Low guanine content and biased nucleotide distribution in vertebrate mtDNA can cause overestimation of non-CpG methylation

ABSTRACT Mitochondrial DNA (mtDNA) methylation in vertebrates has been hotly debated for over 40 years. Most contrasting results have been reported following bisulfite sequencing (BS-seq) analyses. We addressed whether BS-seq experimental and analysis conditions influenced the estimation of the levels of methylation in specific mtDNA sequences. We found false positive non-CpG methylation in the CHH context (fpCHH) using unmethylated Sus scrofa mtDNA BS-seq data. fpCHH methylation was detected on the top/plus strand of mtDNA within low guanine content regions. These top/plus strand sequences of fpCHH regions would become extremely AT-rich sequences after BS-conversion, whilst bottom/minus strand sequences remained almost unchanged. These unique sequences caused BS-seq aligners to falsely assign the origin of each strand in fpCHH regions, resulting in false methylation calls. fpCHH methylation detection was enhanced by short sequence reads, short library inserts, skewed top/bottom read ratios and non-directional read mapping modes. We confirmed no detectable CHH methylation in fpCHH regions by BS-amplicon sequencing. The fpCHH peaks were located in the D-loop, ATP6, ND2, ND4L, ND5 and ND6 regions and identified in our S. scrofa ovary and oocyte data and human BS-seq data sets. We conclude that non-CpG methylation could potentially be overestimated in specific sequence regions by BS-seq analysis.

Myotonic dystrophy type 1 embryonic stem cells show decreased myogenic potential, increased CpG methylation at the DMPK locus and RNA mis-splicing

ABSTRACT Skeletal muscle tissue is severely affected in myotonic dystrophy type 1 (DM1) patients, characterised by muscle weakness, myotonia and muscle immaturity in the most severe congenital form of the disease. Previously, it was not known at what stage during myogenesis the DM1 phenotype appears. In this study we differentiated healthy and DM1 human embryonic stem cells to myoblasts and myotubes and compared their differentiation potential using a comprehensive multi-omics approach. We found myogenesis in DM1 cells to be abnormal with altered myotube generation compared to healthy cells. We did not find differentially expressed genes between DM1 and non-DM1 cell lines within the same developmental stage. However, during differentiation we observed an aberrant inflammatory response and increased CpG methylation upstream of the CTG repeat at the myoblast level and RNA mis-splicing at the myotube stage. We show that early myogenesis modelled in hESC reiterates the early developmental manifestation of DM1.

Developmental and Injury-induced Changes in DNA Methylation in Regenerative versus Non-regenerative Regions of the Vertebrate Central Nervous System

Background Because some of its CNS neurons (e.g., retinal ganglion cells after optic nerve crush (ONC)) regenerate axons throughout life, whereas others (e.g., hindbrain neurons after spinal cord injury (SCI)) lose this capacity as tadpoles metamorphose into frogs, the South African claw-toed frog, Xenopus laevis, offers unique opportunities for exploring differences between regenerative and non-regenerative responses to CNS injury within the same organism. An earlier, three-way RNA-seq study (frog ONC eye, tadpole SCI hindbrain, frog SCI hindbrain) identified genes that regulate chromatin accessibility among those that were differentially expressed in regenerative vs non-regenerative CNS [11]. The current study used whole genome bisulfite sequencing (WGBS) of DNA collected from these same animals at the peak period of axon regeneration to study the extent to which DNA methylation could potentially underlie differences in chromatin accessibility between regenerative and non-regenerative CNS. Results Consistent with the hypothesis that DNA of regenerative CNS is more accessible than that of non-regenerative CNS, DNA from both the regenerative tadpole hindbrain and frog eye was less methylated than that of the non-regenerative frog hindbrain. Also, consistent with observations of CNS injury in mammals, DNA methylation in non-regenerative frog hindbrain decreased after SCI. However, contrary to expectations that the level of DNA methylation would decrease even further with axotomy in regenerative CNS, DNA methylation in these regions instead increased with injury. Injury-induced differences in CpG methylation in regenerative CNS became especially enriched in gene promoter regions, whereas non-CpG methylation differences were more evenly distributed across promoter regions, intergenic, and intragenic regions. In non-regenerative CNS, tissue-related (i.e., regenerative vs. non-regenerative CNS) and injury-induced decreases in promoter region CpG methylation were significantly correlated with increased RNA expression, but the injury-induced, increased CpG methylation seen in regenerative CNS across promoter regions was not, suggesting it was associated with increased rather than decreased chromatin accessibility. This hypothesis received support from observations that in regenerative CNS, many genes exhibiting increased, injury-induced, promoter-associated CpG-methylation also exhibited increased RNA expression and association with histone markers for active promoters and enhancers. DNA immunoprecipitation for 5hmC in optic nerve regeneration found that the promoter-associated increases seen in CpG methylation were distinct from those exhibiting changes in 5hmC. Conclusions Although seemingly paradoxical, the increased injury-associated DNA methylation seen in regenerative CNS has many parallels in stem cells and cancer. Thus, these axotomy-induced changes in DNA methylation in regenerative CNS provide evidence for a novel epigenetic state favoring successful over unsuccessful CNS axon regeneration. The datasets described in this study should help lay the foundations for future studies of the molecular and cellular mechanisms involved. The insights gained should, in turn, help point the way to novel therapeutic approaches for treating CNS injury in mammals.

Hepatitis C Virus Core Protein Down-Regulates Expression of Src-Homology 2 Domain Containing Protein Tyrosine Phosphatase by Modulating Promoter DNA Methylation

Hepatitis C virus (HCV) is the major causative pathogen associated with liver cirrhosis and hepatocellular carcinoma. The main virion component, the core (C) protein, has been implicated in several aspects of HCV pathology including oncogenesis and immune subversion. Here we show that expression of the C protein induced specific tyrosine phosphorylation of the TCR-related signaling proteins ZAP-70, LAT and PLC-γ in the T cells. Stable expression of the C protein specifically reduced Src homology domain 2-containing protein tyrosine phosphatase 1 (SHP-1) mRNA and protein accumulation. Quantitative CpG methylation analysis revealed a distinct CpG methylation pattern at the SHP-1 gene promoter in the C protein expressing cells that included specific hypermethylation of the binding site for Sp1 transcription factor. Collectively, our results suggest that HCV may suppress immune responses and facilitate its own persistence by deregulating phosphotyrosine signaling via repressive epigenetic CpG modification at the SHP-1 promoter in the T cells.

Detection of CpG Methylation in G-Quadruplex Forming Sequences Using G-Quadruplex Ligands

Genomic DNA methylation is involved in many diseases and is expected to be a specific biomarker for even the pre-symptomatic diagnosis of many diseases. Thus, a rapid and inexpensive detection method is required for disease diagnosis. We have previously reported that cytosine methylation in G-quadruplex (G4)-forming oligonucleotides develops different G4 topologies. In this study, we developed a method for detecting CpG methylation in G4-forming oligonucleotides based on the structural differences between methylated and unmethylated G4 DNAs. The differences in G4 topologies due to CpG methylation can be discriminated by G4 ligands. We performed a binding assay between methylated or unmethylated G4 DNAs and G4 ligands. The binding abilities of fluorescent G4 ligands to BCL-2, HRAS1, HRAS2, VEGF G4-forming sequences were examined by fluorescence-based microtiter plate assay. The differences in fluorescence intensities between methylated and unmethylated G4 DNAs were statistically significant. In addition to fluorescence detection, the binding of G4 ligand to DNA was detected by chemiluminescence. A significant difference was also detected in chemiluminescence intensity between methylated and unmethylated DNA. This is the first study on the detection of CpG methylation in G4 structures, focusing on structural changes using G4 ligands.