Altered gene expression in primitive murine hematopoietic progenitors expressing INV16
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ABSTRACT: The role of the INV16 genetic translocation in acute myeloid leukemia may be to alter expression in primitive hematopoietic progenitors of genes important for regulating hematopoiesis. To identify transcriptional targets of INV16 in primitive hematopoietic progenitors, FACS-purified progenitors from murine bone marrow were transduced with retrovirus encoding INV16 and analyzed for alterations in gene expression using whole transcriptome expression arrays. Normal murine bone marrow cells of the Lineage-negative, c-Kit+, Sca-1+, Flt3-negative phenotype (KSLF) were FACS-purified, transduced with retrovirus encoding INV16 (INV), the non-leukemogenic INV16 mutant deleted of the assembly competent domain (ACD) or empty retroviral vector control (MIB). Productively transduced, GFP-positive cells were FACS-sorted 24 hours later, and RNA isolated and analyzed using Affymetrix whole transcriptome expression arrays. Replicate numbers of sorts/transductions/analyses of 4, 3, and 5 were performed for INV, ACD, and MIB, respectively.
Project description:The role of the INV16 genetic translocation in acute myeloid leukemia may be to alter expression in primitive hematopoietic progenitors of genes important for regulating hematopoiesis. To identify transcriptional targets of INV16 in primitive hematopoietic progenitors, FACS-purified progenitors from murine bone marrow were transduced with retrovirus encoding INV16 and analyzed for alterations in gene expression using whole transcriptome expression arrays.
Project description:The involvement of mature hematopoietic cells in disease pathogenesis is well recognized. However it is not clear how if and how primitive progenitors might contribute to inflammatory disease processes. This microarray experiment is used together with data from functional assays to determine how primitive progenitors are altered in a mouse model of autoimmune arthritis and how this in turn might contribute to the disease process. KSL cells were FACS sorted from 7 to 9 6-7 week old arthritic (KRNxG7) mice as well as from two strains of non-arthritic age-matched control mice: KRN and B6xG7 mice. Cells were sorted using identical conditions and identical sorting gates. To verify the primitive status of the KSL cells, Lin+ cells were also MACS sorted from these same mice. All the mice used in this study were C57BL/6 background strain. G7 mice are congenic with C57BL/6 but with MHC II I-Ab replaced with MHC II I-Ag7.
Project description:Reintroduction of CEBPA in MN1-overexpressing hematopoietic cells prevents their hyper-proliferation and restores myeloid differentiation. Forced expression of MN1 in primitive mouse hematopoietic cells causes acute myeloid leukemia and impairs all-trans retinoic acid (ATRA) induced granulocytic differentiation. Here, we studied the effects of MN1 on myeloid differentiation and proliferation using primary human CD34+ hematopoietic cells, lineage depleted mouse bone marrow cells, and bipotential (granulocytic/monocytic) human AML-cell lines. We show that exogenous MN1 stimulated the growth of CD34+ cells, which was accompanied by enhanced survival and increased cell cycle traverse in cultures supporting progenitor cell growth. Forced MN1 expression impaired both granulocytic and monocytic differentiation in vitro in primary hematopoietic cells and AML cell lines. Endogenous MN1 expression was higher in human CD34+ cells compared to both primary and in vitro differentiated monocytes and granulocytes. Microarray and real time RT-PCR analysis of MN1-overexpressing CD34+ cells showed down regulation of CEBPA and its downstream target genes. Re-introduction of conditional and constitutive CEBPA overcame the effects of MN1 on myeloid differentiation and inhibited MN1-induced proliferation in vitro. These results indicate that down regulation of CEBPA activity contributes to MN1-modulated proliferation and impaired myeloid differentiation of hematopoietic cells Human BM derived CD34+ cells (Stemcell Technologies, Vancouver, BC, Canada) were expanded for 2 days and transduced with MSCV-IRES-GFP or MSCV-MN1-IRES-GFP retrovirus for another 2 days. One day later RNA was isolated from GFP+/CD34+ FACS-sorted cells using Trizol (Sigma) and samples were subjected to micro array analysis following Affymetrix protocols (Affymetrix, Santa Clara, CA) using the GeneChip Human U133 Plus 2.0 array
Project description:The bHLH transcription factor stem cell leukemia gene (Scl) is a master regulator for hematopoiesis essential for hematopoietic specification and proper differentiation of the erythroid and megakaryocyte lineages. However, the critical downstream targets of Scl remain undefined. Here, we identified a novel Scl target gene, transcription factor myocyte enhancer factor 2 C (Mef2C) from Sclfl/fl fetal liver progenitor cell lines. Analysis of Mef2C-/- embryos showed that Mef2C, in contrast to Scl, is not essential for specification into primitive or definitive hematopoietic lineages. However, adult VavCre+Mef2Cfl/fl mice exhibited platelet defects similar to those observed in Scl deficient mice. The platelet counts were reduced, while platelet size was increased and the platelet shape and granularity was altered. Furthermore, megakaryopoiesis was severely impaired in vitro. ChIP-on-chip analysis revealed that Mef2C is directly regulated by Scl in megakaryocytic cells, but not in erythroid cells. In addition, an Scl independent requirement for Mef2C in B-lymphoid homeostasis was observed in Mef2C-deficient mice, characterized as severe age-dependent reduction of specific B cell progenitor populations reminiscent of premature aging. In summary, this work identifies Mef2C as an integral member of hematopoietic transcription factors with distinct upstream regulatory mechanisms and functional requirements in megakaryocyte and B-lymphoid lineages. Experiment Overall Design: Sclfl/fl hematopoietic progenitor lines were generated from fetal liver progenitors from E12.5 Sclfl/fl embryos9 by immortalization with Hox11 retrovirus.17 Sclfl/fl progenitor cells were cultured with IL3, and a clonal line containing cells with megakaryocyte morphology and acetylcholinesterase (AchE) activity was selected. The Sclfl/fl cell line was transduced with Cre- GFP retrovirus to generate the Scl Experiment Overall Design: Î?/Î? cell line, and the Scl Î?/Î? cell line was transduced with Scl retrovirus to re-introduce Scl expression (Scl Î?/Î? +Scl cell line). Megakaryocyte differentiation was enhanced by adding Tpo for 5 days before harvesting the cells. RNA was extracted with Trizol (Gibco BRL) and RNEasy (Qiagen) kits. Differential gene expression between Sclfl/fl, Scl Î?/Î? and Scl Î?/Î? +Scl cell lines was analyzed by Affymetrix MOE430_2 microarray in the microarray core facility at the Dana-Farber Cancer Institute.
Project description:Effect of NF-kB inhibition and activation on gene expression in mouse and human lung cancer cell-lines. Cells were transduced with control retrovirus (MiG) or MiG retrovirus expressing IkB alpha super-repressor (SR) mutant (S32, S36 to A mutations) or activated IKK beta (S177, S181 to E mutations). Cells were FACS sorted by GFP expression. RNA isolated from these cells was used for microarray studies.
Project description:HIF-1a and HIF-2a are expressed at high levels in mesenchymal progenitors compared to more committed mesenchymal cells and hematopoietic cells. HIF-factors could therefore have a role in the regulation the biology of mesenchymal progenitors and their functions, like the non cell-autonomous maintenance of hematopoietic progenitors. We used microarrays to detail the global program of gene expression regulated by HIF-1a or HIF-2a in mesenchymal progenitors Mesenchymal progenitors were FACS-sorted and cultured in low oxygen concentration for few days. Once cells started to form CFU-F colonies, we transduced them with shRNAs targeting specifically HIF-1a or HIF-2a. Four days after transduction, cells were collected and RNA extracted for microarray analysis.
Project description:Overexpression of transcription factor Sox17 in human ES cells-derived endothelial cells and hematopoietic cells enhances expansion of hemogenic endothelium-like cells. Human ES cells were differentiated for 6 days, 8 days or 12 days in EBs, then CD34+CD43-CD45- endothelial cells, CD34+CD43+CD45- pre-hematopoietic progenitor cells (HPCs) or CD34+CD43+CD45+ HPCs were isolated by fluorescence activated cell sorting (FACS) and subjected to a microarray analysis.M-cM-^@M-^@Some samples were plated onto OP9 cells after the isolation by FACS, and transduced with the 4OH-tamoxifen-inducible 1M-CM-^WFLAG-tagged Sox17-ERT retrovirus. The cells were cultured with 4OH-tamoxifen. CD34+CD43+CD45low hemogenic endothelium-like cells expanded by Sox17-ERT were collected by magnetic-activated cell sorting (MACS) and subjected to a ChIP-chip analysis.
Project description:We have determined that sustained expression of EBF suppresses alternate lineage genes independently of Pax5. Experiment Overall Design: Pax5-/- pro-B cells transduced with control or EBF retrovirus. Transduced cells were FACS sorted based on GFP expression and total RNA was isolated. RNA isolated from control or EBF transduced cells was analyzed using the Affymetrix Rat 230A microarrays.
Project description:Human pluripotent stem cells were differentiated into hematopoietic progenitors, which were then re-specified using defined transcription factors to resemble hematopoietic stem cells (HSC) We used microarrays to establish the similarity between converted cells and purified human HSCs. The samples analyzed were: starting embryoid body progenitors, transcription factor-converted cells, and primary HSCs and progenitors from fetal liver and cord blood. All samples were flow sorted for CD34+ and CD38- to compare across a similar population of primitive cells.
Project description:MYB is well recognized to be a key regulator of definitive hematopoiesis that plays an important role in the maintenance and multilineage differentiation of hematopoietic stem cells (HSCs). In the vertebrate developmental context, MYB is widely regarded dispensable for primitive hematopoiesis but critically required for the development of definitive hematopoiesis. To explore the role of MYB in human hematopoietic development we have inactivated the gene by bi-allelic TALEN-supported gene targeting in several lines of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), and subjected these cells to hematopoietic differentiation in well-defined cell culture conditions. Venus gene reporter was inserted into the knock-in allele to monitor MYB expression during the course of the hESC/iPSC differentiation. The gene reporter system showed that MYB is specifically expressed during hematopoietic commitment in the earliest primitive blood cells. Moreover, the level of MYB expression was highest at the commitment stage of differentiation and significantly decreased at the maturations stage. We found that MYB was not required for initial hematopoietic commitment of nascent mesoderm and emergence of primitive, yolk sac-type human hematopoietic progenitors. However, inactivation of MYB severely abrogated proliferation of the primitive erythroid and mixed erythroid-macrophage-megakaryocyte progenitors. In addition, MYB-negative hESC/iPSC lines demonstrated major defects in myeloid cell development and completely failed to generate mature granulocytes. Transposon-mediated rescue of MYB expression in MYB-null cells efficiently restored both the primitive hematopoietic progenitors and immature myeloid cells. Our data indicate that in contrast to its previously attributed exclusive role in definitive hematopoiesis, MYB is indispensable for primitive human hematopoiesis.