HOXB4 expression in iRUNX embryoid bodies
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE103627 | GEO | 2018/02/15
REPOSITORIES: GEO
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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: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:Efficient in vitro generation of hematopoietic stem cells (HSCs) from embryonic stem cells (ESCs) holds great promise for cell-based therapies of hematological diseases. To date, HoxB4 remains to be the most effective transcription factor (TF) whose over-expression in ESCs confers long-term repopulating ability to ESC-derived HSCs. Despite its importance, the components and dynamics of the HoxB4 transcriptional regulatory network is poorly understood, hindering efforts to develop a more efficient protocol for in vitro derivation of HSCs. Towards this goal, we performed global gene expression profiling and chromatin immunoprecipitation coupled with deep sequencing (ChIP-Seq) at four stages of the HoxB4-mediated HSC development. Joint analyses of ChIP-Seq and gene expression profiles unveil a number of global features of the HoxB4 regulatory network.
2012-03-23 | GSE34013 | GEO
Project description:Efficient in vitro generation of hematopoietic stem cells (HSCs) from embryonic stem cells (ESCs) holds great promise for cell-based therapies of hematological diseases. To date, HoxB4 remains to be the most effective transcription factor (TF) whose over-expression in ESCs confers long-term repopulating ability to ESC-derived HSCs. Despite its importance, the components and dynamics of the HoxB4 transcriptional regulatory network is poorly understood, hindering efforts to develop a more efficient protocol for in vitro derivation of HSCs. Towards this goal, we performed global gene expression profiling and chromatin immunoprecipitation coupled with deep sequencing (ChIP-Seq) at four stages of the HoxB4-mediated HSC development. Joint analyses of ChIP-Seq and gene expression profiles unveil a number of global features of the HoxB4 regulatory network.
2012-03-23 | GSE33953 | GEO
Project description:Efficient in vitro generation of hematopoietic stem cells (HSCs) from embryonic stem cells (ESCs) holds great promise for cell-based therapies of hematological diseases. To date, HoxB4 remains to be the most effective transcription factor (TF) whose over-expression in ESCs confers long-term repopulating ability to ESC-derived HSCs. Despite its importance, the components and dynamics of the HoxB4 transcriptional regulatory network is poorly understood, hindering efforts to develop a more efficient protocol for in vitro derivation of HSCs. Towards this goal, we performed global gene expression profiling and chromatin immunoprecipitation coupled with deep sequencing (ChIP-Seq) at four stages of the HoxB4-mediated HSC development. Joint analyses of ChIP-Seq and gene expression profiles unveil a number of global features of the HoxB4 regulatory network. Cells from three time points of the developmental processes were collected, including days 6, 16, and 26. Deep sequencing were done for both IP and input DNA from cells from each time point.
2012-03-23 | E-GEOD-34013 | biostudies-arrayexpress
Project description:Efficient in vitro generation of hematopoietic stem cells (HSCs) from embryonic stem cells (ESCs) holds great promise for cell-based therapies of hematological diseases. To date, HoxB4 remains to be the most effective transcription factor (TF) whose over-expression in ESCs confers long-term repopulating ability to ESC-derived HSCs. Despite its importance, the components and dynamics of the HoxB4 transcriptional regulatory network is poorly understood, hindering efforts to develop a more efficient protocol for in vitro derivation of HSCs. Towards this goal, we performed global gene expression profiling and chromatin immunoprecipitation coupled with deep sequencing (ChIP-Seq) at four stages of the HoxB4-mediated HSC development. Joint analyses of ChIP-Seq and gene expression profiles unveil a number of global features of the HoxB4 regulatory network. Cells from four time points of the developmental processes were collected, including days 0, 6, 16, and 26. Triplicate microarray hybridizations were done for cells from each time point.
2012-03-23 | E-GEOD-33953 | 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:Haematopoietic differentiation of embryonic stem (ES) cells in vitro has been used as a model to study early haematopoietic development and it is well documented that haematopoietic differentiation can be enhanced by over-expression of HOXB4. HOXB4 is expressed in haematopoietic progenitor cells (HPC) where it promotes self-renewal, but it is also expressed in the primitive streak of the gastrulating embryo. This led us to hypothesise that HOXB4 might modulate gene expression in pre- haematopoietic mesoderm and that this property might contribute to its pro-haematopoietic effect in differentiating ES cells. To test our hypothesis we developed a conditionally activated HOXB4 expression system using the mutant oestrogen receptor (ERT2) and showed that a pulse of HOXB4 prior to HPC emergence in differentiating ES cells led to an increase in haematopoietic differentiation. Expression profiling revealed an increase in the expression of genes associated with paraxial mesoderm that gives rise to the haematopoietic niche. Cell mixing experiments suggest that HOXB4 activation can indeed have a paracrine, as well as a cell autonomous, effect on haematopoietic differentiation. We provide evidence that this may, in part, be mediated by modulation of the Wnt pathway via the HOXB4 target gene, Frzb. Constructs: HOXB4-ERT2 constructs were generated that utilised the CAG promoter to drive expression of the Hoxb4ERT2 fusion cDNA followed by an IRES element and the puromycin resistance gene (pCAG Hoxb4ERT2IP); CGR8.5 cells bearing Hoxb4VP16 fusion construct. Fusion of HOXB4-ERT2 with the transactivation domain of the Herpes simplex VP16 (Brickman et al. 2000) (Zamparini et al. 2006) was generated by PCR as were 2 mutants: Hoxb4 (N266P)VP16/Pcs2+ and Hoxb4 (d266)VP16/Pcs2+ which had mutations in an asparagine (residue 266) in the third helix of the homeodomain which functions in DNA recognition (Hanes and Brent 1989).
2011-02-05 | E-MTAB-547 | biostudies-arrayexpress
Project description:Oncogenic transformation in Ewing sarcoma tumors is driven by the fusion oncogene EWS-FLI1. The inducible expression of EWS-FLI1 (EF) in embryoid bodies, or collections of differentiating stem cells, generates cells with properties of Ewing sarcoma tumors, including characteristics of transformation. These cell lines exhibit anchorage-independent growth, a lack of contact inhibition and a strong Ewing sarcoma gene expression signature. These cells also demonstrate a requirement for the persistent expression of EWS-FLI1 for cell survival and growth. We used microarrays to detail the effects of doxycycline-inducible expression of EWS-FLI1 on the gene expression signature of cells derived from differentiating stem cells, or embryoid bodies. Triplicate biological replicates were collected and analyzed.
2015-12-31 | E-GEOD-64686 | biostudies-arrayexpress
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