DNA methylation is implicated in a surprising diversity of regulatory, evolutionary processes and diseases in eukaryotes. The introduction of whole-genome bisulfite sequencing has enabled the study of DNA methylation at a single-base resolution, revealing many new aspects of DNA methylation and highlighting the usefulness of methylome data in understanding a variety of genomic phenomena. As the number of publicly available whole-genome bisulfite sequencing studies reaches into the hundreds, reliable and convenient tools for comparing and analyzing methylomes become increasingly important. We present MethPipe, a pipeline for both low and high-level methylome analysis, and MethBase, an accompanying database of annotated methylomes from the public domain. Together these resources enable researchers to extract interesting features from methylomes and compare them with those identified in public methylomes in our database.

Although CpG methylation clearly distributes genome-wide in vertebrate nuclear DNA, the state of methylation in the vertebrate mitochondrial genome has been unclear. Several recent reports using immunoprecipitation, mass spectrometry, and enzyme-linked immunosorbent assay methods concluded that human mitochondrial DNA (mtDNA) has much more than the 2 to 5% CpG methylation previously estimated. However, these methods do not provide information as to the sites or frequency of methylation at each CpG site. Here, we have used the more definitive bisulfite genomic sequencing method to examine CpG methylation in HCT116 human cells and primary human cells to independently answer these two questions. We found no evidence of CpG methylation at a biologically significant level in these regions of the human mitochondrial genome. Furthermore, unbiased next-generation sequencing of sodium bisulfite treated total DNA from HCT116 cells and analysis of genome-wide sodium bisulfite sequencing data sets from several other DNA sources confirmed this absence of CpG methylation in mtDNA. Based on our findings using regionally specific and genome-wide approaches with multiple human cell sources, we can definitively conclude that CpG methylation is absent in mtDNA. It is highly unlikely that CpG methylation plays any role in direct control of mitochondrial function. CpG methylation occurs with a genome-wide distribution in vertebrates and has impacts on transcription, repeat element biology, and recombination. Although the presence of CpG methylation in the mitochondrial genome was reported using restriction digestion (1, 2) and radiolabeling methods (3) a few decades ago, two of our unpublished studies showed no CpG methylation in HEK293 cells by the sodium bisulfite sequencing method. The first of our unpublished studies nearly 2 decades ago did not detect any DNA methylation at CpG sites in the 12S and the 16S regions of mitochondrial DNA (mtDNA) from a limited number of molecules sequenced after sodium bisufite treatment of DNA. Our more recent study of three mtDNA regions (nucleotides 560 to 893, 4250 to 4569, and 16381 to 16470) confirmed an absence of CpG methylation in a total of 1,487 CpG sites from 203 molecules examined from HEK293 cells using the sodium bisulfite genomic sequencing method (see Table S1 in the supplemental material). A limited study using bisulfite-PCR/single-stranded DNA conformation polymorphism analysis failed to detect any cytosine methylation in mtDNA (4). Our interest in mtDNA methylation was then renewed when detection of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) at CpG dinucleotides in mtDNA by immunoprecipitation (IP) (5), mass spectrometry (6), and enzyme-linked immunosorbent assay (ELISA) (7) were recently reported. However, these methods do not provide critical information about the sites and the frequency of methylation at each site that is required to fully assess any putative functional impact of DNA methylation on mtDNA.Shock et al. (5) concluded that the level of 5mC and 5hm...

Motivation: The two major epigenetic modifications of cytosines, 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC), coexist with each other in a range of mammalian cell populations. Increasing evidence points to important roles of 5-hmC in demethylation of 5-mC and epigenomic regulation in development. Recently developed experimental methods allow direct single-base profiling of either 5-hmC or 5-mC. Meaningful analyses seem to require combining these experiments with bisulfite sequencing, but doing so naively produces inconsistent estimates of 5-mC or 5-hmC levels.Results: We present a method to jointly model read counts from bisulfite sequencing, oxidative bisulfite sequencing and Tet-Assisted Bisulfite sequencing, providing simultaneous estimates of 5-hmC and 5-mC levels that are consistent across experiment types.Availability: http://smithlab.usc.edu/software/mlmlContact: andrewds@usc.eduSupplementary information: Supplementary material is available at Bioinformatics online.

Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited disorder characterized by skin fragility, blistering, and multiple skin wounds with no currently approved or consistently effective treatment. It is due to mutations in the gene encoding type VII collagen (C7). Using recombinant human C7 (rhC7) purified from human dermal fibroblasts (FB-rhC7), we showed previously that intravenously injected rhC7 distributed to engrafted RDEB skin, incorporated into its dermal-epidermal junction (DEJ), and reversed the RDEB disease phenotype. Human dermal fibroblasts, however, are not used for commercial production of therapeutic proteins. Therefore, we generated rhC7 from Chinese hamster ovary (CHO) cells. The CHO-derived recombinant type VII collagen (CHO-rhC7), similar to FB-rhC7, was secreted as a correctly folded, disulfide-bonded, helical trimer resistant to protease degradation. CHO-rhC7 bound to fibronectin and promoted human keratinocyte migration in vitro. A single dose of CHO-rhC7, administered intravenously into new-born C7-null RDEB mice, incorporated into the DEJ of multiple skin sites, tongue and esophagus, restored anchoring fibrils, improved dermal-epidermal adherence, and increased the animals' life span. Furthermore, no circulating or tissue-bound anti-C7 antibodies were observed in the mice. These data demonstrate the efficacy of CHO-rhC7 in a preclinical murine model of RDEB.

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