Gene array analysis of human CD34+ hematopoietic cells exposed during 24h to HOXB4 or HOXC4 homeoproteins
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE24379 | GEO | 2010/10/15
SECONDARY ACCESSION(S): PRJNA132863
REPOSITORIES: GEO
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Project description:At the molecular level, the mechanisms of action of Hox genes during hematopoiesis remain elusive. Although reports have described the implication of Hox genes in some molecular pathways in mice, the targets of HOX proteins in human hematopoietic cells are mostly unidentified. Since HOXB4 and HOXC4 are transcription factors encoded by paralogous genes that display similar effects on HSCs and progenitors, we presumed that HOXB4 and HOXC4 regulate the same target genes during human HSC expansion. This hypothesis was supported by the fact that HOXB4 and HOXC4 have similar molecular structure and similar roles and expression patterns during development. To identify early factors and signaling pathways involved in hematopoietic cell expansion consecutive to the action of HOXB4 or HOXC4, we performed a differential transcriptome analysis of RNAs from cord blood-derived CD34+ cells cultured for 24 hours in the presence of MS-5 cells engineered to secrete HOXB4 or HOXC4 proteins. MS-5/GFP cells were used as control in separate co-cultures to exclude nonspecific changes in gene expression that may be associated with the presence of lentivirus-transduced MS-5 cells. Human CD34+ cord blood (4.106) cells were co-cultured for 24 hours with irradiated EGFP-, HOXB4-, or HOXC4-transduced MS-5 cells. 12 arrays were analysed. Three competitive hybridizations to the arrays were performed in duplicate using each time two dye-swap following this experimental design: AVZ056 : CY5 (CD34+/MS5+HOXB4) / CY3 (CD34+/MS5+GFP) AVZ059 : CY5 (CD34+/MS5+GFP) / CY3 (CD34+/MS5+HOXB4) AVZ064 : CY5 (CD34+/MS5+GFP) / CY3 (CD34+/MS5+HOXB4) AVZ067 : CY5 (CD34+/MS5+HOXB4) / CY3 (CD34+/MS5+GFP) AVZ057 : CY5 (CD34+/MS5+GFP) / CY3 (CD34+/MS5+HOXC4) AVZ058 : CY5 (CD34+/MS5+HOXC4) / CY3 (CD34+/MS5+GFP) AVZ060 : CY5 (CD34+/MS5+HOXC4) / CY3 (CD34+/MS5+GFP) AVZ068 : CY5 (CD34+/MS5+GFP) / CY3 (CD34+/MS5+HOXC4) AVZ061 : CY5 (CD34+/MS5+HOXB4) / CY3 (CD34+/MS5+HOXC4) AVZ062 : CY5 (CD34+/MS5+HOXC4) / CY3 (CD34+/MS5+HOXB4) AVZ065 : CY5 (CD34+/MS5+HOXC4) / CY3 (CD34+/MS5+HOXB4) AVZ066 : CY5 (CD34+/MS5+HOXB4) / CY3 (CD34+/MS5+HOXC4)
2010-10-15 | E-GEOD-24379 | biostudies-arrayexpress
Project description:To unravel the molecular mechanism by which HOXB4 promotes the expansion of early hematopoietic progenitors within differentiating ES cells, we analzed the gene expression profiles of embryoid bodies (EBs) in which transcription of HOXB4 had been induced or not induced. A substantial number of the identified HOXB4 target genes are involved in signaling pathways important for controlling self-renewal, maintenance and differentiation of stem cells. Furthermore, we demonstrate that HOXB4 activity and FGF-signaling are intertwined. HOXB4-mediated expansion of ES cell-derived early progenitors was enhanced by specific and complete inhibition of FGF-receptors. In contrast, the expanding activity of HOXB4 on hematopoietic progenitors in day4-6 embryoid bodies was blunted in the presence of basic FGF (FGF2) indicating a dominant negative effect of FGF-signaling on the earliest hematopoietic cells. Taken together, we show that modulation of FGF signaling is an essential feature of HOXB4 activity in the context of embryonic hematopoiesis. Keywords: plus/minus induction of HOXB4 gene expression by treatment with doxycycline (Dox)
2007-09-19 | GSE9044 | GEO
Project description:Enforced expression of the homeobox transcription factor HOXB4 has been shown to enhance hematopoietic stem cell (HSC) self-renewal and expansion ex vivo and in vivo. In order to investigate the largely unknown downstream targets of HOXB4 in hematopoietic progenitor cells, HOXB4 was constitutively overexpressed in the primitive hematopoietic progenitor cell line, EML. Gene expression differences were compared between KLS (c-Kit+, Lin-, Sca-1+)-EML cells that overexpressed HOXB4 (KLS-EML-HOXB4) to control KLS-EML cells that were transduced with vector alone. ChIP-chip was used to identify promoter regions bound by HOXB4.
2010-04-20 | GSE20604 | GEO
Project description:Overexpression of HOXB4 in hematopoietic stem cells (HSCs) leads to increased self-renewal without causing hematopoietic malignancies in transplanted mice. The molecular basis of HOXB4-mediated benign HSC expansion in vivo is not well understood. To gain further insight into the molecular events underlying HOXB4-mediated HSC expansion, we analyzed gene expression changes at multiple time points in Lin-Sca1+c-kit+ (LSK) cells from mice transplanted with bone marrow (BM) cells transduced with a MSCV-HOXB4-ires-YFP vector. A distinct HOXB4 transcriptional program was reproducibly induced and stabilized by 12 weeks after transplant. Dynamic expression changes were observed in genes critical for HSC self-renewal as well as genes involved in myeloid and B cell differentiation. Prdm16, a transcription factor associated with human acute myeloid leukemia (AML), was markedly repressed by HOXB4 but upregulated by HOXA9 and HOXA10, suggesting that Prdm16 downregulation was involved in preventing leukemia in HOXB4 transplanted mice. Functional evidence to support this mechanism was obtained by enforcing co-expression of sPrdm16 and HOXB4, which led to enhanced self-renewal, myeloid expansion, and leukemia. Altogether, these studies define the transcriptional pathways involved in HOXB4 HSC expansion in vivo and identify repression of Prdm16 transcription as a mechanism by which expanding HSCs avoid leukemic transformation.
2014-10-01 | GSE59804 | GEO
Project description:Overexpression of HOXB4 in hematopoietic stem cells (HSCs) leads to increased self-renewal without causing hematopoietic malignancies in transplanted mice. The molecular basis of HOXB4-mediated benign HSC expansion in vivo is not well understood. To gain further insight into the molecular events underlying HOXB4-mediated HSC expansion, we analyzed gene expression changes at multiple time points in Lin-Sca1+c-kit+ (LSK) cells from mice transplanted with bone marrow (BM) cells transduced with a MSCV-HOXB4-ires-YFP vector. A distinct HOXB4 transcriptional program was reproducibly induced and stabilized by 12 weeks after transplant. Dynamic expression changes were observed in genes critical for HSC self-renewal as well as genes involved in myeloid and B cell differentiation. Prdm16, a transcription factor associated with human acute myeloid leukemia (AML), was markedly repressed by HOXB4 but upregulated by HOXA9 and HOXA10, suggesting that Prdm16 downregulation was involved in preventing leukemia in HOXB4 transplanted mice. Functional evidence to support this mechanism was obtained by enforcing co-expression of sPrdm16 and HOXB4, which led to enhanced self-renewal, myeloid expansion, and leukemia. Altogether, these studies define the transcriptional pathways involved in HOXB4 HSC expansion in vivo and identify repression of Prdm16 transcription as a mechanism by which expanding HSCs avoid leukemic transformation. 5-FU treated bone marrow cells from female C57Bl/6L mice were transduced with concentrated supernatant from GPE+86-derived producer cells containing MSCV-HOXB4-ires-YFP or MSCV-ires-GFP retroviral vectors. Transduced cells were transplanted into lethally irradiated C57Bl/6L mice. HOXB4-YFP or GFP expressing LSK cells were sorted from transplanted mice at 6, 12 and 24 weeks post transplantation, of which RNA was extracted for microarray.
2014-10-01 | E-GEOD-59804 | biostudies-arrayexpress
Project description:Enforced expression of the homeobox transcription factor HOXB4 has been shown to enhance hematopoietic stem cell (HSC) self-renewal and expansion ex vivo and in vivo. In order to investigate the largely unknown downstream targets of HOXB4 in hematopoietic progenitor cells, HOXB4 was constitutively overexpressed in the primitive hematopoietic progenitor cell line, EML. Gene expression differences were compared between KLS (c-Kit+, Lin-, Sca-1+)-EML cells that overexpressed HOXB4 (KLS-EML-HOXB4) to control KLS-EML cells that were transduced with vector alone. ChIP-chip was used to identify promoter regions bound by HOXB4. We overexpressed HOXB4 in EML cells. We isolated 3 separate single cell clones as assessed by Southern Blot Analysis (3 clones for EML-HOXB4 and 3 clones for control EML-GFP cells). RNA was isolated from the KLS (c-Kit+, Lin-, Sca-1+) fraction of each single cell clone population and processed for hybridization to array chips using established lab protocols. Chip-Chip analysis of the three HOXB4 overexpressing clones was performed to identify HOXB4 bound promoters.
2010-04-20 | E-GEOD-20604 | biostudies-arrayexpress
Project description:HOXB4 mediates expansion of adult and embryo-derived hematopoietic stem cells (HSCs) when expressed ectopically. To define the underlying molecular mechanisms, we performed gene expression profiling in combination with subsequent functional analysis using enriched adult HSCs expressing inducible HOXB4. A substantial number of the identified HOXB4 target genes are involved in signaling pathways important for controlling self-renewal, maintenance and differentiation of stem cells. Functional assays performed on selected pathways confirmed the biological coherence of the array results. HOXB4 activity protected adult HSCs from the detrimental effects mediated by the proinflammatory cytokine TNF-alpha. Furthermore, we demonstrate that HOXB4 activity and FGF-signaling are intertwined. HOXB4-mediated expansion of adult HSCs was enhanced by specific and complete inhibition of FGF-receptors. Based on our results we propose that HOXB4 governs pivotal cell-intrinsic pathways involved in the regulation of cell cycle, differentiation and apoptosis. Our results strongly suggest that HOXB4 modulates the response of HSCs to multiple extrinsic signals in a concerted manner, thereby shifting the balance towards stem cell self-renewal. Keywords: plus/minus induction of HOXB4 activity by treatment with 4-hydroxytamoxifen (TMX)
2007-09-11 | GSE9010 | GEO
Project description:To unravel the molecular mechanism by which HOXB4 promotes the expansion of early hematopoietic progenitors within differentiating ES cells, we analzed the gene expression profiles of embryoid bodies (EBs) in which transcription of HOXB4 had been induced or not induced. A substantial number of the identified HOXB4 target genes are involved in signaling pathways important for controlling self-renewal, maintenance and differentiation of stem cells. Furthermore, we demonstrate that HOXB4 activity and FGF-signaling are intertwined. HOXB4-mediated expansion of ES cell-derived early progenitors was enhanced by specific and complete inhibition of FGF-receptors. In contrast, the expanding activity of HOXB4 on hematopoietic progenitors in day4-6 embryoid bodies was blunted in the presence of basic FGF (FGF2) indicating a dominant negative effect of FGF-signaling on the earliest hematopoietic cells. Taken together, we show that modulation of FGF signaling is an essential feature of HOXB4 activity in the context of embryonic hematopoiesis. Experiment Overall Design: The Hoxb4i ES cell line (Kyba et al. 2002, Cell 109:29-37) contains an integrated âtet-onâ cassette that allows induction of HOXB4 expression upon treatment with doxycycline. These ES cells can be used to produce hematopoietic cells through the formation of embryoid bodies (EBs). Hematopoiesis starts in these EBs at day 4 and the differentiation into hematopoietic fates can be quantified by colony assays on methyl-cellulose using cells dissociated from EBs at day 6 of incubation. The induction of HOXB4 by incubation with doxycycline increases the production of hematopoietic progenitors within EBs by day 6. Using this specific ES cell line, we compared the transcriptome between embryoid bodies (EBs) in which transcription of HOXB4 had been induced or not induced from day 4 to day 6 (48hours). Experiment Overall Design: Biological replicates: 3
2008-06-16 | E-GEOD-9044 | biostudies-arrayexpress
Project description:HOXB4 mediates expansion of adult and embryo-derived hematopoietic stem cells (HSCs) when expressed ectopically. To define the underlying molecular mechanisms, we performed gene expression profiling in combination with subsequent functional analysis using enriched adult HSCs expressing inducible HOXB4. A substantial number of the identified HOXB4 target genes are involved in signaling pathways important for controlling self-renewal, maintenance and differentiation of stem cells. Functional assays performed on selected pathways confirmed the biological coherence of the array results. HOXB4 activity protected adult HSCs from the detrimental effects mediated by the proinflammatory cytokine TNF-alpha. Furthermore, we demonstrate that HOXB4 activity and FGF-signaling are intertwined. HOXB4-mediated expansion of adult HSCs was enhanced by specific and complete inhibition of FGF-receptors. Based on our results we propose that HOXB4 governs pivotal cell-intrinsic pathways involved in the regulation of cell cycle, differentiation and apoptosis. Our results strongly suggest that HOXB4 modulates the response of HSCs to multiple extrinsic signals in a concerted manner, thereby shifting the balance towards stem cell self-renewal. Experiment Overall Design: To understand the mechanisms of HOXB4 activity, we wished to identify target genes of HOXB4 in adult hematopoietic stem and progenitor cells (HSC/HPCs). We thus transduced murine HSC/HPCs with a retroviral vector that co-expresses EGFP and a tamoxifen-inducible form of HOXB4 (HOXB4-ER). Upon addition of 4-hydroxytamoxifen (TMX), the HOXB4-ER fusion protein translocates from the cytoplasm to the nucleus, consequently being capable of modulating gene expression. Transduced cell populations were expanded for 14 days in the presence of TMX. Thereafter, HOXB4-ER+LSK (GFP+ , lineage negative, Sca1+, ckit+) cells were flow cytometrically isolated and cultivated either with or without TMX for 1 or 4 hours. Inactivation of HOXB4 activity by TMX withdrawal was intended to mimic the naturally occurring down-regulation of HOXB4 in differentiating stem cells. RNA was prepared after the aforementioned times and the transcriptional profiles of HOXB4-ER+LSK +/- TMX analyzed using the Affymetrix⢠platform. As a control, profiling was also performed with LSK cells expressing unmodified constitutively active HOXB4 (HOXB4const) ± TMX, to exclude changes in gene expression due to unknown effects of tamoxifen itself. RNAs from adult LSK cells were processed for use on Affymetrix GeneChips Mouse Genome 430 2.0. All quality parameters for the arrays were confirmed to be in the recommended range.
2008-06-16 | E-GEOD-9010 | biostudies-arrayexpress
Project description:Ectopic expression of homeodomain transcription factor HoxB4 in mouse ES cells enhances in vitro development of hematopoietic stem cells (HSCs).
2011-05-23 | GSE26653 | GEO