Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Downstream Targets of HOXB4 in the primitive hematopoietic progenitor EML Cell line

ABSTRACT: 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.

ORGANISM(S): Mus musculus

SUBMITTER: Vince Schulz

PROVIDER: E-GEOD-20604 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Downstream targets of HOXB4 in a cell line model of primitive hematopoietic progenitor cells.

Lee Han M HM Zhang Hui H Schulz Vincent V Tuck David P DP Forget Bernard G BG

Blood 20100419 5

Enforced expression of the homeobox transcription factor HOXB4 has been shown to enhance hematopoietic stem cell self-renewal and expansion ex vivo and in vivo. To investigate the downstream targets of HOXB4 in hematopoietic progenitor cells, HOXB4 was constitutively overexpressed in the primitive hematopoietic progenitor cell line EML. Two genome-wide analytical techniques were used: RNA expression profiling using microarrays and chromatin immunoprecipitation (ChIP)-chip. RNA expression profili ...[more]

PMID: 20404135

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Project description:Most adult patients have a D816V mutation in phosphotransferase domain (PTD), we have described that half of the children carry mutations in extracellular domain (ECD). KIT-ECD versus IT-PTD-mutants were introduced into rodent Ba/F3, EML, Rat2 and human TF1 cells to investigate their biological effect. ECD- and PTD-mutants also displayed distinct whole-genome transcriptional profiles in EML cells. We observed differences in their signaling properties: they both activated STAT pathways, whereas AKT pathway was only activated by ECD-mutants. Consistently, AKT inhibitor suppressed ECD-mutant-dependent proliferation, clonogenicity and erythroid differentiation. Expression of myristoylated AKT restored erythroid differentiation in EMLPTD cells, suggesting the differential role of AKT in those mutants. Overall, our study implied different pathogenesis of pediatric versus adult mastocytosis, which might explain their diverse phenotypes. Each EML cell line (Del417-419insY mutant and D816V mutant) was cultured with or without 250 ng/mL SCF for 48h. Gene expression profile analysis was performed using whole-genome microarrays. RNA expression profiling of cell lines was done with Affymetrix M430 2.0 mouse oligonucleotide microarrays containing 45.101 probe sets, representing 21.408 transcripts and variants including 17.482 well-characterized mouse genes. Preparation of cRNA, hybridizations, washes, detection and quantification were done as recommended by the supplier. For each sample, synthesis of the first-strand cDNA was done from 3 μg total RNA by T7-oligo(dT) priming, followed by second-strand cDNA synthesis. After purification, in vitro transcription associated with amplification generated cRNA-containing biotinylated pseudouridine. Biotinylated cRNA was purified, quantified and chemically fragmented (95°C for 35 min), then hybridized to microarrays in 200 μL hybridization buffer at 45°C for 16 h. Automated washes and staining with streptavidin-phycoerythrin were done as recommended. Signal amplification was done by biotinylated antistreptavidin antibody with goat-IgG blocking antibody. Scanning was done with Affymetrix GeneArray scanner and quantification with Affymetrix GCOS software.

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

Downstream Targets of HOXB4 in the primitive hematopoietic progenitor EML Cell line

Publications

Downstream targets of HOXB4 in a cell line model of primitive hematopoietic progenitor cells.

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