Comparison of DNA methylation profiles in human fetal and adult red blood cell progenitors

Abstract

BackgroundDNA methylation is an epigenetic modification that plays an important role during mammalian development. Around birth in humans, the main site of red blood cell production moves from the fetal liver to the bone marrow. DNA methylation changes at the β-globin locus and a switch from fetal to adult hemoglobin production characterize this transition. Understanding this globin switch may improve the treatment of patients with sickle cell disease and β-thalassemia, two of the most common Mendelian diseases in the world. The goal of our study was to describe and compare the genome-wide patterns of DNA methylation in fetal and adult human erythroblasts.MethodsWe used the Illumina HumanMethylation 450 k BeadChip to measure DNA methylation at 402,819 CpGs in ex vivo-differentiated erythroblasts from 12 fetal liver and 12 bone marrow CD34+ donors.ResultsWe identified 5,937 differentially methylated CpGs that overlap with erythroid enhancers and binding sites for erythropoiesis-related transcription factors. Combining this information with genome-wide association study results, we show that erythroid enhancers define particularly promising genomic regions to identify new genetic variants associated with fetal hemoglobin (HbF) levels in humans. Many differentially methylated CpGs are located near genes with unanticipated roles in red blood cell differentiation and proliferation. For some of these new candidate genes, we confirm the correlation between DNA methylation and gene expression levels in red blood cell progenitors. We also provide evidence that DNA methylation and genetic variation at the β-globin locus independently control globin gene expression in adult erythroblasts.ConclusionsOur DNA methylome maps confirm the widespread dynamic changes in DNA methylation that occur during human erythropoiesis. These changes tend to happen near erythroid enhancers, further highlighting their importance in erythroid regulation and HbF production. Finally, DNA methylation may act independently of the transcription factor BCL11A to repress fetal hemoglobin production. This provides cues on strategies to more efficiently re-activate HbF production in sickle cell disease and β-thalassemia patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-014-0122-2) contains supplementary material, which is available to authorized users.

Tracing human stem cell lineage during development using DNA methylation

“…+ stem/progenitor cells from fresh umbilical cord blood; 3) GSE56491 (Lessard et al 2015), 12 CD34 + cells from fetal liver and 12 from adult bone marrow, which were differentiated ex-vivo to erythroid cells; 4)…”

An erythroid-specific ATP2B4 enhancer mediates red blood cell hydration and malaria susceptibility

“…The cell culture protocol to proliferate and differentiate human CD34 + HSPCs into erythroblasts has been described before (8,24). We purchased human fetal (fetal liver, n = 12) and adult (bone marrow, n = 12) CD34 + HSCs from DV Biologics and Lonza, respectively.…”

“…This sample size was selected, as it provides 90% power to detect a 3 SD difference in geneexpression levels between fetal and adult erythroblasts at α = 1 × 10 -5 . We have also described elsewhere the protocol for RNA extraction and RNA sequencing (RNA-seq) (8). Briefly, we performed RNA-seq with an Illumina HiSeq2000 sequencer using stranded cDNA library and a paired-ends 100-bp protocol.…”

“…All original microarray data were deposited in the NCBI's Gene Expression Omnibus (GEO GSE90878). Genomic DNA extraction, genotyping on the Illumina HumanOmniExpress-12 v1.1 BeadChip array, and quality control were performed as previously described (8). We imputed genotypes using the Michigan Imputation Server with the Haplotype Reference Consortium (HRC) panel (v. r1.1) (27).…”

“…We mapped expression quantitative trait loci (eQTLs) in ex vivo-differentiated human erythroblasts, the nucleated precursors of mature rbc (8). To increase statistical power, we focused the eQTL search on 479 genes that display allelic imbalance (AI) (P < 2 × 10 -5 ) (Methods and Supplemental Table 1; supplemental material available online with this article; https:// doi.org/10.1172/JCI94378DS1).…”