Global gene expression analysis of human erythroid progenitors

This article is available open access through the publisher’s website. Copyright @ 2011 American Society of Hematology. This article has an erratum: http://bloodjournal.hematologylibrary.org/content/118/26/6993.3.Understanding the pattern of gene expression during erythropoiesis is crucial for a synthesis of erythroid developmental biology. Here, we isolated 4 distinct populations at successive erythropoietin-dependent stages of erythropoiesis, including the terminal, pyknotic stage. The transcriptome was determined using Affymetrix arrays. First, we demonstrated the importance of using defined cell populations to identify lineage and temporally specific patterns of gene expression. Cells sorted by surface expression profile not only express significantly fewer genes than unsorted cells but also demonstrate significantly greater differences in the expression levels of particular genes between stages than unsorted cells. Second, using standard software, we identified more than 1000 transcripts not previously observed to be differentially expressed during erythroid maturation, 13 of which are highly significantly terminally regulated, including RFXAP and SMARCA4. Third, using matched filtering, we identified 12 transcripts not previously reported to be continuously up-regulated in maturing human primary erythroblasts. Finally, using transcription factor binding site analysis, we identified potential transcription factors that may regulate gene expression during terminal erythropoiesis. Our stringent lists of differentially regulated and continuously expressed transcripts containing many genes with undiscovered functions in erythroblasts are a resource for future functional studies of erythropoiesis. Our Human Erythroid Maturation database is available at https://cellline.molbiol.ox.ac.uk/eryth/index.html.National Health Service Blood and Transplant, National Institute for Health Research Biomedical Research Center Program, and National Institute for Health Research

Uncommon mutations and polymorphisms in the hemochromatosis gene

Hereditary hemochromatosis (HH) is a common autosomal recessive disorder of iron metabolism. Iron absorption from the gut is inappropriately high, resulting in increasing iron overload. The hemochromatosis gene (HFE) was identified in 1996 by extensive positional cloning by many groups over a period of about 20 years. Two missense mutations were identified. Homozygosity for one of these, a substitution of a tyrosine for a conserved cysteine (C282Y), has now clearly been shown to be associated with HH in 60-100% of patients. The role of the second mutation, the substitution of an aspartic acid for a histidine (H63D), is not so clear but compound heterozygotes for both these mutations have a significant risk of developing HH. Here we review other putative mutations in the HFE gene and document a number of diallelic polymorphisms in HFE introns

Distinct Mechanisms of Inadequate Erythropoiesis Induced by Tumor Necrosis Factor Alpha or Malarial Pigment

<div><p>The role of infection in erythropoietic dysfunction is poorly understood. In children with <i>P</i>. <i>falciparum</i> 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-α). To gain insight into the common and specific effects of TNF-α and hemozoin on erythropoiesis, we studied the gene expression profile of purified primary erythroid cultures exposed to either TNF-α (10ng/ml) or to hemozoin (12.5μg/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-α were not affected by hemozoin. The genes modulated by TNF-α 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.</p></div

Hierarchical clustering.

<p>Expression of genes modulated >1.5x by hemozoin in erythroid cells before treatment (E1_d0), after incubation for 24 hours with hemozoin (E1H_d1) or TNF-α (E1T_d1) and without treatment in culture medium (E1m_d1) are shown, together with previously described sorted populations of erythroblasts [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119836#pone.0119836.ref026" target="_blank">26</a>]. Hierarchical clustering by gene and experiment (Pearson correlation) is shown centred around these hemozoin- modulated genes (A) or TNF-α- modulated genes (B). CFUE, colony forming unit erythroid FACsorted; ProE, pro- erythroblast FACsorted; Int, Intermediate erythroblast FACsorted; LateE, Late erythroblast sorted; E1, magnetic bead sorted erythroblasts; m, medium; T, TNF-α; H, hemozoin.</p

Representative example of samples used for microarray and rtPCR.

<p>The purity of erythroblast populations treated with either TNF-α or hemozoin was determined by flow cytometry (A). The forward scatter (FSC) and side scatter (SSC) of live cells is shown in red. The proportion of cells (% of total events acquired) expressing lymphoid (CD3) and erythroid markers (CD71^-CD235a⁺ and CD71⁺CD235a⁺) is represented by values to the right of each plot. The average response to treatment of all samples after 24 hours in which viability was assessed (B) or after 6 days when maturation was assessed by comparing the proportion of committed erythroid progenitors (CD235a⁺) that express low levels of CD71 (CD71^lo) (C). Error bars show standard error of the mean of responses from 4 different donors; ND, not determined due to insufficient cell recovery. *p≤0.05 when compared with media alone (two tailed student’s test).</p

Taqman gene expression assays used for RT- PCR.

<p>The inventoried target sequence and NCBI reference sequence (Ref Seq) for each amplicon is shown.</p><p>Taqman gene expression assays used for RT- PCR.</p

TNF-α induces up-regulation of genes distinct to those induced by hemozoin.

<p>Venn analysis illustrates the overlap in genes up-regulated following treatment of primary erythroid cultures. Primary cultures of erythroblasts were established from peripheral blood and basophilic (CD71⁺CD235a^- and CD71⁺CD235a⁺) erythroblasts isolated as described previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119836#pone.0119836.ref023" target="_blank">23</a>]. Erythroblasts were incubated with 10ng/ml TNF-α or 12.5μg/ml hematin units of hemozoin for 24 hours (A). Pie chart showing the number of up-regulated genes associated with biological processes described per concurrent annotation following treatment with TNF-α. The number of genes with annotations that fall within the hypothetical p values shown are listed, together with the proportion that they represent (B). Confirmation of distinct patterns of up-regulation by TNF-α observed in microarray by rtPCR using TaqMan gene expression assays (C). Fold expression compared with cells cultured in media alone is shown. TNF, TNF-α; Hz, hemozoin. Statistical analysis of rtPCR data was performed using the two tailed student’s t test. ** p<0.05 when compared with media or hemozoin. Fold expression determined from the original array data is shown for comparison.</p

Up-regulated genes induced by hemozoin.

<p>Pie chart showing the number of up-regulated genes associated with biological processes following treatment with hemozoin (A). Confirmation of observations in microarray data by rtPCR using TaqMan gene expression assays (B). Fold expression compared with cells cultured in media alone is shown. TNF, TNF-α; Hz, hemozoin. Statistical analysis of rtPCR data was performed using the two tailed student’s t test. * p≤0.05 when compared with media alone. Fold expression determined from the original array data is shown for comparison.</p