DNA methylation shapes the epigenetic state of cells by regulating gene transcription, as the direct executor of life activities, protein expression, and modification are the key downstream effects of methylation regulation. Methylation chip technology can analyze the methylation pattern of CpG loci in the whole genome with high quantity and high specificity, while protein genomics can capture the expression abundance and functional state of proteins in a panoramic way.

The cross-genomics integration of the two provides a unique perspective for analyzing the regulatory axis of methylation-transcription-translation. From the hypermethylation of tumor suppressor gene promoters in cancer to the expression of lineage-specific protein driven by cell-specific methylation profile during development, the dynamic relationship between epigenetics and protein expression has a profound impact on cell fate and disease process. The integration strategy of methylation chip and protein omics, analyzing their synergistic value in revealing disease mechanisms and mining regulatory networks, provides a new paradigm for fully understanding epigenetic regulation of life activities.

The article presents cases to illustrate the value and mechanisms of combining methylation arrays with proteomics in panoramic analysis of epigenetics and protein expression.

Brief Introduction of Methylation Chip and Proteomics

The cross-application of methylation chip and protein genomics provides an innovative perspective for analyzing the relationship between epigenetic regulation and protein expression. The former captures the dynamic changes of genome methylation, and the latter presents the expression map of the protein group. The combination of the two can reveal the regulatory link from DNA modification to functional proteins, provide multi-dimensional evidence for disease mechanism research and marker discovery, and promote the systematic analysis of epigenetic regulation and protein function.

Methylation Chip

The methylation chip is a technical tool to detect the methylation level of genomic DNA by Qualcomm. The methylation status of specific CpG sites can be quantitatively analyzed by probe hybridization combined with bisulfite transformation. The core principle is that the unmethylated cytosine (C) can be transformed into uracil (U) by bisulfite treatment, while the methylated cytosine (5mC) remains unchanged. By designing a specific probe for the transformed sequence, methylated and unmethylated sites can be distinguished, and finally, the methylation level of each site can be reflected by β value (the ratio of methylated signal to total signal) (range 0-1).

common methylation chip platforms include illumina's 935k, 450k, epic (850k), and the latest MSA chip. The number of sites covered ranges from 27,000 to nearly 300,000, focusing on regions with regulatory functions such as promoters, CpG islands, and enhancers. Its technical advantages lie in Qualcomm's quantity, low cost, and standardization. It can detect methylation patterns of thousands of samples at the same time, and is widely used in the fields of epigenome association study (EWAS), disease marker screening (such as early diagnosis of cancer), cell type grouping, and apparent clock construction. For example, hypermethylation sites of tumor suppressor gene promoters can be identified by methylation chip, which provides a basis for noninvasive diagnosis of cancer.

Illustration of sample loading and primer deposition on an individual functional unit (Zhang et al., 2009)

Proteomics

Protein omics is a discipline that studies the composition, expression level, post-translational modification, and interaction of all proteins of an object or cell in a specific state, aiming at analyzing the molecular mechanisms of life activities at the protein level. Its core technologies include quantitative protein omics based on mass spectrometry (MS) (such as iTRAQ and TMT) and protein chip technology. Mass spectrometry-based protein proteomics realizes the identification and quantification of peptide fragments by enzymolysis of protein and liquid chromatography-mass spectrometry (LC-MS/MS), which can simultaneously detect the expression of thousands of proteins and their phosphorylation, acetylation, and other modifications.

A protein chip can detect the presence of specific proteins in the sample by immobilizing known proteins or antibodies (such as the level of cytokines detected by an antibody chip). The advantage of protein omics is that it directly reflects the state of biological function executors, fills the "information gap" between genome and transcriptome and phenotype, and is widely used in disease classification (such as cancer subtype classification), drug target discovery (such as tumor kinesin identification) and signal pathway analysis (such as protein interaction network in immune response).

Smoking: Short-term Innate and Epigenetic Long-term Adaptive Immune Effects

Title: Smoking changes adaptive immunity with persistent effects

Publish Magazine: Nature

Impact Factors: 48.5

Publication Time: 2024.02.22

There are significant differences in individual immune responses. Age, sex, and genetic factors are the main known influencing factors, but the driving factors of cytokine secretion variability are still not completely clear. Environmental factors such as smoking, latent infection of cytomegalovirus (CMV), and body mass index (BMI) are speculated to be involved in regulating immune response, but their specific functions and relative importance with traditional factors are still unclear. In addition, whether smoking has short-term and long-term effects on immunity and its potential molecular mechanisms (such as epigenetic regulation) still needs to be further explored.

Through the Milieu Intérieur cohort (1000 healthy individuals), this study systematically analyzed the secretory relationship between 136 variables and 13 cytokines under 11 kinds of immune stimuli, focusing on the differential effects of smoking on innate and adaptive immunity and its persistence mechanism, to provide a theoretical basis for risk assessment of infection, cancer, and autoimmune diseases.

Smoking effects on innate and adaptive immune responses, represented by E. coli and SEB stimulations, respectively (Saint-André et al., 2024)

In order to explore the key influencing factors of immune stimulus response, principal component analysis (PCA) and hierarchical clustering methods were used to deeply analyze the expression patterns of cytokines induced by different types of immune stimuli. The results showed that innate immune stimulators (such as live Escherichia coli and lipopolysaccharide LPS) and adaptive immune stimulators (staphylococcal enterotoxin B, SEB). The cytokine profile induced by the anti-CD3+CD28 antibody complex showed significant clustering separation characteristics.

Furthermore, through correlation analysis, the key host factors affecting cytokine secretion were explored. The results showed that the effects of smoking status, cytomegalovirus (CMV) infection, and body mass index (BMI) on cytokine secretion were equivalent to those of age, sex, and genetic factors in traditional cognition. Among them, smoking behavior is significantly correlated with the secretion levels of IL-2 and IL-13 under adaptive immune stimulation (SEB) and the release of CXCL5 under innate immune stimulation (E.coli). CMV infection mainly regulates the expression of adaptive immune-related cytokines, such as colony-stimulating factor 2(CSF2) and interferon-γ (IFNγ). BMI is closely related to the secretion level of CXCL5 stimulated by BCG. These findings reveal the multidimensional influence of environmental exposure and physiological state on the immune regulatory network.

Effects of smoking on induced cytokines is modified by blood cell subsets and plasma proteins (Saint-André et al., 2024)

Influence Mechanism of Smoking on Innate Immunity: Further analysis reveals that smoking has a significant double-effect mechanism on the immune system. At the level of innate immunity, it is found that the level of CXCL5, a chemokine secreted by epithelial cells and immune cells in smokers, is abnormally high, and the overexpression of this proinflammatory factor will accelerate the recruitment of neutrophils and form a persistent inflammatory microenvironment. When smokers stop taking nicotine and other tobacco components, the secretion level of CXCL5 can quickly fall back to the baseline level within a few weeks, showing the plasticity of innate immune response.

Long-term effects of smoking on adaptive immunity: In contrast, the change of adaptive immune response is more persistent. The longitudinal follow-up data of smokers for 12 months showed that the secretion levels of key cytokines IL-2 and IL-13 in peripheral blood still deviated significantly from the normal range. In-depth analysis of the changes of immune cell subsets shows that the ability of CD4+ Th1 cells to secrete IL-2 continues to be impaired, while the activity of Th2 cells to secrete IL-13 remains abnormally enhanced, which, together with the changes of B cell memory function, constitute the basis of long-term immune abnormality.

Induced cytokine variance explained (Saint-André et al., 2024)

At the molecular level, plasma protein CEACAM6 plays a key role in the regulation of innate immunity by smoking. As a member of the carcinoembryonic antigen-related cell adhesion molecule family, CEACAM6 can change its chemotaxis, phagocytosis, and cytokine secretion functions by binding to neutrophil surface receptors, thus reshaping the innate immune response pattern. In the field of adaptive immunity, B cells and regulatory T cells (Treg) subsets have become the core mediators of smoking effects. Studies have shown that smoking can induce abnormal activation of B cells, leading to increased secretion of autoantibodies. At the same time, the immunosuppressive function of Treg cells is significantly up-regulated, which may change their transcription activity through Foxp3 methylation modification, and eventually lead to the imbalance of immune homeostasis.

Genetic correlation analysis further revealed the complex interactive network between smoking and immune regulation. In this study, 44 protein quantitative trait loci (pQTLs) related to Toll cytokine response were identified, including toll-like receptor 3(TLR3), immunoglobulin G Fc receptor IIA (FCGR2A), and other key immune genes. It is worth noting that there is a significant interaction between some pQTLs and smoking, suggesting that genetic background may affect the difference in immune response induced by smoking.

Transcriptomics and Proteomics Reveal Key Pro-Inflammatory Pathways Mediating SA and EAA

Title: Subclinical atherosclerosis and accelerated epigenetic age mediated by inflammation: a multi-omics study

Publish Magazine: European Heart Journal

Impact Factors: 35.86

Publication Time: 2023.06.20

As an early stage of cardiovascular disease (CVD), subclinical atherosclerosis (SA) is high in middle-aged people but often has no clinical symptoms. Its impact on life expectancy and its potential mechanism are still unclear. Epigenetic age (such as Grim clock), as an accurate marker of biological age, can predict healthy life span and death risk, but its association with SA and its regulation mechanism still need to be explored. In addition, the role of inflammation in the progression of SA has been of concern, but how to analyze its mediating role between SA and epigenetic aging through multi-group data is still a research blank.

In this study, the progress of early subclinical atherosclerosis (PESA) cohort, combined with the data of methylation group, transcription group and protein group, was used to explore the relationship between SA and accelerated epigenetic age (EAA), and to reveal the mediating role of inflammation in this process, so as to provide theoretical basis for early intervention of CVD.

The accelerated epigenetic age of individuals with subclinicalatherosclerosis is mediated by a low-grade, chronic, systemic inflammation driven by key inflammatory cytokines and pathways (Sánchez-Cabo et al., 2023)

• A. Multivariate analysis of inflammatory pathway
  • a) The results showed that there was a significant correlation between the existence of SA, the range of lesions, the degree of progression, and the accelerated epigenetic age of Grim. It is worth noting that this correlation is only manifested in the Grim epigenetic clock, but there is no significant correlation with other epigenetic clocks such as the Horvath clock and Hannum clock, which are commonly used to predict the actual age.
  • b) From the specific indicators, the increase of plaque load, the increase of coronary artery calcification (CAC) score, and the increase of the number of affected vascular regions, which are accurately measured by 3D vascular ultrasound technology, are positively correlated with the level of Grim's accelerated epigenetic age (Grim EAA). This discovery has important clinical significance, which means that even after excluding the interference of traditional cardiovascular risk factors such as smoking history and diabetes, the correlation between SA and Grimea still exists steadily, highlighting its independent pathophysiological significance.

Progression of Early Subclinical Atherosclerosis participants with larger extension ofsubclinical atherosclerosis have accelerated grim epigenetic age (Sánchez-Cabo et al., 2023)

• c) In the multi-ethnic study of atherosclerosis (MESA) cohort, researchers conducted an in-depth analysis of people with different accelerated epigenetic ages. The results showed that the positive detection rate of coronary artery calcification (CAC) in the Grimea accelerated group was twice as high as that in the normal group. The large sample data of this cohort study further verified the close relationship between SA and epigenetic aging, and provided a new perspective for understanding the interaction between subclinical cardiovascular diseases and the aging process of the body.
• d) Further analysis found that individuals with accelerated Grimea showed significant characteristics in disease progression and risk assessment. The 3-year SA progression rate of these individuals was as high as 43.9%, which was nearly twice as high as that of the normal group (24.1%), and the difference was statistically significant (P<0.05). At the same time, the key cardiovascular risk assessment indicators, such as ASCVD score (from 8.2 2.1 to 13.5 3.4 in the normal group) and cardiovascular age (with an average difference of 7.8 years), all showed a significant upward trend, which strongly suggested that the risk of CVD in the future increased significantly.

Subclinical atherosclerosis association with Grim epigenetic age acceleration in theMulti-Ethnic Study of Atherosclerosis study (Sánchez-Cabo et al., 2023)

• B. Significance of the integration of multi-omics
  • a) Multiomics-mediated analysis revealed that SA affected EAA through inflammatory pathways: at the transcriptome level, key genes such as IL1B, OSM, and TLR5 were significantly enriched in inflammatory corpuscles, IL-6 and IL-10 signaling pathways. The abnormal expression of these genes indicates the cascade activation of the inflammatory reaction. Among them, IL1B, as a core member of pro-inflammatory cytokines, can activate a variety of inflammatory mediators downstream. OSM participates in the process of chronic inflammation by regulating cell proliferation and immune response. As a pattern recognition receptor, TLR5 can recognize the molecular patterns related to pathogens and further aggravate the inflammatory reaction.
  • b) At the level of protein group, CRP, HP, and other classical inflammatory markers showed a significant upward trend. As an acute phase reaction protein, CRP's elevated level can directly reflect the inflammatory state of the body. HP participates in the regulation of the inflammatory microenvironment by binding hemoglobin. These inflammatory molecules lead to significant changes in immune cell subsets through complex immune regulation mechanisms: the abnormal increase of neutrophils indicates that the body is in a state of persistent inflammatory stress; However, the decrease of CD8+T cells may weaken the body's anti-tumor and anti-virus immunity.

Progression of Early Subclinical Atherosclerosis individuals with accelerated grimepigenetic age have a higher predicted risk of having a cardiovascular event in 10 years and anincreased cardiovascular age predicted by traditional cardiovascular risk scores (Sánchez-Cabo et al., 2023)

Conclusion

The integrated application of methylation chip and protein omics provides a multi-dimensional perspective for analyzing the relationship between epigenetic regulation and protein function. By capturing the dynamics of epigenetic modification at the genome level through methylation chip, combined with the functional molecule expression map presented by protein omics, not only can the regulatory link from DNA methylation to protein phenotype be revealed, but also potential epigenetic-protein cross markers can be tapped in the study of disease mechanism. This multi-omics fusion strategy not only makes up for the limitations of a single technology in analyzing complex biological processes, but also provides a more systematic basis for target discovery and curative effect evaluation.

References

1. Saint-André, V., Charbit, B., Biton, A., et al. "Smoking changes adaptive immunity with persistent effects." Nature. 2024 626 827–835 https://doi.org/10.1038/s41586-023-06968-8
2. Sánchez-Cabo F, Fuster V, Silla-Castro JC, et al. "Subclinical atherosclerosis and accelerated epigenetic age mediated by inflammation: a multi-omics study." Eur Heart J. 2023 44(29): 2698-2709 https://doi.org/10.1093/eurheartj/ehad361
3. Zhang Y, Bailey V, Puleo CM, et al. "DNA methylation analysis on a droplet-in-oil PCR array." Lab Chip. 2009 9(8): 1059-1064 https://doi.org/10.1039/b821780g