Dataset Information

HOXB4 target genes in ES cell-derived embryoid bodies (EBs)

ABSTRACT: 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)

ORGANISM(S): Mus musculus

PROVIDER: GSE9044 | GEO | 2007/09/19

SECONDARY ACCESSION(S): PRJNA102549

REPOSITORIES: GEO

ACCESS DATA

Similar Datasets

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 Affymetrixâ¢ platform. As a control, profiling was also performed with LSK cells expressing unmodified constitutively active HOXB4 (HOXB4const) Â± 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.

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)

Dataset Information

HOXB4 target genes in ES cell-derived embryoid bodies (EBs)

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure