Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Genome-scale analysis in blood progenitor and mast cells reveals how lineage-affiliated transcription factors control distinct regulatory programs [Beadchip Experiments]

ABSTRACT: Despite major advances in the generation of genome-wide binding maps, the mechanisms by which transcription factors (TFs) regulate cell type identity have remained largely obscure. Through comparative analysis of 10 key haematopoietic TFs in both mast cells and blood progenitors, we demonstrate that the largely cell-type specific binding profiles are not opportunistic, but instead contribute to cell-type specific transcriptional control, because (1) mathematical modelling of differential binding of shared TFs can explain differential gene expression, (2) cell-type specific binding is largely mediated through consensus binding sites, and (3) knock-down of blood stem cell regulators Gata2 and Erg in mast cells reveals mast cell specific genes as direct targets. Finally we show that the known mast cell regulators Mitf and c-Fos likely contribute to the global reorganisation of TF binding profiles. Taken together therefore, our study elucidates how key regulatory TFs contribute to transcriptional programmes in several distinct mammalian cell types. Total RNA from primary mast cells transfected with retroviral shRNA vectors were compared to negative controls (shRNA against luciferase).

ORGANISM(S): Mus musculus

SUBMITTER: Felicia Ng

PROVIDER: E-GEOD-47988 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Left heart disease (LHD) frequently causes lung vascular remodelling and pulmonary hypertension (PH). Yet, pharmacological treatment for PH in LHD is lacking and its pathophysiological basis remains obscure. We aimed to identify candidate mechanisms of PH in LHD and to test their relevance and therapeutic potential. In rats, LHD was induced by supracoronary aortic banding. Whole genome microarray analyses were performed, candidate genes were confirmed by RT-PCR and Western blots and functional relevance was tested in vivo by genetic and pharmacological strategies. In lungs of LHD rats, mast cell activation was the most prominently upregulated gene ontology cluster. Mast cell gene upregulation was confirmed at RNA and protein levels and remodelled vessels showed perivascular mast cell accumulations. In LHD rats treated with the mast cell stabiliser ketotifen, or in mast cell deficient Ws/Ws rats, PH and vascular remodelling were largely attenuated. Both strategies also reduced PH and vascular remodelling in monocrotaline-induced pulmonary arterial hypertension, suggesting that the role of mast cells extends to noncardiogenic PH. In PH of different aetiologies, mast cells accumulate around pulmonary blood vessels and contribute to vascular remodelling and PH. Mast cells and mast cell-derived mediators may present promising targets for the treatment of PH. Whole rat genome microarray analyses were performed in lung homogenates of three rats with established PH following supracoronary aortic banding and in three sham-operated controls. Out of a total of 28,000 analysed genes, differential expression defined as 2-fold change with p<0.05 was evident for 120 genes. Of these, 76 were upregulated and 44 downregulated in aortic banding compared with control lungs gene. Enrichment analysis revealed regulation of mast cell specific genes - 13 out of 20 genes were significantly upregulated in aortic banding compared with control lungs. To test for a putative functional role of mast cells in lung vascular remodelling and PH in LHD, we applied a pharmacological approach by treatment of aortic banding rats with the mast cell stabiliser ketotifen. Microarray analysis then compared rats that were treated with untreated. In brief, congestive heart failure (CHF) was induced in juvenile rats by supracoronary aortic banding and rats were analyzed 9 weeks thereafter when they had established left heart failure with preserved ejection fraction and secondary pulmonary hypertension. Three heart failure rats were untreated, and 3 received the mast cell stabilizer ketotifen (1 mg/kg-1 bodyweight/day-1) with the drinking water. Sham rats underwent the same surgical procedure but without placement of a clip on the supracoronary aorta. Microarray analyses: Lungs from banded and control rats were excised and total RNA was extracted (Stratagene Absolutely RNA Miniprep Kit; Stratagene, La Jolla, USA). Three Âµg total RNA from three control and three banded rats each were processed according to the One-Cycle Target Labeling protocol (GeneChip Expression Analysis, Affymetrix, Santa Clara, USA). Before and after amplifications, the total RNA/complementary RNA concentrations were checked with NanoDrop ND-1000 (Thermo Scientific, Wilmington, USA) and quality was controlled (Experion electrophoresis station, BioRad; Hercules, USA). Samples were hybridized to GeneChipÂ® Rat Genome 230 2.0 arrays (Affymetrix; Santa Clara, USA) containing 31,000 probe sets covering 28,000 rat genes.

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Genome-scale analysis in blood progenitor and mast cells reveals how lineage-affiliated transcription factors control distinct regulatory programs [Beadchip Experiments]

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