DNA methyltransferases

DNA methylation regulates development and many epigenetic processes in mammals, and it is required for somatic cell growth and survival. In contrast, embryonic stem (ES) cells can self-renew without DNA methylation. 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. In ES cells, DNA methylation prevents differentiation into the trophectodermal fate. 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.

Abnormal enlargement of the alveolar spaces is a hallmark of conditions such as chronic obstructive pulmonary disease and bronchopulmonary dysplasia. Notch signaling is crucial for differentiation and regeneration and repair of the airway epithelium. However, how Notch influences the alveolar compartment and integrates this process with airway development remains little understood. Here we report a prominent role of Notch signaling in the epithelial–mesenchymal interactions that lead to alveolar formation in the developing lung. We found that alveolar type II cells are major sites of Notch2 activation and show by Notch2-specific epithelial deletion (Notch2cNull) a unique contribution of this receptor to alveologenesis. Epithelial Notch2 was required for type II cell induction of the PDGF-A ligand and subsequent paracrine activation of PDGF receptor-α signaling in alveolar myofibroblast progenitors. Moreover, Notch2 was crucial in maintaining the integrity of the epithelial and smooth muscle layers of the distal conducting airways. Our data suggest that epithelial Notch signaling regulates multiple aspects of postnatal development in the distal lung and may represent a potential target for intervention in pulmonary diseases.

DNA methyltransferase 1 (DNMT1) plays an important role in the inheritance of genomic DNA methylation, which is coupled to the DNA replication process. Early embryonic lethality in DNMT1-null mutant (Dnmt1 c ) mice indicates that DNA methylation is essential for mammalian development. DNMT1, however, interacts with a number of transcriptional regulators and has a transcriptional repressor activity independent of its catalytic activity. To examine the roles of the catalytic activity of DNMT1 in vivo, we generated a Dnmt1 ps allele that expresses a point-mutated protein that lacks catalytic activity (DNMT1-C1229S). Dnmt1 ps mutant mice showed developmental arrest shortly after gastrulation, near-complete loss of DNA methylation, and an altered distribution of repressive chromatin markers in the nuclei; these phenotypes are quite similar to those of the Dnmt1 c mutant. The mutant DNMT1 protein failed to associate with replication foci in Dnmt1 ps cells. Reconstitution experiments and replication labeling in Dnmt1 ؊/؊ Dnmt3a ؊/؊ Dnmt3b ؊/؊ (i.e., unmethylated) embryonic stem cells revealed that preexisting DNA methylation is a major determinant for the cell cycledependent localization of DNMT1. The C-terminal catalytic domain of DNMT1 inhibited its stable association with unmethylated chromatin. Our results reveal essential roles for the DNA methylation mark in mammalian development and in DNMT1 localization.The methylation of DNA at the C-5 position of cytosine residues is a heritable epigenetic mechanism that is involved in a broad range of biological processes in vertebrates, plants, and fungi (3). In mammals, DNA methylation is coordinately regulated by three DNA methyltransferases, DNMT1, DNMT3A, and DNMT3B (6), and plays a crucial role in the regulation of gene expression, the silencing of parasitic elements, genomic imprinting, and embryogenesis (3). The hypermethylation of promoter CpG islands in tumor suppressor genes is a wellrecognized feature of many cancers (28). Mammalian genomes are mostly methylated at symmetrical CpG sites, and the pattern of methylated CpGs is thought to propagate through the copying of a template parental strand (3). After semiconservative DNA replication, hemi-methylated CpG sites are recognized and preferentially methylated at cytosine on the opposite, nascent DNA strand.DNMT1 is the major enzyme responsible for the stable inheritance of DNA methylation patterns after DNA replication (21, 27). Inactivation of DNMT1 results in extensive loss of DNA methylation in the mouse (32, 34). DNMT1 has a strong preference for hemi-methylated DNA as a substrate. The major isoform of mouse DNMT1 is a 1,620-amino-acid protein that has a large N-terminal regulatory domain, which contains several functional subdomains, and a C-terminal catalytic domain, separated by a linker region, a Lys-Gly (KG)repeat. The N-terminal regulatory domain and the C-terminal catalytic domain can interact directly with each other (17). This intramolecular interaction seems to be required for the enzymatic function ...

The periodic cartilage and smooth muscle structures in mammalian trachea are derived from tracheal mesoderm, and tracheal malformations result in serious respiratory defects in neonates. Here we show that canonical Wnt signaling in mesoderm is critical to confer trachea mesenchymal identity in human and mouse. At the initiation of tracheal development, endoderm begins to express Nkx2.1, and then mesoderm expresses the Tbx4 gene. Loss of βcatenin in fetal mouse mesoderm causes loss of Tbx4 + tracheal mesoderm and tracheal cartilage agenesis. The mesenchymal Tbx4 expression relies on endodermal Wnt activation and Wnt ligand secretion but is independent of known Nkx2.1-mediated respiratory development, suggesting that bidirectional Wnt signaling between endoderm and mesoderm promotes trachea development. Activating Wnt, Bmp signaling in mouse embryonic stem cell (ESC)-derived lateral plate mesoderm (LPM) generates tracheal mesoderm containing chondrocytes and smooth muscle cells. For human ESC-derived LPM, SHH activation is required along with WNT to generate proper tracheal mesoderm. Together, these findings may contribute to developing applications for human tracheal tissue repair.