both childhood and adult B-cell precursor acute lymphoblastic leukemia. In the last few years, much evidence supporting IKAROS involvement in myeloid differentiation has been reported, suggesting that loss of IKZF1 might also be a determinant of oncogenesis in acute myeloid leukemia. Considering the complicated “social” network that IKAROS manages in hematopoietic cells, we aim to focus on its involvement and the numerous alterations of molecular pathways it can support in acute leukemias.

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Yang, Shangda, Guohuan Sun, Peng Wu, Yijin Kuang, Cong Chen, Zhaofeng Zheng, Quan Gu et al. « A Novel Lncrna, Lncery, Interacts with Wdr82 to Regulate Erythroid Differentiation By Promoting Globin Gene Transcription ». Blood 136, Supplement 1 (5 novembre 2020) : 2. http://dx.doi.org/10.1182/blood-2020-139258.

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Hematopoietic differentiation is controlled by both genetic and epigenetic regulators. Long non-coding RNAs (lncRNAs) have been demonstrated to be important for normal hematopoiesis, but their function in erythropoiesis needs to be further explored. Here, we profiled the transcriptome of 17 murine hematopoietic cell populations by deep sequencing and identified a novel lncRNA, that was highly expressed in erythroid-related progenitors and erythrocytes. For this reason, we named it lncEry. We also identified a novel lncEry isoform, which was the principal transcript and has not been reported before. Furthermore, we found that nearly 90% of lncEry molecules localized to the nucleus. Next, we performed knockdown and knockout assays to study the function of lncEry, and found that lncEry depletion impaired erythroid differentiation. RNA sequencing analysis showed that lncEry depletion decreased the expression of erythrocyte homeostasis or differentiation related genes, including globin genes, thus indicating its important role in regulating erythroid differentiation. Mechanistically, we performed RNA-pulldown assays and found that lncEry could interact with Wdr82, a component of the Set1A histone H3-Lys4 methyltransferase complex. In addition, a series of molecular assays indicated that lncEry could stabilize the localization of Set1A/Wdr82 complex to facilitate H3K4me3 on the promoter region of globin genes and participate in regulating erythropoiesis. These findings identify lncEry as an important player in the transcriptional regulation of globin genes to coordinate erythropoiesis. Disclosures No relevant conflicts of interest to declare.

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Galloway, Jenna L., Christine Thisse, Yi Zhou, Rosanna Beltre, Bernard Thisse et Leonard I. Zon. « Conversion of Erythropoiesis to Myelopoiesis in Gata1-Deficient Zebrafish Embryos. » Blood 104, n^o 11 (16 novembre 2004) : 2774. http://dx.doi.org/10.1182/blood.v104.11.2774.2774.

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Abstract Primitive hematopoiesis in vertebrates initiates in the embryonic yolk sac and yields nucleated erythrocytes and macrophages that later enter circulation. In zebrafish, a group of blood cells contained within the axial vein make up the intermediate cell mass (ICM), the teleost equivalent of the mammalian yolk sac. To identify novel genes involved in hematopoiesis, a high-throughput whole embryo in situ hybridization screen was performed. Examination of the expression pattern of 3700 clones from an adult zebrafish hematopoietic cDNA library discovered 24 genes with expression in the blood during development. Each stage of hematopoiesis was defined by a subset of genes, providing a molecular signature of blood cell maturation, from hematopoietic progenitors to terminally differentiated erythrocytes. By using antisense morpholinos to the transcription factors gata1 and gata2, we were able to dissect the regulation of these 24 genes. Examination of gene expression in Gata1, Gata2, and Gata1/Gata2-deficient animals revealed that most erythroid genes are dependent upon Gata factors for expression. Surprisingly, three novel genes, expressed in hematopoietic progenitors, do not require Gata factors for their expression demonstrating that some erythroid genes are regulated in a Gata-independent manner. During our analysis, we also found persistent ectopic expression of the myeloid transcription factor, PU.1, in the ICM cells and a subsequent expansion of mpo expressing granulocytes and L-plastin expressing macrophages. By utilizing gata1-GFP transgenic zebrafish, we were able to isolate blood cells by flow cytometry and examine their morphology. We discovered that blood cells from the Gata1-deficient animals exhibited features characteristic of myeloid cells when compared to wild-type blood cells. By confocal microscopy, we detected some blood cells in the ICM of Gata1-deficient embryos that co-express globin and PU.1, while blood cells of wild-type embryos never co-express these markers at this stage. These observations demonstrate that in the absence of Gata1 the presumptive erythroid progenitors have transformed into the myeloid lineage, and that a major cell fate alteration has occurred. Ultimately, our studies have molecularly defined blood development by gene expression, and illustrated that Gata1 governs lineage fate decisions of hematopoietic progenitors in the developing embryo.

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Collins, Emma C., Alexandre Appert, Linda Ariza-McNaughton, Richard Pannell, Yoshihiro Yamada et Terence H. Rabbitts. « Mouse Af9 Is a Controller of Embryo Patterning, Like Mll, Whose Human Homologue Fuses with AF9 after Chromosomal Translocation in Leukemia ». Molecular and Cellular Biology 22, n^o 20 (15 octobre 2002) : 7313–24. http://dx.doi.org/10.1128/mcb.22.20.7313-7324.2002.

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ABSTRACT Chromosomal translocation t(9;11)(p22;q23) in acute myeloid leukemia fuses the MLL and AF9 genes. We have inactivated the murine homologue of AF9 to elucidate its normal role. No effect on hematopoiesis was observed in mice with a null mutation of Af9. However, an Af9 null mutation caused perinatal lethality, and homozygous mice exhibited anomalies of the axial skeleton. Both the cervical and thoracic regions were affected by anterior homeotic transformation. Strikingly, mice lacking functional Af9 exhibited a grossly deformed atlas and an extra cervical vertebra. To determine the molecular mediators of this phenotype, analysis of Hox gene expression by in situ hybridization showed that Af9 null embryos have posterior changes in Hoxd4 gene expression. We conclude that the Af9 gene is required for normal embryogenesis in mice by controlling pattern formation, apparently via control of Hox gene regulation. This is analogous to the role of Mll, the murine homolog of human MLL, to which the Af9 gene fuses in acute myeloid leukemias.

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Gao, Hongjuan, Xiaorong Wu et Nancy Fossett. « Upregulation of the Drosophila Friend of GATA Gene u-shaped by JAK/STAT Signaling Maintains Lymph Gland Prohemocyte Potency ». Molecular and Cellular Biology 29, n^o 22 (8 septembre 2009) : 6086–96. http://dx.doi.org/10.1128/mcb.00244-09.

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ABSTRACT Studies using Drosophila melanogaster have contributed significantly to our understanding of the interaction between stem cells and their protective microenvironments or stem cell niches. During lymph gland hematopoiesis, the Drosophila posterior signaling center functions as a stem cell niche to maintain prohemocyte multipotency through Hedgehog and JAK/STAT signaling. In this study, we provide evidence that the Friend of GATA protein U-shaped is an important regulator of lymph gland prohemocyte potency and differentiation. U-shaped expression was determined to be upregulated in third-instar lymph gland prohemocytes and downregulated in a subpopulation of differentiating blood cells. Genetic analyses indicated that U-shaped maintains the prohemocyte population by blocking differentiation. In addition, activated STAT directly regulated ush expression as evidenced by results from loss- and gain-of-function studies and from analyses of the u-shaped hematopoietic cis-regulatory module. Collectively, these findings identify U-shaped as a downstream effector of the posterior signaling center, establishing a novel link between the stem cell niche and the intrinsic regulation of potency and differentiation. Given the functional conservation of Friend of GATA proteins and the role that GATA factors play during cell fate choice, these factors may regulate essential functions of vertebrate hematopoietic stem cells, including processing signals from the stem cell niche.

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Jonsson, JI, Q. Wu, K. Nilsson et RA Phillips. « Use of a promoter-trap retrovirus to identify and isolate genes involved in differentiation of a myeloid progenitor cell line in vitro ». Blood 87, n^o 5 (1 mars 1996) : 1771–79. http://dx.doi.org/10.1182/blood.v87.5.1771.1771.

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Abstract Studies of gene regulation during early hematopoiesis and of the regulatory network that controls differentiation and lineage commitment are hampered by difficulties in isolating and growing stem cells and early progenitor cells. These difficulties preclude the application of standard molecular genetic approaches to these problems. As an alternative approach we have introduced a lacZ-containing promoter-trap retrovirus into hematopoietic cells. We used the interleukin-3- dependent mouse myeloid progenitor cell 32D as a model to identify transcriptionally active genes. The frequency of integrations that led to transcription of the lacZ gene was estimated to be 0.5% of all integrations, of which 14% were downregulated on differentiation of 32D cells towards neutrophils. Thus, one in every 1,000 to 2,000 integrations identified a developmentally regulated gene. Cellular DNA sequences upstream of proviral integrations were isolated by inverse polymerase chain reaction. Five were further characterized and we confirmed by RNA expression analysis that they were downregulated on differentiation. Sequence analysis revealed identification of novel genes with sequence similarity to known genes. Considering the high efficiency of retroviral infection, our study shows the feasibility of using promoter-trap vectors to identity and isolate developmentally regulated genes from early hematopoietic progenitors.

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Jonsson, JI, Q. Wu, K. Nilsson et RA Phillips. « Use of a promoter-trap retrovirus to identify and isolate genes involved in differentiation of a myeloid progenitor cell line in vitro ». Blood 87, n^o 5 (1 mars 1996) : 1771–79. http://dx.doi.org/10.1182/blood.v87.5.1771.bloodjournal8751771.

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Studies of gene regulation during early hematopoiesis and of the regulatory network that controls differentiation and lineage commitment are hampered by difficulties in isolating and growing stem cells and early progenitor cells. These difficulties preclude the application of standard molecular genetic approaches to these problems. As an alternative approach we have introduced a lacZ-containing promoter-trap retrovirus into hematopoietic cells. We used the interleukin-3- dependent mouse myeloid progenitor cell 32D as a model to identify transcriptionally active genes. The frequency of integrations that led to transcription of the lacZ gene was estimated to be 0.5% of all integrations, of which 14% were downregulated on differentiation of 32D cells towards neutrophils. Thus, one in every 1,000 to 2,000 integrations identified a developmentally regulated gene. Cellular DNA sequences upstream of proviral integrations were isolated by inverse polymerase chain reaction. Five were further characterized and we confirmed by RNA expression analysis that they were downregulated on differentiation. Sequence analysis revealed identification of novel genes with sequence similarity to known genes. Considering the high efficiency of retroviral infection, our study shows the feasibility of using promoter-trap vectors to identity and isolate developmentally regulated genes from early hematopoietic progenitors.

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Andina, Nicola Daniele, Mayuresh Sarangdhar, Aubry Tardivel, Giuseppe Bombaci, Mahmoud Hallal, Irene Keller et Ramanjaneyulu Allam. « Higher Vertebrate Specific Gene Ribonuclease Inhibitor (RNH1) Is Essential for Adult Hematopoietic Stem Cell Function and Cell Cycle Regulation ». Blood 134, Supplement_1 (13 novembre 2019) : 273. http://dx.doi.org/10.1182/blood-2019-128647.

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Hematopoietic stem cells (HSC) in higher vertebrate species, especially in mammals, maintain hematopoiesis throughout adult life and require critical cell cycle regulation for their self-renewal and cell fate decisions. Although cell cycle pathways are quite conserved across animal species, it is unknown whether a higher vertebrate specific cell cycle regulation exists in adult mammalian HSCs. Recently, we have published that Ribonuclease inhibitor (RNH1) regulates erythropoiesis by controlling GATA1 mRNA translation. Here, we report that RNH1, which is present only in higher vertebrates regulates HSC cell cycle and HSC function. To study the role of RNH1 in hematopoiesis, we generated hematopoietic-specific knockout mice by backcrossing Rnh1FL/FL mice with Vav1-iCre and Mx1-Cre mice, respectively. Rnh1-deficiency (Rnh1FL/FLVav1-iCre mice) resulted in hematopoietic alterations resembling emergency myelopoiesis. At 15 weeks of age Rnh1-deficient mice had reduced hemoglobin levels (144.4 ± 2.6 vs 165.0 ± 4.2 g/L, p = 0.005), decreased lymphocytes (4.1 ± 0.8 vs 9.6 ± 1.6 K/µL, p = 0.023), increased neutrophils (3.2 ± 0.6 vs 1.5 ± 0.2 K/µL, p = 0.046) and monocytes (0.65 ± 0.05 vs 0.09 ± 0.02 K/µL, p = 0.0001) in the peripheral blood. Total bone-marrow (BM) cellularity was similar in wild type andRnh1-deficient mice, however the number of erythroid cells and lymphoid cells (T and B cells) was significantly decreased, whereas myeloid cells were significantly increased. Rnh1-deficient spleens were significantly larger than wild type controls and showed extramedullary hematopoiesis. Surprisingly, although Rnh1-deficient mice showed myeloproliferation they survived normally and did not show progression to leukemia. However, they did not tolerate even little stress, such as 35 µg LPS administration, which lead to early mortality. We analysed the progenitor populations in the BM. In line with the myelopoiesis dominant phenotype granulocyte-monocyte progenitor (GMP) cell numbers were increased but common lymphoid progenitor (CLP) and megakaryocyte-erythrocyte progenitor (MEP) cell numbers were decreased. Cell extrinsic factors such as growth factors and the bone marrow niche play a critical role in shaping lineage choice. To exclude this, we performed bone marrow transplantation experiments (BMT) by transplanting wild type (Rnh1FL/FL) and Rnh1-deficient (Rnh1FL/FLMx1-Cre+) bone marrow into lethally irradiated CD45.1 congenic mice. After reconstitution Rnh1 was deleted by administration of polyinosinic:polycytidylic acid (polyI:C). We observed a similar myelopoiesis dominant phenotype in Rnh1-deleted mice. Interestingly, we found increased numbers of long term HSCs (LT-HSCs) and short term HSCs (ST-HSCs) in Rnh1-deficient mouse BM, suggesting that RNH1 could affect HSC function. Supporting this Rnh1-deficient HSCs failed to engraft lethally irradiated mice in competitive BMT experiments. Furthermore, Rnh1-deficient HSCs produced significantly less and smaller colonies in in-vitro colony forming cell (CFC) assays. Transcriptome analysis showed increased expression of genes related to cell cycle, kinetochore, DNA damage and decreased expression of genes related to stem cell function in Rnh1-deficient LT-HSCs and ST-HSCs. Corroborating this, Rnh1-deficient LT-HSCs and ST-HSCs showed increased S/G2/M phase in cell cycle analysis. In line with this, at the molecular level, we found that RNH1 directly binds to cell-cycle related proteins such as cyclin-dependent kinase 1 (CDK1), cell-division cycle protein 20 (CDC20) and mitotic checkpoint protein BUB3, suggesting direct involvement of RNH1 in cell cycle regulation. Confirming this, pharmacological inhibition of CDK1 (RO-3306, 10 µM) in Rnh1-deficinet ST-HSCs restored colony size in CFC assays, suggesting that RNH1 and CDK1 inhibition have a synergistic effect in ST-HSCs. In summary, our results demonstrate that RNH1, which is present only in higher vertebrates, is essential for HSC cell cycle regulation and steady state hematopoiesis. Disclosures No relevant conflicts of interest to declare.

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Trombetti, Silvia, Nunzia Iaccarino, Patrizia Riccio, Raffaele Sessa, Rosa Catapano, Marcella Salvatore, Stelina Luka et al. « Over-Expressed GATA-1S, the Short Isoform of the Hematopoietic Transcriptional Factor GATA-1, Inhibits Ferroptosis in K562 Myeloid Leukemia Cells by Preventing Lipid Peroxidation ». Antioxidants 12, n^o 3 (21 février 2023) : 537. http://dx.doi.org/10.3390/antiox12030537.

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Ferroptosis is a recently recognized form of regulated cell death involving lipid peroxidation. Glutathione peroxidase 4 (GPX4) plays a central role in the regulation of ferroptosis through the suppression of lipid peroxidation generation. Connections have been reported between ferroptosis, lipid metabolism, cancer onset, and drug resistance. Recently, interest has grown in ferroptosis induction as a potential strategy to overcome drug resistance in hematological malignancies. GATA-1 is a key transcriptional factor controlling hematopoiesis-related gene expression. Two GATA-1 isoforms, the full-length protein (GATA-1FL) and a shorter isoform (GATA-1S), are described. A balanced GATA-1FL/GATA-1S ratio helps to control hematopoiesis, with GATA-1S overexpression being associated with hematological malignancies by promoting proliferation and survival pathways in hematopoietic precursors. Recently, optical techniques allowed us to highlight different lipid profiles associated with the expression of GATA-1 isoforms, thus raising the hypothesis that ferroptosis-regulated processes could be involved. Lipidomic and functional analysis were conducted to elucidate these mechanisms. Studies on lipid peroxidation production, cell viability, cell death, and gene expression were used to evaluate the impact of GPX4 inhibition. Here, we provide the first evidence that over-expressed GATA-1S prevents K562 myeloid leukemia cells from lipid peroxidation-induced ferroptosis. Targeting ferroptosis is a promising strategy to overcome chemoresistance. Therefore, our results could provide novel potential therapeutic approaches and targets to overcome drug resistance in hematological malignancies.

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Tarrant, Jacqueline M., Joanna Groom, Donald Metcalf, Ruili Li, Bette Borobokas, Mark D. Wright, David Tarlinton et Lorraine Robb. « The Absence of Tssc6, a Member of the Tetraspanin Superfamily, Does Not Affect Lymphoid Development but Enhances In Vitro T-Cell Proliferative Responses ». Molecular and Cellular Biology 22, n^o 14 (15 juillet 2002) : 5006–18. http://dx.doi.org/10.1128/mcb.22.14.5006-5018.2002.

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ABSTRACT The tetraspanins are a family of integral membrane proteins with four transmembrane domains. These molecules form multimolecular networks on the surfaces of many different cell types. Gene-targeting studies have revealed a role for tetraspanins in B- and T-lymphocyte function. We have isolated and deleted a novel tetraspanin, Tssc6, which is expressed exclusively in hematopoietic and lymphoid organs. Using a gene-trapping strategy, we generated an embryonic stem (ES) cell line with an insertion in the Tssc6 locus. Mice were derived from these ES cells and, using RNase protection and reverse transcription-PCR, we demonstrated that the insertion resulted in a null mutation of the Tssc6 allele. Mice homozygous for the gene trap insertion (Tssc6 gt/gt mice) were viable and fertile, with normal steady-state hematopoiesis. Furthermore, responses to hemolysis and granulocyte colony-stimulating factor-induced granulopoiesis were equivalent to those of wild-type mice. Lymphoid development was normal in Tssc6 gt/gt mice. Whereas Tssc6 gt/gt B cells responded normally to lipopolysaccharide, anti-CD40, and anti-immunoglobulin M stimulation, Tssc6 gt/gt T cells showed enhanced responses to concanavalin A, anti-CD3, and anti-CD28. This increased proliferation by Tssc6-deleted T lymphocytes was due to increased interleukin 2 production following T-cell receptor stimulation. These results demonstrate that Tssc6 is not required for normal development of the hematopoietic system but may play a role in the negative regulation of peripheral T-lymphocyte proliferation.

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de Alava, Enrique, et William L. Gerald. « Molecular Biology of the Ewing’s Sarcoma/Primitive Neuroectodermal Tumor Family ». Journal of Clinical Oncology 18, n^o 1 (1 janvier 2000) : 204. http://dx.doi.org/10.1200/jco.2000.18.1.204.

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ABSTRACT: Ewing’s sarcoma (ES) and primitive neuroectodermal tumor (PNET) are members of a tumor family consistently associated with chromosomal translocation and functional fusion of the EWS gene to any of several structurally related transcription factor genes. Similar gene fusion events occur in other mesenchymal and hematopoietic tumors and are tumor-specific. The resulting novel transcription factor–like chimeric proteins are believed to contribute to tumor biology by aberrant regulation of gene expression altering critical controls of cell proliferation and differentiation. These tumor-specific molecular rearrangements are useful for primary diagnosis, may provide prognostic information, and present potential therapeutic targets. The recent advances in our understanding of the molecular biology of ES and PNET represent a paradigm for the application of the basic biology of neoplasia to clinical management of patients.

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Schwarzenberger, Paul, et Jay K. Kolls. « Interleukin 17 : An example for gene therapy as a tool to study cytokine mediated regulation of hematopoiesis ». Journal of Cellular Biochemistry 85, S38 (2002) : 88–95. http://dx.doi.org/10.1002/jcb.10054.

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Raman, Rachna, Ashwini Hinge, Rupali Kumar, Juying Xu, Kathleen Szczur et Marie-Dominique Filippi. « P190-B RhoGAP Is Critical for Hematopoietic Stem Cell Niche Regulation ». Blood 118, n^o 21 (18 novembre 2011) : 221. http://dx.doi.org/10.1182/blood.v118.21.221.221.

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Abstract Abstract 221 Hematopoiesis is regulated by components of the stromal microenvironment, so-called niche. Although the concept of hematopoietic stem and progenitor cell (HSC/P) niche is well known, its molecular regulation remains ill-defined. Here, we provide evidence that p190-B GTPase Activating Protein (p190-B), a negative regulator of Rho activity, is a regulator of mesenchymal/stromal cell functions necessary for normal hematopoiesis during fetal development. Mice lacking p190-B die before birth. At day 14.5 post coitum, p190-B−/− embryos are paler than WT embryos with a 40% lower hematocrit. Cellularity and numbers of burst forming-unit (BFU) erythroid (E), colony forming-unit (CFU)-granulo-monocytic and CFU-erythroid per p190-B−/− fetal livers (FL) were 50% lower than WT. In addition, the frequency of LongTerm-HSC (LinnegScaposKitposCD150posCD48neg), ShortTerm-HSC (LSK-CD150posCD48pos), and progenitors (LK) appeared significantly reduced in the bone marrow of e17.5/18 embryos. These data suggest a defect in fetal hematopoiesis. However, p190-B−/− FL cells were able to fully reconstitute hematopoiesis of adult irradiated recipients (Xu, Blood 2009). Mice reconstituted with p190-B−/− FL cells exhibited bone marrow content, white blood count, red blood count and hematocrit similar to those reconstituted with WT cells. Furthermore, hematopoietic recovery of p190-B−/− reconstituted animals following 5-Fluorouracil or phenylhydrazine-induced stress was comparable to that of WT. Therefore, FL HSC/P retain hematopoietic potential, which suggests a non-cell autonomous defect in p190-B−/− embryos. In the present study, we examined in more detail this possibility. The numbers of CFU-fibroblast (CFU-F) were 2-fold lower in p190-B−/− FL compared to WT FL. We next examined the hematopoietic supportive capacity of p190-B−/− FL microenvironment using stromal cell cultures derived from e14.5 FL. No noticeable differences were observed during the establishment of the stromal cell cultures. p190-B−/− stromal cells exhibited a spindle-like shape and expressed CD90, CD44 and Sca-1 but not CD45, CD31 or CD11b similarly to WT stromal cells. Cobblestone Area Forming Cell assays (CAFC) was performed in co-culture between various number of WT BM cells and p190-B−/− and WT stromal cells. We examined 5 independent stromal cell cultures from both genotypes. Frequency of CAFC was 75% lower on p190-B−/− stroma than WT stroma at both 1 week (1 in 5 × 10 3 versus 1 in 21 × 10 3, n = 5 p < 0.01) and 2 weeks (1 in 21 × 10 3 versus 1 in 87 × 10 3, n = 4 p < 0.01) in culture. CFU with cells recovered from one week coculture was also performed. Cocultures with p190-B−/− stroma gave rise to 2-fold less CFU than with WT stroma (538+90 versus 255+81, p < 0.01, one representative experiment of 4). Using competitive repopulation assay, we then assessed the in vivo repopulation ability of CD45.2+ hematopoietic cells co-cultured for one week on each stroma. Fifteen weeks following transplantation, the frequency of CD45.2+ cells in the peripheral blood of mice that received cells cocultured on p190-B−/− stroma was dramatically reduced compared to mice that received cells cocultured on WT stroma (0.43% + 0.37 versus 32.4% + 7.5, n = 6, p < 0.01), equivalent to a 100-fold difference in calculated competitive repopulation unit. Therefore, p190-B−/− stroma exhibited defective hematopoietic supportive activity. Interestingly, p190-B−/− stromal cells, like WT, showed characteristic of mesenchymal stem cells (MSC), i.e. ability to give rise to CFU-F and to differentiate to adipocytes and osteoblasts. They express relatively high level of nestin and osteopontin. At a mechanistic level, gene expression analysis of molecules known to play a critical role in HSC maintenance in the niche indicated 200-fold downregulation of Kit-ligand and Wnt3a in p190-B−/− stroma compared to WT stroma (p<0.05). Surprisingly, BMP4, angiopoietin and CXCL12 were upregulated in p190-B−/− stroma, 35-fold, 14-fold and 16-fold, respectively (p<0.05). Hence, p190-B expression in MSC/stromal cell microenvironment fine-tunes niche regulatory pathways to maintain normal hematopoiesis. Therefore, p190-B may be a critical regulator of the mesenchymal stem/hematopoietic stem and progenitor niche. Disclosures: No relevant conflicts of interest to declare.

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Konantz, Martina, Sarah Grzywna, Tamara C. Pereboom, Kelli J. Carroll, Virginie Esain, Lothar Kanz, Trista E. North et Claudia Lengerke. « Multiple Roles for the Zebrafish Homologue of the Murine Evi1 Gene during Primitive Myelopoiesis and HSC Development ». Blood 124, n^o 21 (6 décembre 2014) : 2901. http://dx.doi.org/10.1182/blood.v124.21.2901.2901.

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Abstract The Evi1 locus was originally identified as a common site of retroviral integration in murine myeloid tumors. Several reports associate Evi1 expression with aggressiveness in myeloid leukemia. Since developmental pathways often reactivate in cancer, we hypothesized that Evi1 also plays critical roles during developmental hematopoiesis. Here, we employ the zebrafish model to study how evi1 modulates early blood development. We find that indeed zebrafish evi1 co-localizes with the hematopoietic markers scl, gata1, pu.1 and gata2 in the posterior lateral mesoderm and the rostral blood islands, indicating involvement in primitive hematopoiesis. Knockdown of evi1 via three independent Morpholino Oligonucleotides impairs embryonic myelopoiesis as shown by reduced pu.1, mpo and l-plastin staining, while not affecting hemangioblast formation and primitive erythropoiesis. Additionally, we observe reduced levels of cd41 expression upon evi1 knockdown, indicating that megakaryopoiesis is also impaired. Notably, at later time-points, evi1 is also expressed in the AGM region and evi1 morphants show strong reduction of runx1/c-myb expression in this region, demonstrating an additional role in hematopoietic stem cell (HSC) formation. Consistently, evi1 morphants lack ikaros+ lymphocyte precursor cells and rag1+ T-lymphocytes, and less circulating globin+, lyz+ and cd41+ cells are detected in transgenic fish analyzed by flow cytometry at 5 days post fertilization. To dissect the mechanisms by which evi1 regulates HSC formation, we furthermore analyzed genes specifically expressed in the dorsal aorta, where HSCs emerge from the hemogenic endothelium. We hypothesize that evi1 is indeed regulating HSC specification from hemogenic endothelial cells, since detailed analyses show defective aortic expression of efnb2a and dlc. How this effect is mediated is currently being investigated. Furthermore, TUNEL, anti-activated Caspase-3, anti-phospho Histone H3 and BrdU assays show both increased apoptosis and decreased proliferation in the AGM region of evi1 morphants as compared to control injected fish, suggesting important additional effects of evi1 on HSC biology beyond the specification step. Similar effects are seen in the rostral blood islands and are possibly responsible for the observed reduction of primitive myeloid progenitors. We also examined potential downstream targets of evi1. Previous reports in adult murine hematopoietic cells suggest that Evi1 affects HSC proliferation through regulation of Gata2. Indeed, gata2 co-injection enhances hematopoietic cell viability also in zebrafish embryos, and co-injection of gata2 mRNA is able to fully rescue both primitive myelopoiesis and HSC formation. We are currently investigating whether gata2 expression can compensate evi1 requirement during HSC specification or, alternatively, might rescue HSC numbers by amplifying residual HSCs that escape evi1 inhibition. Taken together, our data demonstrate that evi1 plays multiple roles during hematopoietic development, critically regulating the biology of primitive myeloid progenitors and pre-formed HSCs, as well as HSC specification from the hemogenic endothelium. Currently, we analyze the molecular mechanisms that mediate evi1 effects during these different regulatory steps and use the CRISPR/Cas9 system to generate evi1 mutant fish for further validation of our findings. Disclosures No relevant conflicts of interest to declare.

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Staeble, Sina, Stephen Kraemer, Jens Langstein, Ruzhica Bogeska, Mark Hartmann, Maximilian Schoenung, Melinda Czeh et al. « Deconvolution of Hematopoietic Commitment Decisions By Genome-Wide Analysis of Progressive DNA Methylation Changes ». Blood 134, Supplement_1 (13 novembre 2019) : 1179. http://dx.doi.org/10.1182/blood-2019-124429.

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Recent advances in single cell transcriptome analyses have resulted in the derivation of new models to describe the hierarchical organization of the mammalian hematopoietic system. While such an approach appears to be effective at discerning the trajectory of differentiation from hematopoietic stem cells (HSCs) to a given mature lineage, it remains a challenge to identify definitive points where specific lineage fates become restricted. The characterization of molecular events that correspond to such commitment decisions is critical to our interrogation of the existence and nature of serial bifurcation steps that are hypothesized to underlie the process of hematopoiesis. 5-Methylcytosine is a stable epigenetic modification, whose remodeling at specific CpG residues appears to be integral to the process of enforcing lineage-restricted gene expression programs. We have previously observed that the remodeling of the DNA methylome appears to be both progressive and irreversible during the process of hematopoietic differentiation, suggesting that this modification could be used to unambiguously identify molecular marks of lineage commitment. In order to pursue this concept further, we used tagmentation-based whole-genome bisulfite sequencing to generate a genome-wide DNA methylation map of murine hematopoiesis. This map encompasses 26 different FACS-purified populations, ranging from LT-HSCs through to terminally differentiated blood cell lineages. Across all cell populations studied, we identified 147,232 differentially methylated regions (DMRs). In line with our previous data, hierarchical clustering of these DMRs revealed coordinately regulated regions that show progressive and unidirectional lineage-specific DNA methylation dynamics during hematopoietic differentiation that would be indicative of a molecular mechanism of cell fate restriction. Single cell DNA methylome analysis indicated that methylation programming may be exclusive for a specific lineage within each cell analyzed, supporting the use of this data to establish the discreet points at which lineage commitment occurs. Along these lines, lineage-specific DMRs could already be identified within the early hematopoietic stem and multipotent progenitor compartments, supporting the concept that lineage restriction occurs early during differentiation and providing a potential molecular basis for so called lineage-priming/bias. Indeed, a phylogenetic tree of the entire hematopoietic system could be constructed purely based on methylation remodeling events that took place in the Lin-, Sca-1+, c-Kit+ compartment. To gain further insight into how the DNA methylation programming relates to regulation of gene expression, we generated a comprehensive single cell transcriptome map encompassing the entire hematopoietic component of the bone marrow. Integration of DNA methylation dynamics with single cell gene expression dynamics provided evidence for anti-correlation between the transcriptional program and DNA methylation. However, loss of DNA methylation was not invariably associated with an increase in gene expression, suggesting that DNA methylation has more a permissive rather than an instructive role in regulating gene expression programs. We next applied our data set to the exploration of the phenomenon of myeloid lineage bias that has been described in aged mice, by investigating the methylomes of young and aged HSCs. Compared to young HSCs, we identified 3,275 DMRs in aged HSCs, which were predominantly associated with loss of DNA methylation and affected genes involved in HSC adhesion and migration, such as Vwf and ITGB3. Remarkably, 46 % of aging DMRs overlapped with the methylome programs identified in hematopoietic differentiation and were enriched for genes involved in integrin signaling, platelet activation and aggregation. This data suggests that HSC aging results in remodeling of the DNA methylome in vivo, in a manner that is associated with a differentiation bias towards the megakaryocytic lineage. Together, our work provides a rich resource to investigate DNA methylation changes in normal or diseased hematopoiesis, across a broad range of conditions such as aging. Disclosures Lipka: InfectoPharm GmbH: Employment.

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Xiong, Qian, Zhaojun Zhang, Hongzhu Qu, Xiuyan Ruan, Hai Wang, Qian Zhang, Heyuan Qi et al. « Deciphering the Cis- and Trans-regulatory Roles of KLF6 in Primitive Hematopoiesis ». Blood 120, n^o 21 (16 novembre 2012) : 4730. http://dx.doi.org/10.1182/blood.v120.21.4730.4730.

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Abstract Abstract 4730 Krüppel-like factors (KLFs) are a conserved family of Cys2His2 zinc finger proteins which are important components of eukaryotic cellular transcriptional machinery that controls many biological processes including erythroid differentiation and development. As a transcriptional activator and a tumor suppressor, KLF6 was also involved in hematopoiesis. Klf6−/− mice is embryonic lethal by embryonic day 12.5 and associated with markedly reduced hematopoiesis as well as poorly organized yolk sac vascularization. Moreover, the expression of erythroid differentiation markers including Klf1, Gata1 and Scl are delayed and hematopoietic differentiation is impaired in klf6−/− ES cells. However, the detailed mechanism that KLF6 regulates hematopoiesis is not fully understood. To characterize the role of KLF6 in hematopoiesis, we firstly detected the dynamic expression pattern of KLF6 during erythroid differentiation by mRNA-seq in undifferentiated human embryonic stem cells (hESC), three primary erythroid cells at different developmental stages including ES-derived erythroid cells (ESER), fetal- and adult-type erythroid cells (FLER, PBER). The transcriptome analysis showed that KLF6 expressed at significantly higher level in ESER cells compared with that in other cells. Meanwhile, chromatin immunoprecipitation (ChIP) studies in human K562 cells demonstrated the enrichment of KLF6 on the promoter region of embryonic epsilon-globin gene. These results probably indicate that KLF6 play an important role in primitive hematopoiesis. To clarify whether the erythroid-specific enhancers in the genomic region of KLF6 participate in the regulation of primitive hematopoiesis, we extensively screened the erythroid-specific DNaseI hypersensitive sites (DHSs) in the KLF6 locus, from 70 kb upstream of the transcription start site to 20 kb downstream of the poly(A) site, from DNase-seq data in four erythroid cells including ESER, FLER, PBER, K562 and seven non-erythroid cells. The enhancer activity of these erythroid-specific DHSs was comprehensively characterized by dual-luciferase reporter assay in K562 cells as well as non-erythroid HeLa and HEK293 cells. Three erythroid-specific enhancers located 18–24 kb upstream of human KLF6 were finally characterized, which not only helps to understand the higher expression of KLF6 in ESER, but also hints that KLF6 could participate in primitive hematopoiesis through erythroid-specific enhancers. In conclusion, we depicted the dynamic expression pattern of KLF6 during erythroid differentiation, characterized three erythroid-specific enhancers in KLF6 gene locus, and disclosed the potential role of KLF6 in primitive hematopoiesis. Next, the overexpression and depletion of KLF6 in K562 cells will be executed to further explore whether the abnormal KLF6 will affect the expression and functions of globin genes as well as erythroid-specific transcription factors. Chromosome conformation capture (3C) analysis will be performed to evaluate the interactions between the erythroid-specific enhancers and the cis-regulatory elements of hematopoiesis related genes. Moreover, we will establish morpholino-based klf6 knockdown zebrafish model and study the target genes, interacting networks and pathways in which KLF6 involved. Collectively, these results will address the detailed cis- and trans- regulatory functions and molecular mechanism of KLF6 in regulating hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

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