DNA Methylation: Roles and Implications
DNA methylation, a widespread phenomenon in bacteria, plants, and animals, participates in various biological processes. In plants, DNA methylation exhibits species-, tissue-, organ-, and age-specific characteristics, regulating genetic functions including transcription, replication, DNA repair, transgene behavior, and cellular differentiation. It plays a critical regulatory role in plant growth, development, and evolution. Due to the specificity of methylation, different genomic regions, such as TSS1500, TSS200, 5'UTR, 1st Exon, Gene body, and 3'UTR, exhibit varying degrees of methylation, which perform distinct functions.
In the promoter regions of tumor cells, abnormal DNA methylation is closely associated with tumorigenesis and progression. Hypomethylation of proto-oncogenes can induce abnormal cell proliferation, eventually leading to tumor formation, while hypermethylation of tumor suppressor gene promoters can result in gene silencing, thereby promoting tumorigenesis.
The transcriptional activity of genes dictates gene expression, with DNA methylation playing a pivotal role in transcriptional regulation. Genes in an active state typically exhibit low methylation levels. As development progresses and gene silencing becomes necessary, methylation occurs in the promoter or coding regions, inhibiting transcription and rendering the gene inactive. Conversely, genes that are initially inactive may be activated through demethylation of their promoter or coding regions, facilitating transcription and expression.
In plants, methylation predominantly occurs within the nuclear genome, although it also occurs in the genomes of organelles such as chloroplasts and mitochondria. For instance, in carrot mitochondria, methylation occurs at CNG and CCGG nucleotide sequences. Research suggests that mitochondrial methylation may be related to the regulation of mitochondrial gene expression. The methylation ratio in higher plants is significantly higher than in animals, with approximately 80% of CG sites and 50% of CNG sites being methylated. However, a substantial portion of CG islands remains unmethylated, indicating that the methylation percentage in organisms is never absolute. Many unmethylated genes are involved in gene expression, and these non-expressed methylated genes become potential metabolic regulators, forming new methylation targets, which is of great significance for biodiversity.
Advantages and Framework of Integrative Analysis of DNA Methylation and Transcriptome Sequencing
The integration of transcriptomic and methylation sequencing data primarily encompasses two approaches:
- Establishing correlations between transcriptomic data and methylation sequencing data based on specific genes;
- Utilizing genes as intermediaries to integrate data from both omics.
The intricate regulatory relationship between DNA methylation and gene expression necessitates an in-depth correlation analysis to discern the overarching relationship between DNA methylation modifications and RNA expression within a given sample. This analysis further aims to identify specific genes whose transcriptional expression is influenced by DNA methylation modifications and to elucidate how these methylation-regulated genes subsequently impact downstream functions.
Integrative Framework for DNA Methylation and Transcriptome Sequencing Analysis
Case Study of Integrated Analysis of DNA Methylation and Transcriptome Sequencing
Case (I)
Multi-faceted Epigenetic Dysregulation of Gene Expression Promotes Esophageal Squamous Cell Carcinoma
Research Findings
98% of the CpG sites in the esophageal squamous cell carcinoma (ESCC) genome exhibit hypomethylation.
Hypomethylated regions are enriched in areas with heterochromatin-associated markers (H3K9me3, H3K27me3), whereas hypermethylated regions are enriched in regions recognized by the Polycomb Repressive Complex (EZH2 / SUZ12).
A correlation between differential methylation and patient prognosis in ESCC was identified; hypomethylation is associated with favorable prognosis, whereas hypermethylation correlates with poor prognosis.
The impact of methylation alterations on gene expression was analyzed across multiple patient cohorts and human ESCC cell lines.
An atypical regulatory signaling pathway was identified, namely the epigenetically mediated upregulation of the Wnt2/β-catenin/MMP axis, which promotes ESCC growth, invasion, and migration.
Advantages of Integrated Analysis
The combined analysis of whole-genome bisulfite sequencing (WGBS) and RNA sequencing (RNA-seq) elucidated various methylation-mediated gene regulatory relationships. This integrative Multi-omics approach enables the delineation of the methylome and characterization of oncogenic drivers in esophageal squamous cell carcinoma (ESCC).
This methodology enhances the understanding of how epigenetic modifications influence cancer pathogenesis and provides a valuable resource for the identification of biomarkers and therapeutic targets.
Case(II)
Identification of Subtypes of Barrett's Esophagus and Esophageal Adenocarcinoma Based on DNA Methylation Profiles and Integration of Transcriptome and Genome Data
Research Findings
- Epigenetic analyses were conducted on Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC) tissues, integrating these data with transcriptomic and genomic data to elucidate the mechanisms regulating gene expression and genomic integrity.
- Compared to normal tissues from the same patients (esophagus, stomach, and duodenum; controls), BE and EAC samples exhibited distinct genome-wide methylation patterns and were categorized into four subtypes. Each subtype was analyzed for methylation status, gene expression, genomic structure, and copy number variations.
- Subtype 1 was characterized by high DNA methylation, a high mutation burden, and multiple mutations in genes associated with cell cycle regulation and receptor tyrosine kinase signaling pathways.
- Subtype 2 exhibited gene expression patterns related to metabolic processes (ATP synthesis and fatty acid oxidation) and lacked methylation at specific transcription factor binding sites.
- Subtype 3 displayed methylation patterns similar to control tissues but had a gene expression profile indicative of immune cell infiltration; this tumor type was associated with the shortest patient survival time.
- Subtype 4 was distinguished by low DNA methylation associated with structural rearrangements and copy number alterations.
Advantages of Integrated Analysis
The integrative analysis of whole-genome bisulfite sequencing (WGBS) and RNA sequencing (RNA-seq) data provided a comprehensive methylation profile of Barrett's esophagus and esophageal adenocarcinoma. This approach enabled the characterization of methylation, genomic, and expression features within each subtype. A thorough integrated analysis of methylation, transcriptomic, genomic profiles, and clinical data from Barrett's esophagus and esophageal adenocarcinoma tissues identified four subtypes associated with patient prognosis and potential therapeutic responses.
Case Study of Integrated Analysis (III)
Integrated Transcriptomics and Epigenomics Reveal Chamber-Specific and Species-Specific Characteristics of Human and Mouse Hearts
Research Findings
- Conserved region-specific patterns in transcriptomes and DNA methylation levels were observed in mammalian hearts.
- Distinct features in the DNA methylome, chromatin accessibility, and transcriptome were evident in adult and fetal human hearts.
- Novel long non-coding RNAs were identified in the human heart, displaying gene expression profiles associated with major cardiovascular diseases.
- Cross-species comparisons revealed species-specific differentially expressed genes between atria and ventricles in humans and mice. Relationships among Multi-omics data were reported, demonstrating an association between gene body methylation and gene expression in the human heart. Overall, this study provides comprehensive spatiotemporal and evolutionary insights into the regulation of gene expression in the heart.
Advantages of Integrated Analysis
The integrative analysis utilizing whole-genome bisulfite sequencing (WGBS) and RNA sequencing (RNA-seq) revealed significant differences between the four chambers of the adult heart, as well as between fetal and adult hearts. DNA methylation, chromatin accessibility, and gene expression represent different levels of information in biological processes. However, comprehensive Multi-omics analyses of mammalian hearts are lacking. By employing nucleosome occupancy and methylome sequencing, this study simultaneously assessed DNA methylation and chromatin accessibility, alongside RNA-seq, to conduct multi-omics analyses of the four chambers in adult and fetal hearts and the adult mouse heart.
Case Study (IV)
Foetal hypoxia impacts methylome and transcriptome in developmental programming of heart disease
Research Findings
- Methylation data indicated a pronounced decrease in CpG methylation centered around transcription start sites (TSS). Compared to intragenic CpG islands (CGI) and CpG shores (CGS), CpG islands and shores within 10 kb upstream of TSS exhibited lower methylation levels.
- Integration of transcriptomic data revealed a negative correlation between gene expression and CpG methylation surrounding TSS. Notably, prenatal hypoxia induced divergent methylation patterns in foetal and adult hearts, characterized by hypermethylation in foetuses and hypomethylation in adults.
- Significant sex-specific differences were observed in the gene expression patterns of adult hearts. Pathway analysis demonstrated a greater enrichment of inflammation-related pathways in adult male hearts compared to female hearts.
Advantages of Integrated Analysis
Prenatal hypoxia negatively impacts foetal heart development and increases the risk of future cardiovascular diseases. The molecular mechanisms underlying these effects remain largely unexplained. The authors conducted a comprehensive genome-wide analysis to investigate the effects of prenatal hypoxia on DNA methylation and transcriptomic profiles in foetal and adult hearts.
