Molecular Mechanism Of STAT5/TET2Regulating DNA Methylation Of FCER1G Promoter In Monocyte And Its Role In The Pathogenesis Of Atopic Dermatitis

Atopic Dermatitis (AD) is a highly pruritic chronic inflammatory skin disorder affecting10-20%of children worldwide. Symptoms can persist or begin in adulthood. Prevalence of this disease has increased by two to three-fold during the past three decades in industrialised countries. AD is a major public-health problem worldwide with a lifetime prevalence of10-20%in children and1-3%in adults.The pathogenesis of AD is unclear, the complex interrelation of genetic, immunological, and environmental factors contributes to its pathogenesis. The steadily increasing prevalence of AD is suggestive of an important role played by enviromental factors in its pathogenesis. Epigenetics as a mechanism mediating gene-environment interaction offers new opportunities to advance novel concepts about this disease. Epigenetics is the study of heritable changes in gene expression that occur without DNA sequence alteration. Epigenetic regulation consists mainly of DNA methylation, histone modification, and microRNA expression. In this study, we found that CD14+monocytes from patients with AD had global DNA hypomethylation relative to healthy controls. DNA methylation within the FCERIG promoter was significantly reduced in monocytes from patients with AD, and this was associated with upregulation of FcRyand its related receptors (FcεR1and Dectin-2). Furthermore, we also found that proinflammatory cytokines GM-CSF and TSLP activate the STAT5pathway which promotes DNA demethylase TET2binding to the FCER1G promoter. This leads to demethylation of the FCER1G promoter sequence and upregulation of FcRyand its related receptors (FcεR1and Dectin-2) in monocytes. In addition, pretreatment of monocyte-derived dendritic cells (MoDC) with GM-CSF increased Dectin-2expression, strengthened the ability of allergen uptake, and induced Th2differentiation.Therefore, we propose the pathogenesis of AD as follows:GM-CSF and TSLP lead to DNA demethylation of the FCER1G promoter sequence through activation of the STAT5pathway which promotes TET2binding on the FCER1G promoter in monocytes. TET2causes FCER1G promoter demethylation which enhances the expression of FcRyand its associated receptors (FcεRl and Dectin-2). The end result is increased Th2differentiation which induces hypersensitivity reactions and finally causes AD.Part I Methylation status of the FCERIG promoter and its correlation with FcRy expression in monocytes of AD patientsObjective:To investigate FCER1G promoter methylation status and how it correlates with FcRy expression in monocytes of patients with AD.Methods:CD14+monocytes were isolated from10AD patients and10age and sex matched healthy controls. Genomic DNA was isolated from monocytes using magnetic beads. Global DNA methylation was measured using the MethylampTM Global DNA Methylation Quantification Kit. Bisulfite sequencing was performed to determine the methylation status of the FCERIG promoter region. FcRy mRNA and protein levels were detected by real-time RT-PCR and western blotting, respectively. The expression of Dectin-2in peripheral blood monocytes of AD patients and healthy controls was detected by flow cytometry.Results:There was a significant decrease in global DNA methylation in CD14+monocytes from AD patients compared to healthy controls. Monocytes from AD patients showed a locus-specific hypomethylation at the FCERIG promoter when compared to healthy controls. In addition, the methylation status of the FCERIG promoter was inversely correlated with FcRy expression. Furthermore, CD14+Dectin-2+double positive monocytes were increased in peripheral blood of AD patients compared with healthy controls.Conclusion:There is global DNA hypomethylation in CD14+monocytes of AD patients. Demethylation of specific regulatory elements within the FCERIG promoter promotes overexpression of FcRy and its associated receptor Dectin-2in monocytes of patients with atopic dermatitis.Part II Construction of a special reporter through patch methylation to detect regulatory activity of DNA methylation in the promoter region of the FCER1G geneObjective:To construct a special luciferase reporter to detect regulatory activity of DNA methylation in the FCER1G gene promoter.Methods:We constructed fully-and mock-methylated FCER1G gene promoter luciferase reporters by Patch methylation. Regulatory activity of DNA methylation was detected by comparing luciferase activity of the fully-methylated luciferase reporter with the mock-methylated reporter.Results:Fully-and mock-methylated FCER1G gene promoter luciferase reporters were constructed. The ratio of luciferase activity between fully-methylated and mock-methylated reporters was0.36±0.07:1,(P<0.001).Conclusion:FCER1G promoter activity is methylation-sensitive and regulated by DNA methylation.PART III Molecular mechanism by which STAT5/TET2regulates DNA methylation and transcriptional activity of the FCER1G promoter in monocytesObjective:To investigate the molecular mechanism by which DNA methylation and transcriptional activity of the FCER1G promoter is regulated by STAT5/TET2in monocytes.Methods:CD14+monocytes were isolated from healthy subjects using magnetic beads. They were then treated by GM-CSF and TSLP, respectively. Bisulfite sequencing was done to determine the methylation status of the FCER1G promoter region. Detection of FcRy protein expression and its associated receptors Dectin-2and FcεR1was done using flow cytometry. Immunoprecipitation (IP) was done to determine whether phosphorylated STAT5(pSTAT5) recruites the DNA-binding demethylase TET2. CHIP-PCR was done to determine whether p-STAT5binds to the FCER1G promoter region.Results:GM-CSF and TSLP led to demethylation of the FCER1G promoter and upregulation of FcRyand its associated receptors (Dectin-2and FcεRl). IP confirmed that pSTAT5promotes recruitment of the DNA-binding demethylase TET2. CHIP analysis confirmed that pSTAT5binds to the FCER1G promoter.Conclusions:GM-CSF and TSLP can lead to demethylation of the FCER1G promoter by activating STAT5which recruites DNA demethylase TET2and binds to the FCER1G promoter in monocytes. The end result is upregulated expression of FcR FcRyand its associated receptors (Dectin-2and FcεR1) Part IV Dectin-2’s effect on allergen uptake by MoDCs and induction Th2differentiationObjectives:To investigate the effect of overexpressed Dectin-2on MoDC allergen uptake and Th2differentiation.Methods:CD14+monocytes were isolated from healthy subjects using magnetic beads. The monocytes were then pretreated with GM-CSF for3days. Differentiation into MoDC was then induced by treatment of the cells with GM-CSF and IL-4for7days. This was followed by incubation of MoDCs with CFSE-labeled CD4+T cells and stimulation with OVA for3days. T cell proliferation was analyzed by flow cytometry. The mRNA levels of IL-4, IL-13, and IFN-y in T cells were measured using RT-PCR. Finally, MoDCs were incubated with OVA-FITC or anti Dectin-2-PE for30minutes, followed by determination of OVA uptake and Dectin-2expression using fluorescence microscopy.Results:There was high Dectin-2expression and increased OVA uptake in MoDCs pretreated with GM-CSF compared to controls. T cell proliferation and the mRNA expression of IL-4and IL-13were enhanced in MoDCs pretreated with GM-CSF compared to controls. Conclusion:GM-CSF leads to aberrant up-regulation Dectin-2in MoDCs and promotes allergen uptake and Th2differentiation.(27pictures,25tables,150references)...