DNA methylation is the most studied epigenetic modification with a wide range of regulatory functions in mammalian genomes. It almost exclusively resides on CpG dinucleotides and, among others, plays important roles in early embryo development, onset, and maintenance of cancer. During the past 3 decades, many approaches have been developed to discriminate methylated from unmethylated DNA including antibody-based enrichment of methylated DNA, restriction enzyme-based, or hybridization-based methods. The conversion of unmethylated cytosines to uracils by sodium or ammonium bisulfite is regarded as golden standard as this approach requires no enzymatic reaction and provides deep and reliable insight in methylation patterns at single-base resolution. Nowadays, there are many commercial kits for bisulfite conversion available but they perform differently and also vary in protocols and chemicals used. Here, we provide the first comprehensive and comparative evaluation of bisulfite conversion kits observing major differences in conversion efficiency and DNA degradation which greatly affect the performance of downstream applications, ie, polymerase chain reactions (PCRs). Moreover, deep sequencing of amplicons containing oxidized derivates of 5ʹ-methylC shows that none of the tested kits efficiently converts 5ʹ-formylC without substantial conversion of 5ʹ-methylC or 5ʹ-hydroxymethylC. Consequently, we developed a robust and easy-to-use protocol that allows maximal discrimination between 5ʹ-formylC and 5ʹ-methylC/5ʹ-hydroxymethylC with low DNA degradation and high PCR efficiency on the bisulfite-treated DNA. We highly recommend to use our time- and cost-efficient protocol for any genome-wide or local high-resolution bisulfite sequencing application to minimize conversion-dependent error rates.
In the wild-type allele, DNA methylation levels of 10 consecutive CpG sites adjacent to the upstream 5′-breakpoint of α-thalassemia Southeast Asian (SEA) deletion are not different between placenta and leukocytes. However, no previous study has reported the map of DNA methylation in the SEA allele. This report aims to show that the SEA mutation is associated with DNA methylation changes, resulting in differential methylation between placenta and leukocytes. Methylation-sensitive high-resolution analysis was used to compare DNA methylation among placenta, leukocytes, and unmethylated control DNA. The result indicates that the DNA methylation between placenta and leukocyte DNA is different and shows that the CpG status of both is not fully unmethylated. Mapping of individual CpG sites was performed by targeted bisulfite sequencing. The DNA methylation level of the 10 consecutive CpG sites was different between placenta and leukocyte DNA. When the 10th CpG of the mutation allele was considered as a hallmark for comparing DNA methylation level, it was totally different from the unmethylated 10th CpG of the wild-type allele. Finally, the distinct DNA methylation patterns between both DNA were extracted. In total, 24 patterns were found in leukocyte samples and 9 patterns were found in placenta samples. This report shows that the large deletion is associated with DNA methylation change. In further studies for clinical application, the distinct DNA methylation pattern might be a potential marker for detecting cell-free fetal DNA.
Colorectal cancer (CRC) is the third most common cause of cancer mortality globally. Development of CRC is closely associated with lifestyle, and diet may modulate risk. A Western-style diet is characterised by a high intake of red meat but low consumption of fruit, vegetables, and whole cereals. Such a diet is associated with CRC risks. It has been demonstrated that butyrate, produced by the fermentation of dietary plant fibre, can alter both genetic and epigenetic expressions. MicroRNAs (miRNAs) are small non-coding RNAs that are commonly present in both normal and tumour cells. Aberrant miRNA expression is associated with CRC initiation, progression, and metastasis. In addition, butyrate can modulate cell proliferation, differentiation, apoptosis, and miRNA expression in CRC. In this review, the effects of butyrate on modulating miRNA expression in CRC will be discussed. Furthermore, evidence on the effect of butyrate on CRC risk through reducing oncogenic miRNA expression will be presented.
Prior DNA methylation (DNA-m) analyses have identified cytosine-phosphate-guanine (CpG) sites, which show either a significant change or consistency during lifetime. However, the proportion of CpGs that are neither significantly different nor consistent over time (indifferent CpGs) is unknown. We investigated the methylation dynamics, both longitudinal changes and consistency, in women from preadolescence to late pregnancy using DNA-m of peripheral blood cells. Consistency of cell type-adjusted DNA-m between paired individuals was assessed by regressing CpGs of subsequent age on the prior, stability by intraclass correlation coefficients (>0.5), and changes by linear mixed models. In the first 2 transitions (10-18 years and 18 years to early pregnancy), 19.5% and 20.9% CpGs were consistent, but only 0.35% in the third transition (from early to late pregnancy). Significant changes in methylation were found in 0.7%, 5.6%, and 0% CpGs, respectively. Functional enrichment analyses of genes with significant changes in DNA-m in early pregnancy (5.6%) showed that the maternal DNA-m seems to reflect signaling pathways between the uterus and the trophoblast. The transition from early to late pregnancy showed low consistency/stability and no changes, suggesting the presence of a large proportion of indifferent CpGs in late pregnancy.
The evolution process includes genetic alterations that started with prokaryotes and now continues in humans. A distinct difference between prokaryotic chromosomes and eukaryotic chromosomes involves histones. As evolution progressed, genetic alterations accumulated and a mechanism for gene selection developed. It was as if nature was experimenting to optimally utilize the gene pool without changing individual gene sequences. This mechanism is called epigenetics, as it is above the genome. Curiously, the mechanism of epigenetic regulation in prokaryotes is strikingly different from that in eukaryotes, mainly higher eukaryotes, like mammals. In fact, epigenetics plays a significant role in the conserved process of embryogenesis and human development. Malfunction of epigenetic regulation results in many types of undesirable effects, including cardiovascular disease, metabolic disorders, autoimmune diseases, and cancer. This review provides a comparative analysis and new insights into these aspects.
