Regulation of Lineage Specific DNA Hypomethylation in Mouse Trophectoderm

<div><p>Background</p><p>DNA methylation is reprogrammed during early embryogenesis by active and passive mechanisms in advance of the first differentiation event producing the embryonic and extraembryonic lineage cells which contribute to the future embryo proper and to the placenta respectively. Embryonic lineage cells re-acquire a highly methylated genome dependent on the DNA methyltransferases (DNMTs) Dnmt3a and Dnmt3b that are required for <i>de novo</i> methylation. By contrast, extraembryonic lineage cells remain globally hypomethylated but the mechanisms that underlie this hypomethylation remain unknown.</p> <p>Methodology/Principal Findings</p><p>We have employed an inducible system that supports differentiation between these two lineages and recapitulates the DNA methylation asymmetry generated <i>in vivo</i>. We find that <i>in vitro</i> down-regulation of <i>Oct3/4</i> in ES cells recapitulates the decline in global DNA methylation associated with trophoblast. The <i>de novo</i> DNMTs Dnmt3a2 and Dnmt3b are down-regulated during trophoblast differentiation. Dnmt1, which is responsible for maintenance methylation, is expressed comparably in embryonic and trophoblast lineages, however importantly in trophoblast giant cells Dnmt1fails to be attracted to replication foci, thus allowing loss of DNA methylation while implicating a passive demethylation mechanism. Interestingly, Dnmt1 localization was restored by exogenous Np95/Uhrf1, a Dnmt1 chaperone required for Dnmt1-targeting to replication foci, yet DNA methylation levels remained low. Over-expression of <i>de novo</i> DNMTs also failed to increase DNA methylation in target sequences.</p> <p>Conclusions/Significance</p><p>We propose that induced trophoblast cells may have a mechanism to resist genome-wide increases of DNA methylation, thus reinforcing the genome-wide epigenetic distinctions between the embryonic and extraembryonic lineages in the mouse. This resistance may be based on transcription factors or on global differences in chromatin structure.</p> </div

Differential expression of DNA methyltransferases in the first two lineages.

<p>mRNA expression of DNMTs and Np95 in E9.5 conceptus (Em: embryo proper, Tr: trophoblast cells), ZHBTc4 ES cells (ES), ZHBTc4-derived trophoblast cells (+Dx) and ZHBTc4-derived embryonic cells (-Lf). The value of ES cell is set to 1.0. Values are means ± SD of biological replicates (n=3-4). ***: <i>p</i><0.001, **: <i>p</i><0.01, *: <i>p</i><0.05, ns: not significant; Mann-Whitney and ANOVA followed by Tukey HSD post-hoc tests when appropriate. The data for +Dx and –Lf were collected at day 2 for <i>Dnmt3a2</i> and at day 4 for <i>Dnmt3a1</i>, <i>Dnmt3b</i>, <i>Dnmt1</i> and <i>Np95</i>. Data for marker gene expression and the time course experiments are shown in supporting information (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068846#pone.0068846.s003" target="_blank">Figure S3</a>).</p

Asymmetric DNA methylation in the first two lineages.

<p>(A) The percentage of CpG methylation analyzed by Sequenom which averages the methylation of CpG methylation across each region. Embryo proper (Em) and trophoblast cells (Tr) are from E9.5 conceptus. ZHBTc4 ES-derived trophoblast cells (+Dx) and embryonic cells (-Lf) are differentiated by addition of doxycycline or removal of LIF respectively. Genomic DNA was collected at day 4 after differentiation. Values are means ± standard deviation (SD) of biological replicates (n=3-4). ***: p<0.001, **: <i>p</i><0.01, *: <i>p</i><0.05, ns: not significant; t-test and ANOVA followed by Tukey HSD post-hoc tests when appropriate. (B) DNA methylation of minor satellite and MMLV analyzed by southern blotting. Genomic DNA was collected from undifferentiated ES cells (0), trophoblast cells day 4 (4) or day 9 (9) after doxycycline treatment (+Dx), and embryonic cells day 5 (5) after the removal of LIF (-Lf). Genomic DNA was digested with methylation sensitive restriction enzyme HpaII and analyzed with each probe. Digestion with methylation-insensitive restriction enzyme MspI (M) is a control.</p

Overexpression of the Np95 gene fails to restore somatic levels of DNA methylation in trophoblast differentiation.

<p>(A) Immunostaining analysis of ZHBTc4-derived trophoblast cells (+Dx), Np95-KO ES cells, ZHBTc4 ES cells overexpressing exogenous Np95 (+MycNp95 ES) and ZHBTc4-derived trophoblast cells overexpressing exogenous Np95 (+MycNp95+Dx) using antibodies against Dnmt1, Np95 or Myc. DNA and replication sites were visualized with DAPI and EdU respectively. Scale bar, 10 µm. (B) mRNA expression of <i>Np95</i>, <i>Oct3/4</i>, <i>Rhox6</i> and Plate <i>1</i> genes during ZHBTc4 trophoblast differentiation with (+Np95) or without (ZH) overexpression of exogenous Np95. A representative clone for Np95-overexpressing ZHBTc4 cells is shown in the figure. Values are means ± SD of technical replicates (n=3). Other independent clones also showed similar results for marker gene and Np95 expression. (C) Dnmt1 localization at replication site in Np95-overexpressing ZHBTc4-derived trophoblast cells at day 4 after differentiation. The control data in ZHBTc4-derived trophoblast cells (ZH+Dx) is identical to ZHBTc4+Dx of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068846#pone-0068846-g004" target="_blank">Figure 4C</a>. Values are means ± SD of biological replicates (n=3). ***: <i>p</i><0.001, **: <i>p</i><0.01, ns: not significant; ANOVA and Bonferroni’s multiple comparison test. (D) DNA methylation analysis by Sequenom in ZHBTc4 ES (Day0) and ZHBTc4-derived trophoblast cells (Day4+Dx) with (+Np95) or without (ZH) overexpression of exogenous Np95. The control data (+emp) is identical to the data of +emp in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068846#pone-0068846-g003" target="_blank">Figure 3B</a>. Values are means ± SD of biological replicates (n=3-5). ***: <i>p</i><0.001, **: <i>p</i><0.01; paired t-tests.</p

Overexpression of the Dnmt3 genes fail to restore somatic level of DNA methylation in trophoblast differentiation.

<p>(A) mRNA expression of <i>Dnmt3</i> genes, <i>Oct3/4</i>, <i>Rhox6</i> and Plate <i>1</i> during ZHBTc4 trophoblast differentiation with/without overexpression of exogenous Dnmt3a1 (3a1), Dnmt3a2 (3a2) or Dnmt3b (3b). A representative clone from each group of Dnmt3-expressing stable clone is shown in the figure. Values are means ± SD of technical replicates (n=3). Other clones in each group showed similar levels of expression of Dnmt3 genes and marker genes. (B) DNA methylation analysis by Sequenom in ZHBTc4 ES cells and trophoblast cells with/without overexpression of exogenous Dnmt3a1 (3a1), Dnmt3a2 (3a2) or Dnmt3b (3b). emp: empty vector. A gray column indicates ES cell data (Day 0). A black column indicates data from trophoblast cells induced by the addition of doxycycline (Day4+Dx). Values are means ± SD of biological replicates (n=3-5) except for the value of major satellite for empty vector day 4 whose value is shown as the mean of biological duplicate. So, there is no stats for the value of major satellite at day 4. ****: p<0.0001, ***: <i>p</i><0.001, **: <i>p</i><0.01, *: <i>p</i><0.05, ns: not significant; paired t-tests.</p

DNA Methylation Restricts Lineage-specific Functions of Transcription Factor Gata4 during Embryonic Stem Cell Differentiation

<div><p>DNA methylation changes dynamically during development and is essential for embryogenesis in mammals. However, how DNA methylation affects developmental gene expression and cell differentiation remains elusive. During embryogenesis, many key transcription factors are used repeatedly, triggering different outcomes depending on the cell type and developmental stage. Here, we report that DNA methylation modulates transcription-factor output in the context of cell differentiation. Using a drug-inducible Gata4 system and a mouse embryonic stem (ES) cell model of mesoderm differentiation, we examined the cellular response to Gata4 in ES and mesoderm cells. The activation of Gata4 in ES cells is known to drive their differentiation to endoderm. We show that the differentiation of wild-type ES cells into mesoderm blocks their Gata4-induced endoderm differentiation, while mesoderm cells derived from ES cells that are deficient in the DNA methyltransferases Dnmt3a and Dnmt3b can retain their response to Gata4, allowing lineage conversion from mesoderm cells to endoderm. Transcriptome analysis of the cells' response to Gata4 over time revealed groups of endoderm and mesoderm developmental genes whose expression was induced by Gata4 only when DNA methylation was lost, suggesting that DNA methylation restricts the ability of these genes to respond to Gata4, rather than controlling their transcription <i>per se</i>. Gata4-binding-site profiles and DNA methylation analyses suggested that DNA methylation modulates the Gata4 response through diverse mechanisms. Our data indicate that epigenetic regulation by DNA methylation functions as a heritable safeguard to prevent transcription factors from activating inappropriate downstream genes, thereby contributing to the restriction of the differentiation potential of somatic cells.</p></div

Gata4-induced primitive endoderm differentiation from <i>Dnmt3a</i>^−/−<i>Dnmt3b</i>^−/− (DKO) Flk1(+) mesoderm cells derived from OP9 co-culture conditions.

<p>(<i>A</i>) Experimental strategy for isolating mesoderm progenitors from ES cells and the subsequent activation of Gata4. Wild-type (WT) or DKO ES cells stably expressing Gata4GR were differentiated on OP9 stromal cells for 4 days. The Flk1(+) mesoderm cells (Me) were sorted and cultured on type IV collagen with or without dexamethasone (Dex) to activate Gata4GR. ES cells were also directly differentiated into primitive endoderm (PE) by adding Dex to ES maintenance medium containing LIF. (<i>B</i>) Flow cytometry profiles of Flk1 and E-cadherin in ES cells differentiated on OP9 stromal cells. The percentages of Flk1(+)/E-cadherin(−) cells are indicated. (<i>C</i>) Phase-contrast photomicrographs of differentiated Flk1(+) mesoderm cells. WT or DKO Flk1(+) cells were cultured with or without Dex for 4 days. (<i>D</i>, <i>E</i>) Immunofluorescence analysis of the mural cell marker SMA (<i>D</i>) or endoderm marker Dab2 (<i>E</i>) (green) in WT or DKO Flk1(+) mesoderm cells cultured for 4 days with or without Dex. DNA was stained with Hoechst 33342 (blue). All experiments were performed three times. Scale bar, 50 µm.</p

Time course analysis of transcriptome changes in response to Gata4.

<p>(<i>A</i>) Cluster heat map representing the temporal transcriptional changes for Gata4 response genes in Flk1(+) mesoderm or ES cells. WT or DKO Flk1(+) mesoderm cells or ES cells expressing Gata4GR were cultured for 72 hr in the presence or absence of Dex, and expression microarray data were obtained at several time points (0, 12, 24, 36, 48, and 72 hr for Flk1(+) mesoderm cells; 0, 3, 6, 12, 24, 48, and 72 hr for ES cells). Ninety-four genes whose responses to Gata4 were higher in DKO than WT Flk1(+) mesoderm cells at 24 hr were extracted as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003574#pgen-1003574-g002" target="_blank">Figure 2E</a>. The clustering of these 94 genes was based on their temporal expression profiles in Flk1(+) mesoderm and ES cells, and the resulting dendrogram is shown at left. Genes used in (<i>B</i>) and (<i>C</i>) are highlighted as blue circles and red triangles, respectively. Relative gene expression values (log2) are represented as colors, from lowest (blue) to highest (yellow). (<i>B</i>, <i>C</i>) Line graphs of the gene expression values (linear) from the microarray data. The mean values of triplicates (Flk1+, 0 hr and Dex+) or duplicates (others) with their standard deviations are shown. (<i>B</i>) Ectopic expression of endoderm genes in response to Gata4 in DKO mesoderm cells. These genes responded to Gata4 in WT and DKO ES cells, but not in WT mesoderm cells. Note that smaller scales are used for the expression signal for Flk1(+) mesoderm cells compared to those for ES cells. (<i>C</i>) Precocious expression of cardiac genes in response to Gata4 in DKO mesoderm cells. These genes did not respond to Gata4 in WT or DKO ES cells. “WT+Gata4GR Dex+” and “WT+Gata4GR Dex−”, WT cells expressing Gata4GR with and without Dex, respectively; “DKO+Gata4GR Dex+” and “DKO+Gata4GR Dex−”, DKO cells expressing Gata4GR with and without Dex, respectively.</p

Transcriptome analysis of Gata4-induced DKO Flk1(+) mesoderm cells.

<p>(<i>A</i>) Experimental scheme to examine transcriptome changes in response to Gata4 in mesoderm or ES cells. WT or DKO ES cells stably expressing Gata4GR were differentiated on OP9 stromal cells for 4 days, then the Flk1(+) mesoderm cells (Me) were sorted and cultured with or without Dex to activate Gata4GR (top). The same WT and DKO ES cells were also cultured with Dex in ES culture conditions (bottom). The expression microarray data were obtained at several time points (up to 72 hr) after Gata4GR activation in both mesoderm and ES cells. (<i>B</i>) Numbers of genes with a more than 4-fold difference between the indicated cell conditions 72 hr after Gata4GR activation by the addition of Dex in ES or Flk1(+) mesoderm cells. In each comparison, ‘<i>up</i>’ represents genes expressed higher in Cell-2 than Cell-1, and ‘<i>down</i>’ represents genes expressed lower in Cell-2 than Cell-1. Representative gene ontology (GO) terms at Biological Process level 4 (BP4) for differentially expressed genes in Flk1(+) mesoderm cells are shown in the right column. (<i>C</i>) Venn diagram representing the overlap between (i) the genes expressed 2-fold higher in DKO Flk1(+) mesoderm with Dex at 72 hr than WT cells under the same conditions (WT Dex+Tables S1 and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003574#pgen.1003574.s014" target="_blank">S2</a>. (<i>D</i>) Extraction of Gata4-hyper-responsive genes in Dnmt3a/Dnmt3b-deficient Flk1(+) mesoderm cells from transcriptome data. Venn diagrams of the 2-fold upregulated genes in DKO mesoderm with Gata4 activation at 72 hr compared to (i) WT cells under the same conditions (WT Dex+E) Venn diagram of the Gata4-responsive genes in DNA-hypomethylated mesoderm cells at 72 hr and 24 hr identified in (<i>D</i>) and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003574#pgen.1003574.s005" target="_blank">Figure S5</a>. The 94 overlapping genes were used for the analysis in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003574#pgen-1003574-g003" target="_blank">Figure 3</a>, while the 320 genes at 72 hr were used in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003574#pgen.1003574.s006" target="_blank">Figure S6</a>.</p

Immediate response to Gata4 in DKO Flk1(+) mesoderm cells.

<p>ES cells differentiated on OP9 stromal cells were treated with Dex for 0, 1, 2 or 3 hr. The Flk1(+) mesoderm cells were then sorted by flow cytometry, and their gene expression was analyzed using microarrays. The mean values of duplicates for gene expression values (linear) with their standard deviations are shown. (<i>A</i>) Endodermal Gata4-responsive genes. (<i>B</i>) Cardiac Gata4-responsive genes. “WT+Gata4GR Dex+”, WT cells expressing Gata4GR with Dex; “DKO+Gata4GR Dex+”, DKO cells expressing Gata4GR with Dex; “DKO Dex+”, DKO cells without the Gata4GR transgene with Dex.</p