DNA Methylation Is Dispensable for the Growth and Survival of the Extraembryonic Lineages

SummaryDNA methylation regulates development and many epigenetic processes in mammals [1], and it is required for somatic cell growth and survival [2, 3]. In contrast, embryonic stem (ES) cells can self-renew without DNA methylation [4–6]. It remains unclear whether any lineage-committed cells can survive without DNA-methylation machineries. Unlike in somatic cells, DNA methylation is dispensable for imprinting and X-inactivation in the extraembryonic lineages [7–12]. In ES cells, DNA methylation prevents differentiation into the trophectodermal fate [13]. Here, we created triple-knockout (TKO) mouse embryos deficient for the active DNA methyltransferases Dnmt1, Dnmt3a, and Dnmt3b (TKO) by nuclear transfer (NT), and we examined their development. In chimeric TKO-NT and WT embryos, few TKO cells were found in the embryo proper, but they contributed to extraembryonic tissues. TKO ES cells showed increasing cell death during their differentiation into epiblast lineages, but not during differentiation into extraembryonic lineages. Furthermore, we successfully established trophoblastic stem cells (ntTS cells) from TKO-NT blastocysts. These TKO ntTS cells could self-renew, and they retained the fundamental gene expression patterns of stem cells. Our findings indicated that extraembryonic-lineage cells can survive and proliferate in the absence of DNA methyltransferases and that a cell's response to the stress of epigenomic damage is cell type dependent

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

Gata4-binding site and DNA-methylation analyses of Gata4-response genes.

<p>(<i>A</i>) Motif discovery of transcription-factor-binding motifs by the DREME algorithm using all the peaks of the ChIP-seq data for WT or DKO Flk1(+) cells in which Gata4GR was activated by Dex addition. Logos for the most enriched motif identified by DREME and its reverse complement sequence, motif IDs, and E-values are shown. (<i>B,C</i>) Gata4 ChIP-seq enrichment at the Gata4-response gene (<i>B</i>) <i>Aqp8</i> and (<i>C</i>) <i>Sox7</i> loci in WT or DKO Flk1(+) cells in which Gata4GR was activated by Dex, and the DNA methylation state at the transcription-start sites and Gata4-binding sites. Tracks represent the mapped read enrichment as determined by DNAnexus software. Blue arrowheads mark Gata4 peaks enriched in DKO Flk1(+) cells compared to WT Flk1(+) cells. Above the peak profiles, the nucleotide positions and Refseq genes are indicated. Horizontal bars represent the genomic regions subjected to DNA methylation analysis by bisulfite sequencing. Open circles represent unmethylated CpGs, and filled circles represent methylated CpGs. The percentage of total CpGs that were methylated is shown below each bisulfite sequencing profile. “WT”, WT Flk1(+) cells; “DKO”, DKO Flk1(+) cells; “ES”, WT ES cells.</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

Mycophagy among Japanese macaques in Yakushima: fungal species diversity and behavioral patterns.

Mycophagy (fungus-feeding) by Japanese macaques (Macaca fuscata yakui) in Yakushima has been observed by many researchers, but no detailed information is available on this behavior, including which fungal species are consumed. To provide a general description of mycophagy and to understand how and whether macaques avoid poisonous fungi, we conducted behavioral observation of wild Japanese macaques in Yakushima and used molecular techniques to identify fungal species. The results indicate that the diet of the macaques contains a large variety of fungal species (67 possible species in 31 genera), although they compose a very small portion of the total diet (2.2 % of annual feeding time). Fungi which were eaten by macaques immediately after they were picked up were less likely to be poisonous than those which were examined (sniffed, nibbled, carefully handled) by macaques. However, such examining behaviors did not appear to increase the macaques' abilities to detect poisonous fungi. Fungi that were only partially consumed included more poisonous species than those fully consumed with/without examining behavior, yet this was not significant. Taste, therefore, might also play an important role in discriminating poisonous from non-poisonous