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. 2011 Nov 11;334(6057):799-802.
doi: 10.1126/science.1207306.

Global DNA demethylation during mouse erythropoiesis in vivo

Jeffrey R Shearstone  1 , Ramona Pop, Christoph Bock, Patrick Boyle, Alexander Meissner, Merav Socolovsky
Affiliations

Global DNA demethylation during mouse erythropoiesis in vivo

Jeffrey R Shearstone et al. Science. .

Abstract

In the mammalian genome, 5'-CpG-3' dinucleotides are frequently methylated, correlating with transcriptional silencing. Genome-wide demethylation is thought to occur only twice during development, in primordial germ cells and in the pre-implantation embryo. These demethylation events are followed by de novo methylation, setting up a pattern inherited throughout development and modified only at tissue-specific loci. We studied DNA methylation in differentiating mouse erythroblasts in vivo by using genomic-scale reduced representation bisulfite sequencing (RRBS). Demethylation at the erythroid-specific β-globin locus was coincident with global DNA demethylation at most genomic elements. Global demethylation was continuous throughout differentiation and required rapid DNA replication. Hence, DNA demethylation can occur globally during somatic cell differentiation, providing an experimental model for its study in development and disease.

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Figures

Figure 1
Figure 1. Global DNA demethylation in erythropoiesis
A Erythoid differentiation subsets in freshly isolated fetal liver, defined by the CD71/ Ter119 flow cytometric profile. Box on right shows examples of cytospin cells from each subset, stained with Giemsa (blue) and diaminobenzidine (for hemoglobin, brown). B DNA methylation of individual CpGs (columns) in individual clones (rows) at the H19 DMR, in genomic DNA from sorted S0 and S4/5 subsets. The median clone is indicated (red line). Methylation fell from 61% (S0) to 42% (S4/5, p=0.007, two-tailed Mann-Whitney test). C Methylation at the 5’ tandem repeat of LINE-1 retrotransposons. A genomic map (not to scale) shows tandem repeats in red, and numbered CpGs within one expanded repeat sequence. Data points are methylation level of individual CpGs in specific differentiation subsets within multiple replicate experiments. ***p<0.001, **p<0.01, *p<0.05 (linear mixed model). See also Fig. S2B.
Figure 2
Figure 2. RRBS analysis shows global demethylation
A DNA methylation of 5kb non-overlapping tiles across the genome in increasingly mature S1, S3 and S4/5 erythroblasts plotted against S0 progenitors. Data are mean of 2 experiments, shown for tiles with sufficient RRBS coverage (≥5 CpGs, with ≥5 valid sequencing reads per CpG, Fig. S3A). B Linear correlation across S0 to S4/5 between global methylation at various genomic elements, and methylation at the β-globin LCR. ‘P’, pyrosequencing; ‘R’, RRBS. Data pooled from Figures 1C, 2A, S1C, S1E, S2A, S2B, S3B, S3C. C DNA methylation at 5kb tiles associated with genes that are either upregulated or downregulated with the transition from S1 to S3, by gene expression microarrays. Boxplots correspond to center quartiles, with indicated median (black bar). Whiskers extend to the most extreme data point, which is no more than 1.5 times the interquartile range from the box.
Figure 3
Figure 3. Decreased Dnmt3 expression with differentiation
Western blotting for Dnmts in freshly sorted erythroid subsets (left panels), with corresponding quantitation (right panels). An equal mass of cell lysate was loaded in each lane. Phoenix cells (derived from Large T antigen-transformed human embryonic kidney ‘293T’ cells), were either untransfected (Ctrl) or transduced with vectors encoding Dnmt3a1, Dnmt3a2, Dnmt3b1, or Dnmt3b3. Representative of two independent experiments.
Figure 4
Figure 4. Global demethylation is dependent on rapid DNA replication
A Cell number in S phase (%) in subsets S0 to S4/5, and corresponding intra-S phase DNA synthesis rate as measured by BrdU Median Fluorscence Intensity (MFI) in the S phase gate. Pregnant mice were pulsed with BrdU 30' prior to harvest of fetal livers. Data in lower panels are mean ± s.e.m of 8 -15 replicates. B Global methylation (RRBS, 5kb tiles, lower panels) of S1 cells following differentiation in Epo ± Aphi (0.1 μM) for t=24 hr, compared with their methylation levels at the time of sorting (t=0). Data are mean of duplicate experiments with sufficient RRBS coverage. Upper panel: a representative cell cycle analysis of S1 cells cultured in Epo ± Aphi (0.1 μM, 16hr) showing the fraction of cells in S phase (%) and intra-S phase DNA synthesis (BrdU MFI). See also Fig. S9C, D.
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