Expression data from murine cell line transduced with epitope tagged forms of Hoxa9
ABSTRACT: Importantly increasing evidence shows that Hox genes such as Hoxa9 are key regulators of stem cell self-renewal and hematopoiesis. Hoxa9 is expressed in early hematopoietic progenitor cells and promotes stem cell expansion. In contrast Hoxa9 down regulation is associated with hematopoietic differentiation. In addition to its role in development, HOXA9 has been intensively studied because of its central role in human acute leukemias. Despite their obvious biomedical importance, the mechanisms through which Hoxa9 and its partner proteins exert their downstream functions are poorly understood. Using whole-genome gene expression profiling, we identified direct targets of Hoxa9 in murine MHPs after 4-OHT withdraw, resulting in cell differentiation. Bone marrow cells were harvested from 5-Fluorouracil treated female 6-8 week old C57BL/6 mice and transduced with an MSCV-based retrovirus expressing Hoxa9 fused to a modified estrogen receptor ligand binding domain (Hoxa9-ER). Hoxa9-ER cells were washed 3x and resuspended in IL-3+ media with/without 100 nM 4-OHT (Sigma). At selected intervals, cells were removed for flow cytometric analysis using anti-Gr1 and anti-Mac1 antibodies (BD biosciences), morphologic assessment by cytocentrifugation and staining with Diff-Quick reagents (Intl. Med. Equip.), and RNA collection. For RNA, Pellets were lysed in Trizol reagent (Invitrogen) and RNA was extracted following manufacturer's instructions until phase separation, after which RNeasy columns (Qiagen) were employed for further purification. cRNA probes were synthesized at the University of Michigan microarray core. Probes were hybridized to Affymetrix Mouse 430 2.0 array.
Project description:Characterization of gene expression changes 72 hours after withdrawal of tamoxifen in murine hematopoietic progenitors transformed by Hoxa9-ER/Meis1 using RNAseq. In the presence of tamoxifen (4OHT), Hoxa9-ER localizes to the nucleus of cells allowing for transformation, while withdrawal of 4OHT (culture in EtOH) leads to loss of nuclear Hoxa9-ER. Loss of Hoxa9-ER leads to a decrease in cellular proliferation and differentiation along the myeloid lineage. Examination of gene expression by RNAseq in two conditions in biological replicates.
Project description:Relative overexpression of HOXA9 is a key feature of aggressive AML (acute myeloid leukemia). Hoxa9 responsive genes were identified by nascent RNA sequencing (4-thio-uridine labeled RNA - sequencing) in samples of primary hematopoietic precursor cells from mice transformed by an tamoxifen inducible version of Hoxa9 (Hoxa9-ER). Samples were generated in the presence of active Hoxa9 (0h) and in a time series after Hoxa9 was inactivated at 8h, 16h, 24h, 48h, and 72h.
Project description:Lysozyme-GFP ER-HoxA9 cells were cultured in the presence of estradiol (active ER-HoxA9) or in the absence of estradiol (inactive ER-HoxA9). Samples were taken at 10 time points over a 120 hour time course of myeloid differentiation to examine those gene expression changes that accompany differentiation upon the release of HoxA9 differentiation arrest. Overall design: RNA Sequencing at 10 different time points done in duplicate
Project description:To explore the molecular mechanisms induced by the leukemic fusion protein NUP98-HOXA9, we performed gene expression analysis of human hematopoietic progenitors and primary samples of patients that express NUP98-HOXA9. By combining these data with ChIP-seq results, we observed that the fusion protein is able to both activate and repress the expression of its target genes. Overall design: Three independent experiments were performed using different clones of the human hematopoietic progenitor model (hHP-NH) and control model (hHP-empty vector). On the other hand, RNA from seven primary samples was analyzed.
Project description:Relative overexpression of HOXA9 is a key feature of aggressive AML (acute myeloid leukemia). Here we determined genome wide binding sites of Hoxa9 in primary murine cells transformed by Hoxa9 and in a human AML cell line. In addition global H3K4 monomethylation and H3K27acetylation levels were determined in cells transformed by an inducible Hoxa9-ER construct in Hoxa9-active conditions and 72h after Hoxa9 was inactivated.
Project description:During hematopoietic differentiation of hESCs, HOXA9 expression parallels hematopoietic development but is restricted to the hemogenic precursors (HEP, CD31+CD34+CD45-), and diminishes as HEPs differentiate into blood cells (CD45+). Enforced expression of Hoxa9 in hESCs robustly promoted differentiation into primitive (CD34+CD45+) and total (CD45+) blood cells with higher clonogenic (CFU) potential. To identify patterns of gene expression that could explain at the molecular level the developmental impact of HOXA9 in hematopoietic commitment of hESCs, we performed gene expression profile in FACS-purified EV- and HOXA9-HEPs. 10^5 HoxA9 over expressing and EV control HEPs purified by FACS from H9 and AND1 hEBs at day 15 of hematopoietic differentiation were used for gene expression analysis using Whole Human Genome Oligo Microarray chips (Agilent Technologies).
Project description:This SuperSeries is composed of the following subset Series: GSE21299: Expression data from murine cell line transduced with epitope tagged forms of Hoxa9 GSE33509: Identification and Characterization of Hoxa9 Binding Sites in Hematopoietic Cells GSE33517: Epigenetic profiling of histone H3K4me1, H3K4me3, H3K27me3, H3ac, H4ac, CBP and P300 using ChIP-chip Refer to individual Series
Project description:Leukemia is a complex malignancy with hundreds of distinct mutations associated with disease development. Studies have shown that oncogenes cooperate to promote leukemia transformation, however, the downstream effectors of this cooperation are largely unknown. Using a genetically defined mouse model of acute leuekmia, we investigated the regulated of genes downstream of the cooperative oncogenic interaction between BCR-ABL and NUP98-HOXA9 and identified a unique gene signature abberrantly expression in leukemia. Total RNA was isolated from hematopoietic cells transduced with BCR-ABL and Nup98-HOXA9 retroviruses and transplanted into recipient mice. Bone marrow cells were purified by GFP (BCR-ABL) and YFP (NUP98-HOXA9) using FACS.
Project description:SEM cells were established from the peripheral blood of a 5-year-old girl in relapse with acute lymphoblastic leukaemia (ALL). SEM cells exhibit the t(4;11) chromosomal rearrangement, which leads to production of the MLL-AF4 fusion protein. Hematopoietic transcription factors including HOXA9 and MEIS1 are highly expressed in ALL. ChIP-seq was performed against HoxA9 and MEIS1 in SEM cells. DNA was enriched by chromatin immunoprecipitation (ChIP) and analyzed by Solexa sequencing.
Project description:Primary murine hematopoietic cells were retrovirally transduced with Hoxa9 cDNA, expanded in vitro before undergoing secondary infection with the cDNA of Meis1, Prep1 and the Prep1-MC mutant. RNA was extracted and transcriptomes were analyzed. Background corrections and normalisations were performed using RMA in NimbleScan 2.5