Unknown,Transcriptomics,Genomics,Proteomics

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High resolution methylome analysis reveals widespread functional hypomethylation during adult human erythropoiesis

ABSTRACT: Differentiation of hematopoietic stem cells to red cells requires coordinated expression of numerous erythroid genes. To understand the regulatory mechanisms governing lineage commitment we conducted a high resolution methylomic and transcriptomic analysis of six major stages of human erythroid differentiation. We observed widespread epigenetic differences between early and late stages of erythropoiesis with progressive loss of methylation being the dominant change during differentiation. Gene bodies, intergenic regions and CpG shores were preferentially demethylated during erythropoiesis. Epigenetic changes at transcription factor binding sites correlated significantly with changes in gene expression and were enriched for binding motifs for SCL, MYB, GATA and other factors not previously implicated in erythropoiesis. Demethylation at gene promoters was associated with increased expression of genes, while epigenetic changes at gene bodies correlated inversely with gene expression. Important gene networks encoding erythrocyte membrane proteins, surface receptors and heme synthesis proteins were found to be regulated by DNA methylation. Furthermore, integrative analysis enabled us to identify novel, potential regulatory areas of the genome that underwent differential methylation and correlated with corresponding changes in gene expression, as evident by epigenetic changes in a predicted PU.1 binding site in intron 1 of the GATA1 gene. This intronic site was found to be conserved across species and was validated to be a novel PU.1 binding site by qCHIP in erythroid cells. Altogether, our study provides a comprehensive analysis of methylomic and transcriptomic changes during erythroid differentiation and demonstrates that hypomethylation of the genome during erythroid differentiation has implications in modulating gene expression. We examined changes in methylation on days 0, 3, 7, 10, 13 and 16 of human erythroid culture. Two independent sets of experiments were performed.

ORGANISM(S): Homo sapiens

SUBMITTER: Yiting Yu

PROVIDER: E-GEOD-44054 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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High resolution methylome analysis reveals widespread functional hypomethylation during adult human erythropoiesis.

Yu Yiting Y Mo Yongkai Y Ebenezer David D Bhattacharyya Sanchari S Liu Hui H Sundaravel Sriram S Giricz Orsolya O Wontakal Sandeep S Cartier Jessy J Caces Bennett B Artz Andrew A Nischal Sangeeta S Bhagat Tushar T Bathon Kathleen K Maqbool Shahina S Gligich Oleg O Suzuki Masako M Steidl Ulrich U Godley Lucy L Skoultchi Art A Greally John J Wickrema Amittha A Verma Amit A

The Journal of biological chemistry 20130110 13

Differentiation of hematopoietic stem cells to red cells requires coordinated expression of numerous erythroid genes and is characterized by nuclear condensation and extrusion during terminal development. To understand the regulatory mechanisms governing these widespread phenotypic changes, we conducted a high resolution methylomic and transcriptomic analysis of six major stages of human erythroid differentiation. We observed widespread epigenetic differences between early and late stages of ery ...[more]

PMID: 23306203

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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).

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High resolution methylome analysis reveals widespread functional hypomethylation during adult human erythropoiesis

Publications

High resolution methylome analysis reveals widespread functional hypomethylation during adult human erythropoiesis.

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