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.