Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of human CD34++ hematopoietic cells reveals vitamin D3/Hoxa10 pathway supports Mafb function during monocyte differentiation

ABSTRACT: Although a considerable number of reports indicate an involvement of the Hox-A10 gene in the molecular control of hematopoiesis, the conclusions of such studies are quite controversial since they support, in some cases, a role in the stimulation of stem cell self-renewal and myeloid progenitor expansion while, in others, implicate this transcription factor in the induction of monocyte - macrophage differentiation. To clarify this issue we analyzed the biological effects and the transcriptome changes determined in human primary CD34+ hematopoietic progenitors by retroviral transduction of a full length Hox-A10 cDNA. The results obtained clearly indicated that this homeogene is an inducer of monocyte differentiation, at least partly acting through the up-regulation of MafB gene, recently identified as master regulator of such maturation pathway. By using a combined approach based on computational analysis, EMSA experiments and luciferase assays, we were able to demonstrate the presence of a Hox-A10 binding site in the promoter region of the MafB gene, which suggested the likely molecular mechanism underlying the observed effect. Interestingly, stimulation of the same cells with the Vitamin D3 monocyte differentiation inducer resulted in a clear increase of Hox-A10 and MafB transcripts, indicating the existence of a precise transactivation cascade involving VDR, Hox-A10 and MafB transcription factors. Altogether these data allow to conclude that the Vitamin D3 / Hox-A10 pathway supports MafB function during the induction of monocyte differentiation. Experiment Overall Design: RNA pools (100 ng) of LXIDN and LHoxA10IDN transduced CD34+ cells, obtained from three independent experiments, were converted in labelled cRNA according with the âTwo cycleâ protocol advised by Affymetrix. cRNA has been used to hybridise Affymetrix HG-U133A GeneChip arrays. Images obtained by scanning chips of LXIDN and LHoxA10IDN transduced CD34+ cells were processed using the GeneChip Operating Software. Microarray analysis of Hox-A10 transduced CD34+ cells provided a substantial contribute for a better comprehension of the biological effects driven by this transcription factor in human primary hematopoietic stem / progenitor cells. Results of this analysis confirmed the stimulatory effect exerted by Hox-A10 on monocytopoiesis, disclosing an up-regulated expression of transcription factors and differentiation markers (CD antigens, granule proteins, cytokines / chemokines) that are typically associated with this maturation lineage. In addition, they also evidenced a decreased expression of genes related to erythroid and granulocyte differentiation programs. This last effect was also confirmed by cytochemical and morphological evaluation of Hox-A10 transduced CD34+ cells.

ORGANISM(S): Homo sapiens

SUBMITTER: CLAUDIA GEMELLI

PROVIDER: E-GEOD-12396 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The occurrence of clonal perturbations and leukemia in patients transplanted with retrovirally-transduced autologous hematopoietic stem and progenitor cells (HSPCs) has stimulated extensive investigation, demonstrating that proviral insertions perturb adjacent proto-oncogene expression. Although enhancer-deleted lentiviruses are less likely to result in insertional oncogenesis, there is evidence that they may perturb transcript splicing, and one patient with a benign clonal expansion of lentivirally-transduced HPSC has been reported. The rhesus macaque model provides an opportunity for informative long-term analysis to ask whether transduction impacts on long-term HSPC properties. We utilized two techniques to examine whether lentivirally-transduced HSPCs from eight rhesus macaques transplanted 1-13.5 years previously are perturbed at a population level, comparing telomere length as a measure of replicative history and gene expression profile of vector positive versus vector negative cells. There were no differences in telomere lengths between sorted GFP+ and GFP- blood cells, suggesting that lentiviral transduction did not globally disrupt replicative patterns. Bone marrow GFP+ and GFP- CD34+ cells showed no differences in gene expression using unsupervised and principal component analysis. These studies did not uncover any global long-term perturbation of proliferation, differentiation, or other important functional parameters of transduced HSPCs in the rhesus macaque model. CD34+ hematopoietic stem and progenitor cells were purified from bone marrow aspirates obtained from 4 rhesus macaques 3-9 years following transplantation with lentivirally-transduced autologous hematopoietic stem and progenitor cells. All lentiviral vectors contained the GFP marker gene. The CD34+ cells from each animal were sorted via flow cytometry into GFP+ and GFP- fractions, and RNA from these cells was used for Affymetrix gene expression analysis as detailed below. There were two samples, GFP+ and GFP-, from each of 4 animals.

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Transcription profiling of human CD34++ hematopoietic cells reveals vitamin D3/Hoxa10 pathway supports Mafb function during monocyte differentiation

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