Provide a comprehensive overview of methylation mechanisms, including DNA, RNA, histone, and protein methylation.

Comprehensive Overview of Methylation Mechanisms

Methylation is a universal biochemical process that covalently adds methyl groups (-CH3) from S-adenosylmethionine (AdoMet) to diverse molecular targets including DNA, RNA, histones, and proteins, serving as a fundamental regulatory mechanism across all domains of life. 1, 2

Biochemical Foundation

Methylation represents one of the most ancient and conserved biochemical modifications, with evidence suggesting its origins in prebiotic chemistry and early evolution of life. 3 The process involves the enzymatic transfer of a methyl group from the universal methyl donor S-adenosylmethionine (AdoMet, also called SAM) to specific target molecules. 1, 2

Methyl Donor Metabolism

• S-adenosylmethionine synthesis requires essential nutrients including vitamin B9 (folate), vitamin B12, methionine, and choline, which power a complex metabolic pathway that generates the methyl donor for over 200 substrate-specific methyltransferases in humans. 3

• Methyl metabolism shows profound interconnections with fundamental cellular processes, including circadian rhythms, where global inhibition affects biological timing in both prokaryotes and eukaryotes. 3

DNA Methylation Mechanisms

Basic Biochemistry

DNA methylation involves the addition of a methyl group to the 5th carbon of the pyrimidine ring of cytosine bases, typically at CpG dinucleotides (cytosine-guanine linked by phosphate), forming 5-methylcytosine (5mC). 4

Enzymatic Machinery

• DNA methyltransferases (DNMTs) catalyze the transfer of methyl groups from AdoMet to cytosine residues at CG sites (also designated CpG). 1, 5

• Ten eleven translocation (TET) enzymes provide dynamic regulation by mediating demethylation processes, allowing methylation patterns to be reversibly modified. 5

Functional Roles

DNA methylation serves multiple critical functions across organisms:

• In prokaryotes: Protects against phage attack and regulates chromosome replication and repair. 6

• In mammals: Controls transposable element silencing, gene expression regulation, genomic imprinting, and X-chromosome inactivation. 5, 6

• Gene expression regulation: Methylation at promoter sites generally suppresses transcription by blocking transcription factor binding, though the relationship is complex and context-dependent. 4

Complexity of DNA Methylation-Gene Expression Relationships

The canonical inverse association (higher DNA methylation correlating with lower gene expression) holds for approximately two-thirds of differentially methylated CpG sites, but numerous exceptions exist depending on genomic context. 4 Methylation can also increase transcription in certain contexts, and effects vary based on location relative to transcription start sites. 4

Histone Methylation Mechanisms

Target Residues and Enzymes

Histone methylation occurs primarily on lysine and arginine residues, catalyzed by two major enzyme families. 4

Protein Lysine Methyltransferases (PKMTs)

Two structural classes exist:

1. SET domain-containing PKMTs (Su(var)3-9, Enhancer-of-zeste, Trithorax domain): Originally described as histone-specific but now known to methylate non-histone substrates. 4

2. Seven β-strand (7BS) methyltransferases: Large enzyme family including Dot1L (methylates histone H3 at K79) and numerous enzymes targeting DNA, RNA, small molecules, and proteins at arginine, lysine, or glutamine residues. 4, 2

Functional Significance

• Epigenetic signaling: Histone methylation works alongside DNA methylation and non-coding RNAs to regulate chromatin structure and gene expression. 4

• Developmental regulation: Histone modifications often precede DNA methylation changes during normal development, suggesting methylation may be downstream of histone modifications rather than the primary regulatory event. 4

Protein Methylation Beyond Histones

Emerging General Post-Translational Modification

Protein lysine methylation is now recognized as a general post-translational modification regulating protein stability, activity, and protein-protein interactions, extending far beyond histones. 4

Substrate Diversity

• Over 120 human 7BS methyltransferases collectively target nucleic acids, proteins, small metabolites, and signaling molecules, playing essential roles in gene regulation, protein synthesis, metabolism, and neurotransmitter synthesis. 2

• Widespread lysine methylation has been identified in proteomic studies across diverse proteins, though connecting specific methylation events to responsible enzymes remains challenging. 4

Validation Challenges and Guidelines

The field has faced significant issues with non-reproducible substrate assignments. Seven critical biochemical rules have been established for validating protein methyltransferase substrates: 4

1. Include positive controls with validated substrates
2. Use target lysine mutations as negative controls
3. Use inactive enzyme variants as negative controls
4. Report quantitative methylation data
5. Consider enzyme specificity profiles
6. Validate methyl-lysine antibodies rigorously
7. Connect cellular and in vitro results

RNA Methylation

While less extensively detailed in the provided evidence, RNA methylation represents another substrate category for 7BS methyltransferases, with AdoMet-dependent enzymes targeting various RNA species. 4, 2

Detection and Measurement Methods

Technical Approaches

Three principal assay categories exist for detecting methylation: 4

1. Radioactive AdoMet labeling: Direct, sensitive, and quantitative detection via radioactivity transfer, though subject to non-specific binding and automethylation artifacts.

2. Mass spectrometry: Directly detects mass differences from methylation, distinguishing methylation states, but may struggle differentiating trimethylation (+42.081 Da) from acetylation (+42.037 Da) without high-resolution instruments.

3. Antibody-based methods: Widely used for histone methylation but require rigorous validation of specificity for each antibody batch and generally lack quantitative precision.

DNA Methylation Assessment

• Clinical and research methods include microarrays, pyrosequencing, and whole-genome bisulfite sequencing for quantifying site-specific methylation percentages. 4

• Quality control considerations include filtering based on coverage levels, correction for SNP genotypes, and appropriate statistical methods accounting for overdispersion. 7

Clinical and Biological Significance

Epigenetic Clocks

DNA methylation patterns at specific CpG sites track closely with chronological aging, creating "epigenetic clocks" that are consistent across tissues, individuals, and populations. 4 Discrepancies between chronological and epigenetic age predict mortality risk and age-related disease development. 4

Disease Implications

• Cancer: Abnormal DNA methylation patterns are connected to various cancers, with specialized testing recommended at academic tertiary centers for diagnosis and subtyping, particularly for brain tumors. 7, 6

• Developmental disorders: Methylation deficiencies contribute to pathology etiology, affecting placental development and connecting to autoimmune diseases and rheumatoid arthritis. 1, 6

Reproductive Biology

Methylation determines complementary regulatory characteristics of male and female genomes, regulating gametogenesis and embryo development through epigenetic modifications and genomic imprinting. 1

Environmental and Transcriptional Interactions

Environmental Induction

DNA methylation can be induced by genetic variation, spontaneous epimutations, and environmental factors, providing organisms the ability to develop adaptive phenotypes in response to environmental cues—a mechanism underlying phenotypic plasticity. 4

Transcription Factor Involvement

The sequence-specificity of methylation changes suggests transcription factor involvement, where exposures may activate or repress transcription factors that facilitate or hinder gene-specific differential methylation. 4

Critical Methodological Considerations

Common Pitfalls

• PCR bias, low read coverage, and SNPs at CpG sites can affect DNA methylation testing results. 7

• Different algorithms for detecting differentially methylated regions can yield different values even from identical sequencing data. 7

• Antibody specificity problems remain widespread, requiring experimental validation before use. 4

Multi-Omic Integration

Methylation represents only one aspect of the epigenome, with regulatory RNA and histone modifications providing additional layers that require integrated analysis for complete mechanistic understanding. 4 Incorporating gene expression, genetics, metabolomics, and proteomics data improves biological interpretation of methylation findings. 4