Unknown,Transcriptomics,Genomics,Proteomics

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Altered gene expression in primitive murine hematopoietic progenitors expressing INV16

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.

ORGANISM(S): Mus musculus

SUBMITTER: Chris Klug

PROVIDER: E-GEOD-85659 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.

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Altered gene expression in primitive murine hematopoietic progenitors expressing INV16

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