Unknown,Transcriptomics,Genomics,Proteomics

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Genome-wide ChIP-Seq reveals a dramatic shift in the binding of the transcription factor erythroid Kruppel-like factor during erythrocyte differentiation

ABSTRACT: Erythropoiesis is dependent on the activity of transcription factors, including the erythroid-specific erythroid Kruppel-like factor (EKLF). ChIP followed by massively parallel sequencing (ChIP-Seq) is a powerful, unbiased method to map transfactor occupancy. We used ChIP-Seq to study the interactome of EKLF in mouse erythroid progenitor cells and more differentiated erythroblasts. We correlated these results with the nuclear distribution of EKLF, RNA-Seq analysis of the transcriptome, and the occupancy of other erythroid transcription factors. In progenitor cells, EKLF is found predominantly at the periphery of the nucleus, where EKLF primarily occupies the promoter regions of genes and acts as a transcriptional activator. In erythroblasts, EKLF is distributed throughout the nucleus, and erythroblast-specific EKLF occupancy is predominantly in intragenic regions. In progenitor cells, EKLF modulates general cell growth and cell cycle regulatory pathways, whereas in erythroblasts EKLF is associated with repression of these pathways. The EKLF interactome shows very little overlap with the interactomes of GATA1, GATA2, or TAL1, leading to a model in which EKLF directs programs that are independent of those regulated by the GATA factors or TAL1. (Blood.2011;118(17):e139-e148) We used ChIP-Seq to study the interactome of EKLF in mouse erythroid progenitor cells and more differentiated erythroblasts and RNA-Seq analysis of the transcriptome.

ORGANISM(S): Mus musculus

SUBMITTER: Ross Hardison

PROVIDER: E-GEOD-48020 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Genome-wide ChIP-Seq reveals a dramatic shift in the binding of the transcription factor erythroid Kruppel-like factor during erythrocyte differentiation.

Pilon Andre M AM Ajay Subramanian S SS Kumar Swathi Ashok SA Steiner Laurie A LA Cherukuri Praveen F PF Wincovitch Stephen S Anderson Stacie M SM Mullikin James C JC Gallagher Patrick G PG Hardison Ross C RC Margulies Elliott H EH Bodine David M DM

Blood 20110906 17

Erythropoiesis is dependent on the activity of transcription factors, including the erythroid-specific erythroid Kruppel-like factor (EKLF). ChIP followed by massively parallel sequencing (ChIP-Seq) is a powerful, unbiased method to map trans-factor occupancy. We used ChIP-Seq to study the interactome of EKLF in mouse erythroid progenitor cells and more differentiated erythroblasts. We correlated these results with the nuclear distribution of EKLF, RNA-Seq analysis of the transcriptome, and the ...[more]

PMID: 21900194

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Project description:Global, genomic responses of erythrocytes to infectious agents have been difficult to measure, because these cells are e-nucleated. We have previously demonstrated that in vitro matured, nucleated erythroblast cells at the orthochromatic stage can be efficiently infected by the human malaria parasite Plasmodium falciparum. We now show that infection of orthochromatic cells induces change in 609 host genes. 592 of these transcripts are up-regulated and associated with metabolic and chaperone pathways unique to P. falciparum infection, as well as a wide range of signaling pathways that are also induced in related apicomplexan infections of mouse hepatocytes or human fibroblast cells. Our data additionally show that polychromatophilic cells, which precede the orthochromatic stage and are not infected when co-cultured with P. falciparum, up-regulate a small set of 35 genes, 9 of which are associated with pathways of hematopoiesis and/or erythroid cell development. These data unexpectedly predict that blood stage P. falciparum may induce host responses common to infections of other pathogens. Further P. falciparum may modulate gene expression in bystander erythroblasts and thus influence pathways of erythrocyte development. Human primary erythroid cells were differentiated from CD34+ hematopoietic stem cells isolated from growth factor-mobilized peripheral blood (ALL Cells, Inc.). Cells from five donors were cultured until polychromatophilic and orthochromatophilic stages of differentiation and served as uninfected control samples. Of the five donors, three were used to initiate Plasmodium falciparum (3D7) infection at a multiplicity of infection = 5. Infected cells were harvested 24 hours post-infection, and RNA was isolated with Trizol (Invitrogen) and purified with RNeasy columns (QIAGEN) according to manufacturer recommendations. Microarray labeling and hybridizations were done according to Affymetrix protocols using HG U133 plus 2.0 chips. For GenePattern analysis, all samples (5 control and 3 infected samples) were analyzed; for Dchip analysis, only three samples were analyzed (the same 3 donors served as control and infected samples).

Project description:The role of infection in erythropoietic dysfunction is poorly understood. In children with P. falciparum malaria, the by-product of hemoglobin digestion in infected red cells (hemozoin) is associated with the severity of anemia which is independent of circulating levels of the inflammatory cytokine tumor necrosis alpha (TNF-alpha). To gain insight into the common and specific effects of TNF-alpha and hemozoin on erythropoiesis, we studied the gene expression profile of purified primary erythroid cultures exposed to either TNF-alpha (10ng/ml) or to hemozoin (12.5microgram/ml heme units) for 24 hours. Perturbed gene function was assessed using co-annotation of associated gene ontologies and expression of selected genes representative of the profile observed was confirmed by real time PCR (rtPCR). The changes in gene expression induced by each agent were largely distinct; many of the genes significantly modulated by TNF-alpha were not affected by hemozoin. The genes modulated by TNF-alpha were significantly enriched for those encoding proteins involved in the control of type 1 interferon signalling and the immune response to viral infection. In contrast, genes induced by hemozoin were significantly enriched for functional roles in regulation of transcription and apoptosis. Further analyses by rtPCR revealed that hemozoin increases expression of transcription factors that form part of the integrated stress response which is accompanied by reduced expression of genes involved in DNA repair. This study confirms that hemozoin induces cellular stress on erythroblasts that is additional to and distinct from responses to inflammatory cytokines and identifies new genes that may be involved in the pathogenesis of severe malarial anemia. More generally the respective transcription profiles highlight the varied mechanisms through which erythropoiesis may be disrupted during infectious disease. Erythroblasts derived from PBMCs were enriched to 97% purity for proerythroblast and intermediate/normoblast phases of differentiation using magnetic bead isolation. There were three treatment groups: media, P.falciparum hemozoin (12.5 microgram /ml hematin units) and tumour necrosis factor alpha(10ng/ml). Total RNA using 7.5 x105 cells per treatment from one donor was extracted using Trizol. Highly significant gene ontological findings (where p> x10^-17 but < x10^-7) were confirmed by quantitative PCR.

Dataset Information

Genome-wide ChIP-Seq reveals a dramatic shift in the binding of the transcription factor erythroid Kruppel-like factor during erythrocyte differentiation

Publications

Genome-wide ChIP-Seq reveals a dramatic shift in the binding of the transcription factor erythroid Kruppel-like factor during erythrocyte differentiation.

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