WGBS Application Summary: Deep Insights in Life Science Fields

DNA methylation, a crucial epigenetic modification, modulates gene expression without altering the DNA sequence. It plays a fundamental role in numerous biological processes such as organism growth and development, cell differentiation, and disease occurrence and progression. Genome-wide bisulfite sequencing (WGBS) technology has emerged as a powerful tool in this context. By treating unmethylated cytosine with bisulfite to convert it into uracil while leaving methylated cytosine unchanged, and integrating with high-throughput sequencing, WGBS enables the construction of methylation maps with single-base resolution across the entire genome. This technology has opened new avenues for in-depth exploration of DNA methylation functions and mechanisms, demonstrating extensive application potential across various life science disciplines.

WGBS Applications in Biological Development

Embryonic development: In the process of embryonic development, DNA methylation plays a vital role, which is involved in key processes such as cell fate determination and lineage differentiation. WGBS technology can map the whole genome methylation at different stages of embryonic development and reveal the dynamic change law of methylation. For example, in the study of mouse embryo development, WGBS found that large-scale demethylation occurred in the genome after fertilization, and then there was a process of demethylation before and after implantation. These methylation changes are closely related to the totipotency, pluripotency and differentiation of embryonic cells. By analyzing the methylation status of specific gene promoter region, we can deeply understand the mechanism of gene expression regulation during embryonic development, and provide theoretical basis for studying diseases caused by abnormal embryonic development.

Cell differentiation: Cell differentiation is the basis of individual development of multicellular organisms, and DNA methylation plays a key role in it. WGBS can be used to study the methylation differences of different types of cells during differentiation. Taking the differentiation of hematopoietic stem cells as an example, through WGBS analysis of hematopoietic stem cells and various blood cells formed by differentiation, it was found that the methylation pattern of genome changed significantly with the differentiation. The promoter region of some genes related to stem cell self-renewal gradually methylates during the differentiation process, which leads to gene expression silence, while the methylation level of genes related to specific functions of blood cells decreases correspondingly, which promotes gene expression. This indicates that WGBS can reveal the regulatory network of gene expression mediated by methylation in the process of cell differentiation, which is helpful to understand the molecular mechanism of cell differentiation.

WGBS Insights into Disease Development

Research on Tumor

• Early diagnosis marker identification: The occurrence of tumors is usually accompanied by changes in DNA methylation patterns. WGBS technology can analyze the whole genome methylation of tumor tissues and normal tissues, and screen out tumor-specific methylation sites. For example, in the study of lung cancer, it was found by WGBS that the promoter region of some tumor suppressor genes, such as RASSF1A gene, was hypermethylated in lung cancer tissues, which led to gene silencing and lost its inhibitory effect on tumor cell growth. These specific methylation sites are expected to be biomarkers for early diagnosis of lung cancer, which may provide the possibility for early detection of tumors.

Subtype-specific differentially methylated regions in esophageal cancer (Zheng et al., 2023)

• Tumor molecular typing and personalized treatment: Different types of tumors and different subtypes of the same tumor have different methylation patterns. WGBS can classify tumors according to methylation patterns. Taking colorectal cancer as an example, it can be divided into different subtypes according to methylation characteristics, and each subtype has differences in clinical characteristics, treatment response and prognosis. Through this precise classification, doctors can make personalized treatment plans for patients. For some tumors with specific methylation subtypes, they may be more sensitive to specific chemotherapy drugs or targeted drugs, thus improving the therapeutic effect and reducing unnecessary side effects.
• Treatment effect monitoring and recurrence prediction: In the process of tumor treatment, the methylation state of tumor cells will change dynamically. WGBS can monitor these changes in real time and evaluate the therapeutic effect. For example, during the endocrine therapy for breast cancer patients, through WGBS analysis of tumor tissues or circulating tumor DNA, if the methylation level of some genes related to treatment sensitivity changes, the treatment strategy can be adjusted in time. In addition, continuous monitoring of methylation status after treatment is helpful to predict tumor recurrence. If the methylation pattern of a specific gene is abnormal again, it may suggest that the risk of tumor recurrence is increased, so that doctors can take intervention measures in advance.

WGBS reveals that MBC methylation profiles differ from DFS and H (Christophe et al., 2015)

Neurodevelopmental diseases

• In the study of autism, WGBS was carried out on brain tissues of autistic patients and normal controls, and it was found that the methylation patterns of several genes related to neural development changed, such as aberrant methylation of UBE3A gene in brain tissues of patients, which affected its expression, which may be related to the pathogenesis of autism. In the research of schizophrenia, used WGBS to analyze the DNA methylation of peripheral blood and brain tissue of patients and healthy controls, and identified a number of differential methylation regions, involving neurotransmitter metabolism, synaptic function and other related pathways, providing new ideas for the pathogenesis of schizophrenia.

Differentially methylated regions nearest to the CYP1A1 gene (Adrienne R et al., 2021)

• Alzheimer's disease (AD) is a common neurodegenerative disease, and its pathogenesis is closely related to abnormal DNA methylation. Genome-wide methylation analysis of brain tissues of AD patients and healthy controls by WGBS showed that there were methylation differences in promoter regions of several genes related to neuroinflammation, neurotransmitter metabolism and amyloid precursor protein processing. For example, in AD patients, some gene promoters involved in the regulation of neuroinflammation are hypomethylated, which leads to the up-regulation of gene expression, triggering excessive neuroinflammatory response, and then damaging neurons. Through in-depth study of these methylation changes, it is helpful to reveal the pathogenesis of AD and provide theoretical basis for developing new therapeutic targets.

Enrichment of genomic features among the differentially methylated regions (Li et al., 2020)

• Epigenetic study of Parkinson's disease Abnormal DNA methylation also exists in Parkinson's disease (PD). WGBS study showed that the methylation status of genes related to dopaminergic neuron function, mitochondrial function, protein folding and degradation in PD patients' brain tissue changed. These methylation changes may affect gene expression, leading to the degeneration and death of dopaminergic neurons, which may lead to the symptoms of PD. In addition, WGBS analysis of peripheral blood lymphocytes of PD patients found some disease-related methylation markers, which provided a new way for early diagnosis and disease monitoring of PD.

Cardiovascular disease

• Atherosclerosis methylation regulation mechanism: Atherosclerosis is an important pathological basis of cardiovascular diseases. WGBS study found that the methylation level of genes related to inflammatory reaction, lipid metabolism, proliferation and migration of vascular smooth muscle cells changed significantly in atherosclerotic vascular tissues. For example, the promoter region of some inflammation-related genes is hypomethylated, which enhances gene expression, promotes inflammatory response and accelerates the process of atherosclerosis. Through in-depth study of these methylation regulation mechanisms, it is helpful to develop new therapeutic strategies for atherosclerosis, such as inhibiting inflammatory response and lipid deposition by regulating the methylation level of specific genes.

Methylation sites in heart and vasculature-related studies (Mykhailo et al., 2023)

• Coronary heart disease Epigenetic study: Coronary heart disease is a cardiovascular disease caused by many factors, and DNA methylation also plays an important role in its pathogenesis. Using WGBS to analyze the methylation of genomes of patients with coronary heart disease and healthy people, it was found that there were differences in methylation patterns of some genes related to coronary heart disease, such as those involved in vascular endothelial function, platelet activation, coagulation and fibrinolysis system. These methylation changes may affect gene expression and function, and then affect the occurrence and development of coronary heart disease. In-depth study of these epigenetic changes will help to better understand the pathogenesis of coronary heart disease and provide new targets for early diagnosis and treatment of coronary heart disease.

WGBS for Agricultural Breeding

Crop genetic improvement: In crop breeding, it is of great significance to understand the methylation pattern of crop genome for genetic improvement. WGBS can be used to analyze the methylation differences of different crop varieties or the same variety under different environmental conditions. For example, in rice research, through WGBS of different rice varieties, it was found that the methylation status of some genes related to important agronomic traits such as yield, quality and stress resistance was different. These methylation differences may affect gene expression, and then lead to trait differences. Through the study of these methylation sites, we can develop molecular markers closely linked with excellent traits for molecular marker-assisted breeding and improve breeding efficiency. In addition, studying the methylation changes of crops in response to adversity (such as drought, salinity, etc.) is helpful to explore the genes related to stress resistance and their regulatory mechanisms, and provide theoretical support for cultivating crop varieties with strong stress resistance.

Variation in DNA methylome profiles and correlations with gene expression (Chen et al., 2022)

Livestock and poultry genetic breeding: In livestock and poultry breeding, WGBS technology can also be used in genetic breeding research. Through WGBS analysis of genomes of different breeds of livestock and poultry or different individuals of the same breed, methylation sites related to important economic traits such as growth performance, meat quality and reproductive performance can be found. For example, in the breeding research of pigs, it is found that the methylation level of some genes related to muscle growth and fat metabolism is closely related to the growth speed and meat quality of pigs. Using these methylation information, we can establish a new method for genetic evaluation of livestock and poultry, and realize accurate selection and genetic improvement of excellent traits of livestock and poultry.

Schematic representation of the two-step interaction between PGPB and plants mediated by DNA methylation and root recruitment (Chen et al., 2022)

WGBS in Microbiology Research

Microbial infection and host interaction: In the process of pathogen infecting host, the DNA methylation of microorganism itself will change dynamically. Taking Helicobacter pylori infection in human stomach as an example, the researchers found that the methylation status of some virulence-related genes of Helicobacter pylori changed during the infection process by using WGBS technology. The methylation level of promoter regions of some genes decreased, which increased the expression of these genes and enhanced the colonization ability and pathogenicity of Helicobacter pylori. At the same time, the DNA methylation pattern of host cells will also be affected. Through WGBS analysis of gastric mucosal cells infected by Helicobacter pylori, it was found that the methylation level of some genes related to immune response and cell proliferation changed, which further affected the immune response and cell physiological function of the host.

Heat map and hierarchical cluster analysis of the top 100 differentially methylated CpG identified in the study (Basavaraj et al., 2019)

Symbiotic microorganism-host epigenetic relationship: There are complex interactions between symbiotic microorganisms and their hosts. For example, beneficial bacteria such as Bifidobacterium in the intestine form a symbiotic relationship with the host. Using WGBS technology to study the intestinal epithelial cells and symbiotic microorganisms of the host, it is found that symbiotic microorganisms can affect the physiological processes such as metabolism and immunity of the host by regulating the DNA methylation pattern of the host cells. At the same time, the physiological state of the host will also feedback the DNA methylation of symbiotic microorganisms, thus regulating their growth, metabolism and interaction with the host.

Environmental microbial community methylation: In complex environments such as soil and ocean, the function and ecology of microbial communities are very important for maintaining ecological balance. WGBS technology can be used to analyze the DNA methylation characteristics of environmental microbial communities. For example, in the study of soil microbial community, through WGBS of microorganisms in different fertility soils, it is found that there are specific methylation patterns of microbial groups related to nutrient cycle and environmental adaptation. In high fertility soil, the methylation level of some key genes of microorganisms involved in nitrogen and phosphorus cycling is low, and the gene expression activity is high, which is beneficial to improve the availability of soil nutrients.

Industrial fermentation microbial community epigenetic regulation: In the process of industrial fermentation, the synergistic effect of microbial communities determines the fermentation efficiency and product quality. WGBS technology is helpful to reveal the epigenetic regulation mechanism among members of industrial fermentation microbial community. Taking alcohol production by Saccharomyces cerevisiae as an example, it was found that the DNA methylation pattern of yeast cells changed at different stages of fermentation. At the initial stage of fermentation, the methylation level of genes related to sugar metabolism is low, which promotes the uptake and metabolism of sugar by yeast. In the late stage of fermentation, the methylation status of some genes related to cell stress response changed to adapt to the changes of fermentation environment.

Pyrosequencing validation of CpG sites identified in the genome wide methylation including SENP6 (cg90300054), SDF4 (cg63545568), JAK2 (cg217001619), SRXN1 (cg34587223), ZMYM2 (c651169) (Basavaraj et al., 2019)

Despite the remarkable achievements of WGBS in various research fields, it still faces several challenges. Firstly, the high cost associated with the technology, from sample processing to sequencing analysis, requires professional equipment and a large amount of reagents, limiting its large-scale application. Secondly, the complexity of data analysis demands professional bioinformatics knowledge and high-performance computing resources to interpret the massive data generated. Additionally, strict sample requirements imply that the quality and quantity of samples can impact the accuracy of experimental results.

However, with the continuous development of technology, these challenges are expected to be gradually overcome. In the future, WGBS technology may make breakthroughs in the following aspects. On the one hand, technical optimization will reduce the cost and improve the efficiency and accuracy of the experiment. On the other hand, the integration of multi-omics will become a trend, and the combination of WGBS with transcriptomics, protein omics and other technologies can reveal the molecular mechanism of diseases more comprehensively. In addition, the development of single cell WGBS technology will contribute to the in-depth study of cell heterogeneity and provide more powerful support for accurate diagnosis and treatment of diseases.

In conclusion, WGBS technology has demonstrated substantial application potential in biological development, disease research, agricultural breeding, and microbiology due to its high resolution and genome-wide coverage. Despite the existing challenges of high cost and complex data analysis, continuous technological progress and innovation are expected to position WGBS as an indispensable tool in life science research in the future. It will provide crucial technical support for unraveling the mysteries of life, combating major diseases, and promoting agricultural development, thereby opening a new chapter in life science research.

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