Bisulfite sequencing, a standard method for DNA methylation profile analysis, is widely used in basic and clinical studies. This method is limited, however, by the time-consuming data analysis processes required to obtain accurate DNA methylation profiles from the raw sequence output of the DNA sequencer, and by the fact that quality checking of the results can be influenced by a researcher's bias. We have developed an interactive and easy-to-use web-based tool, QUMA (quantification tool for methylation analysis), for the bisulfite sequencing analysis of CpG methylation. QUMA includes most of the data-processing functions necessary for the analysis of bisulfite sequences. It also provides a platform for consistent quality control of the analysis. The QUMA web server is available at http://quma.cdb.riken.jp/.

In the brain, enormous numbers of neurons have functional individuality and distinct circuit specificities. Clustered Protocadherins (Pcdhs), diversified cell-surface proteins, are stochastically expressed by alternative promoter choice and affect dendritic arborization in individual neurons. Here we found that the Pcdh promoters are differentially methylated by the de novo DNA methyltransferase Dnmt3b during early embryogenesis. To determine this methylation's role in neurons, we produced chimeric mice from Dnmt3b-deficient induced pluripotent stem cells (iPSCs). Single-cell expression analysis revealed that individual Dnmt3b-deficient Purkinje cells expressed increased numbers of Pcdh isoforms; in vivo, they exhibited abnormal dendritic arborization. These results indicate that DNA methylation by Dnmt3b at early embryonic stages regulates the probability of expression for the stochastically expressed Pcdh isoforms. They also suggest a mechanism for a rare human recessive disease, the ICF (Immunodeficiency, Centromere instability, and Facial anomalies) syndrome, which is caused by Dnmt3b mutations.

DNA methylation is a major epigenetic mechanism that has been suggested to control developmental gene regulation during embryogenesis, but its regulatory mechanisms remain unclear. In this report, we show that CpG islands associated with the X-linked homeobox gene cluster Rhox, which is highly expressed in the extraembryonic trophectoderm, are differentially methylated in a stage-and lineage-specific manner during the post-implantation development of mice. Inactivation of both Dnmt3a and Dnmt3b, DNA methyltransferases essential for the initiation of de novo DNA methylation, abolished the establishment of DNA methylation and the silencing of Rhox cluster genes in the embryo proper. The Dnmt3-dependent CpG-island methylation at the Rhox locus extended for a large genomic region (∼1 Mb) containing the Rhox cluster and surrounding genes. Complementation experiments using embryonic stem (ES) cells deficient in the DNA methyltransferases suggested that the CpG-island methylation by Dnmt3a and Dnmt3b was restricted within this large genomic region, and did not affect the neighboring genes outside it, implicating the existence of region-specific boundaries. Our results suggest that DNA methylation plays important roles in both long-range gene silencing and lineage-specific silencing in embryogenesis.[Keywords: DNA methylation; Dnmt3a; Dnmt3b; Rhox] Supplemental material is available at http://www.genesdev.org.

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.

Protective activities of the filamentous hemagglutinin (FHA) and the lymphocytosis-promoting factor (LPF) of Bordetella pertussis were compared by active and passive protection tests with intracerebral or respiratory challenge in mice. Mice immunized twice by intraperitoneal injection of 8 micrograms of FHA or glutaraldehyde-inactivated LPF were protected after aerosol challenge. One intraperitoneal injection of inactivated LPF also protected mice from intracerebral challenge; the dose protecting 50% of the mice was 8.5 micrograms. However, one intraperitoneal injection of 48 micrograms of FHA or two weekly intraperitoneal injections of 20 micrograms did not protect mice from death after intracerebral challenge. Injection of affinity-purified antibody to LPF from mouse hybridomas or from goats gave a dose-dependent protection against aerosol challenge. The smallest dose giving protection was 80-90 micrograms. Polyclonal or monoclonal antibody to FHA at doses of 1,440 micrograms or 360 micrograms, respectively, gave very little protection from disease after respiratory challenge. These data indicate that active immunization of mice followed by respiratory challenge with B. pertussis is a useful model to identify protective antigens.