Cytosine DNA methylation is a stable epigenetic mark that is crucial for diverse biological processes, including gene and transposon silencing, imprinting and X chromosome inactivation. Recent findings in plants and animals have greatly increased our understanding of the pathways used to accurately target, maintain and modify patterns of DNA methylation and have revealed unanticipated mechanistic similarities between these organisms. Key roles have emerged for small RNAs, proteins with domains that bind methylated DNA and DNA glycosylases in these processes. Drawing on insights from both plants and animals should deepen our understanding of the regulation and biological significance of DNA methylation.

Establishing, maintaining and modifying DNA methylation patterns in plants and animals

DNA methylation is among the best studied epigenetic modifications and is essential to mammalian development. Although the methylation status of most CpG dinucleotides in the genome is stably propagated through mitosis, improvements to methods for measuring methylation have identified numerous regions in which it is dynamically regulated. In this Review, we discuss key concepts in the function of DNA methylation in mammals, stemming from more than two decades of research, including many recent studies that have elucidated when and where DNA methylation has a regulatory role in the genome. We include insights from early development, embryonic stem cells and adult lineages, particularly haematopoiesis, to highlight the general features of this modification as it participates in both global and localized epigenetic regulation.

DNA methylation: roles in mammalian development

The prevalence of obesity has increased worldwide in the past ~50 years, reaching pandemic levels. Obesity represents a major health challenge because it substantially increases the risk of diseases such as type 2 diabetes mellitus, fatty liver disease, hypertension, myocardial infarction, stroke, dementia, osteoarthritis, obstructive sleep apnoea and several cancers, thereby contributing to a decline in both quality of life and life expectancy. Obesity is also associated with unemployment, social disadvantages and reduced socio-economic productivity, thus increasingly creating an economic burden. Thus far, obesity prevention and treatment strategies - both at the individual and population level - have not been successful in the long term. Lifestyle and behavioural interventions aimed at reducing calorie intake and increasing energy expenditure have limited effectiveness because complex and persistent hormonal, metabolic and neurochemical adaptations defend against weight loss and promote weight regain. Reducing the obesity burden requires approaches that combine individual interventions with changes in the environment and society. Therefore, a better understanding of the remarkable regional differences in obesity prevalence and trends might help to identify societal causes of obesity and provide guidance on which are the most promising intervention strategies.

Obesity: global epidemiology and pathogenesis.

The recent discovery of reversible mRNA methylation has opened a new realm of post-transcriptional gene regulation in eukaryotes. The identification and functional characterization of proteins that specifically recognize RNA N6-methyladenosine (m6A) unveiled it as a modification that cells utilize to accelerate mRNA metabolism and translation. N6-adenosine methylation directs mRNAs to distinct fates by grouping them for differential processing, translation and decay in processes such as cell differentiation, embryonic development and stress responses. Other mRNA modifications, including N1-methyladenosine (m1A), 5-methylcytosine (m5C) and pseudouridine, together with m6A form the epitranscriptome and collectively code a new layer of information that controls protein synthesis.

/pdf/post-transcriptional-gene-regulation-by-mrna-modifications-5984s6cty9.pdf

Post-transcriptional gene regulation by mRNA modifications

Cellular RNAs carry diverse chemical modifications that used to be regarded as static and having minor roles in 'fine-tuning' structural and functional properties of RNAs. In this Review, we focus on reversible methylation through the most prevalent mammalian mRNA internal modification, N(6)-methyladenosine (m(6)A). Recent studies have discovered protein 'writers', 'erasers' and 'readers' of this RNA chemical mark, as well as its dynamic deposition on mRNA and other types of nuclear RNA. These findings strongly indicate dynamic regulatory roles that are analogous to the well-known reversible epigenetic modifications of DNA and histone proteins. This reversible RNA methylation adds a new dimension to the developing picture of post-transcriptional regulation of gene expression.

http://basicmed.med.ncku.edu.tw/public/study/7-1417587520-1.pdf

Gene expression regulation mediated through reversible m 6 A RNA methylation

Immune response to seasonal influenza A virus infection: a proteomic approach.

During analysis of the proteome in the cerebrospinal fluid (CSF) of the Caucasian form of moyamoya disease (MMD), a novel post-translational modification of human transthyretin was observed. Two-dimensional electrophoresis and subsequent peptide sequencing with ESI-MS/MS were performed to discover the gamma-carboxylation of the Glu-42 (Gla-42).

Detection of a gamma-carboxy-glutamate as novel post-translational modification of human transthyretin.

For the identification of proteins from biological samples by mass spectrometry there are two scenarios. Proteins are pre-fractionated in some way or are analysed as a complex mixture (shotgun). Shotgun proteomics recently became more popular, because of technological developments on the mass spectrometer side which now allows the identification of several thousand proteins from a complex biological matrix at one go. However, in many cases it is still useful to separate proteins first in a gel. But not only mass spectrometer technology made progress. Recently, new protocols and techniques which make the analysis of starting material in the low microgram range possible, and also simplify the whole procedure, were developed. In this small review detailed protocols will be described which also allow inexperienced beginners to get good results.

https://www.tandfonline.com/doi/pdf/10.3109/13813455.2014.955032

Up to date sample preparation of proteins for mass spectrometric analysis.

Separation or prefractionation of proteins by gel electrophoresis, followed by in-gel proteolytic digest and analysis by MS is a typical standard application in proteomics. For many laboratories, it is not cost-effective to use a robot with all its drawbacks. On the other hand, manual digest is time consuming and not free of error if many samples are processed at the same time. The OASIS® HLB μElution plate allows the handling of 96 samples for protein in-gel digest and peptide desalting in a semiautomatic way. Using multichannel pipettes, solutions are added quickly, and with the use of the positive pressure-96 stand all solutions are removed simultaneously. In this article, a complete and detailed protocol for the use of the OASIS® HLB μElution plate is introduced and its effectiveness is shown with 2D- and 1D-gel samples.

The OASIS® HLB μElution plate as a one‐step platform for manual high‐throughput in‐gel digestion of proteins and peptide desalting

Two-dimensional gel electrophoresis (2-DE) is one of the most powerful methods for studying global protein profiles. However, due to the multiple manual steps involved in gel based processing it is challenging to achieve the necessary overall reproducibility for a reliable comparative analysis, especially between different laboratories. To improve the 2-DE technique for quantitative analyses we have set up a robust 2-DE workflow, called 2D-ToGo, which utilizes latest innovations concerning instrumentation, consumables and protocols. Quantitative data analyses indicate the high reproducibility between replicate gels processed at a single site (intra-laboratory variation: CV 20%). The data-sets of the inter-laboratory comparison revealed similar results displaying a variation of CV 23%. The technical improvements given by our 2-DE workflow have a positive impact on process robustness and most importantly, reproducibility. Accordingly, many of the well-known challenges for resolving and quantitating up to thousands of different protein components in a given biological sample are minimized.

Thomas Franz

Papers

Immune response to seasonal influenza A virus infection: a proteomic approach.

Detection of a gamma-carboxy-glutamate as novel post-translational modification of human transthyretin.

Up to date sample preparation of proteins for mass spectrometric analysis.

The OASIS® HLB μElution plate as a one‐step platform for manual high‐throughput in‐gel digestion of proteins and peptide desalting

2D-ToGo workflow: increasing feasibility and reproducibility of 2-dimensional gel electrophoresis.