3.3. Alterations in CpGs methylation pattern potentially disrupt binding affinity of transcription factors
Lengthwise sequences including CpGs at HLA-G promoter and CpG41 of HLA-DRB1 gene, transcription factor (TF) binding sites were appointed to specified positions utilizing the JASPAR CORE open-source database 2020 26, a collection of all most recently defined transcription factor binding sites for eukaryotes. Figure 1 illustrates curated TFs occupying either forward (+) or reverse (-) DNA strand, limited only to those comprising CpG sites in their consensus motif. Methylation status at specific Cytosines could potentially affect their recognition capacity and/or binding affinity and to uncover such disruptions we have emphasized only on the CpG areas of genes that manifested statistically significant results in the present study.
Analysis revealed different sets of TFs, mainly composed of factors with zinc finger motifs (ZIC1, ZIC4, ZIC5, ZBTB7A, ZNF281 ), subunits of NFKB factor (REL, RELA , p100 and p105 subunits) and TFs from the Helix-Loop-Helix family (NHLH2, TCF3 ), known to influence essential biological functions.
Related epigenetic research has shown that HLA-G is demethylated and expressed in various types of tumors and further correlated with immune tolerance to malignancy, tumor escape and poor prognosis27. Predicted TF binding sites within the HLA-G promoter include zinc finger proteins ZIC1, ZIC4 and ZIC5 and NF-kappa-B p100 and p105 subunits, among which ZIC5, a known transcriptional repressor recognizes the CpG site 2 exhibiting the highest demethylating value with statistical significance among the FA group (Figure 1).
ZBTB7A and ZNF281 acquiring zinc finger motifs, REL and RELA subunits of NFKB factor and NHLH2 (Helix-Loop-Helix) are among TFs with recognition binding sites residing across CpG41 of HLA-DRB1 gene (Figure 1). Also, co-existence of genetic variants clearly showed an impact towards DNA methylation spread (Figure 4). These results reflect a regulatory role for this gene’s area, located within HLA-DRB1 gene body. Potential roles for the TFs binding are downstream enhancer and/or insulator and/or nucleosome modulatory properties.
Discussion
DNA methylation signatures across specific genetic-loci emerge as essential diagnostic and disorder-monitoring tools for FA. Furthermore, DNA methylation could distinguish allergy-risk epigenetic profiles, predict responses to therapy or inception of the natural desensitization process.
Previous studies have validated DNA methylation across the top-ten gene loci and showed altered hyper- or hypo-methylation profiles among children suffering from cow’s milk allergy (CMA) (IgE and non IgE-mediated), relative to controls. Methylation disruptions were sex-dependent and disappeared upon developing tolerance to CMA28, confirming the plasticity of epigenetic events. Results were not replicated by other epigenetic studies in CMA29,30, herein differences can be attributed to cell source subjected to analyses and the sort of FA exhibited, either IgE- or non ΙgE-mediated CMA. Also, specific DNA methylation signatures have been underscored in twelve gene loci between peanut allergic versus nonallergic participants. DNA methylation status of BDNF and CXCL12genes exhibited the most superior diagnostic potential compared to serum peanut–specific IgE detection for discriminating allergy31.
Our study revealed similar methylation profiles of FOXP3 TSDR and CpG73 of HLA-DQB1 in both IgE-mediated food allergic samples and controls; FOXP3 TSDR exhibited a hypermethylated profile andHLA-DQB1 an unmethylated (Figures 2A, 2B). In this context, results from HLA-DQB1 gene are not further discussed. Contrariwise, results from CpGs at HLA-G promoter region and CpG island 41 of HLA-DRB1 gene in food allergic samples were deviated from controls (Figures 2C, 2D, 4) and high methylation status was significantly assisted by the presence of SNPs located at CpG island 41. On the contrary, rs1233333, located within HLA-G promoter region had an apparent effect on DNA methylation protection.
Tolerance acquisition in children with IgE-mediated CMA has been shown to involve epigenetic regulation of FOXP3 gene28,32, which has been also suggested as a biomarker of oral tolerance33. Data derived from our study showed insignificant methylation level differences inFOXP3 TSDR between the 64 subjects with IgE-mediated FA versus the 44 clinically healthy children (in both male and female donors), underlying the importance and the great bias potential of epigenetic, tissue and sex related heterogeneity, which should be accounted for the contradictory results obtained between parallel epigenetic studies. However, higher methylation at FOXP3 TSDR CpG sites 1, 5, 6 and 9, both in control and food allergic groups (p=0.004, p=0.004, p=0.011 and p=0.023 respectively) was attributed to parents’ smoking habit (Figure 3A), confirming results from other pediatric disorders deriving from parental burdened epigenetic background22. Analogous results were displayed from methylation analyses at the HLA-G promoter region (Figure 3B).
Methylation status of CpG41, located at exon 2 of HLA-DRB1 and CpGs lying at promoter region of HLA-G manifested their contingent nature to FA predisposition. Genetic and epigenetic contribution of HLA-DRB1 has been established in multiple sclerosis, and rheumatoid arthritis2425, diseases with evident autoimmune features. Recent findings from GWAS in an Australian population trying to decode the genetic background association between IgE-mediated peanut allergy and HLA-DRB1, have established an amino acid variant located at position 71 within the peptide-binding groove 34. In the present study, methylation status and SNPs residing within GpC island 41 highlighted their capacity to influence IgE-mediated FA development, however SNPs detected (Figure 1) failed to be significantly corelated with DNA methylation status along the same genetic area. Nevertheless, it was significantly demonstrated that high DNA cytosine methylation is accompanied with genetic SNPs (Figure 4). Our results suggest that critical SNPs that would differentiate the challenge-proven food allergic samples from controls, potentially reside within the genetic loci surroundings and a broader genetic area needs to be investigated to endorse more accurate results.
The attempt to investigate possible disruptions caused in transcription factor (TF) binding affinity by the differential methylation status of CpG 41, revealed a variety of critical factors predicted to have recognition sites (Figure 1). These TFs potentially co-operate with factors acting as downstream enhancers (probably the NFKB subunits) and/or may accomplish insulator power.
To the best of our knowledge results from this study are the first which show the potential involvement of CpG rich areas of HLA-Gpromoter and HLA-DRB1 , with simultaneous coexistence and cooperation of genetic variants, in FA. Demethylation of the HLA-G promoter region among FA samples potentially induces differentiation of a subset of CD4+T cells towards TregHLA-G+/FOXP3- with potent immunosuppression functions14, similar to the natural, thymus-derived Tregs, as a premature immune response towards the desensitization process. Another important result from the present study was the significant correlation of rs1233333, residing inHLA-G promoter region, with DNA methylation levels, exerting its modulatory effect in gene’s expression by preventing DNA methylation spreading (Figure 2).
Allergic reactions should be considered in terms of complex pathways’ cooperation anticipating to decode synergistic effects towards FA onset and thus, an augmented analysis should be conducted. Obstacles to overcome are the insufficient quantities of pediatric samples, which pose limitations for extended analysis requirements. Other limitations are the inconsistent time points of specimen collection, the wide range of FA symptoms’ manifestation or the drug regimens utilized in clinical research studies, underlying discrepancies in results among different research conducted. Nevertheless, targeted research holds great promise for decoding the FA complex genetic/epigenetic background and offer new insights on molecular events contributing to FA development.
Acknowledgments: Authors wish to thank all patients and donors for providing samples for the completion of this research.