Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Jun 17;9(1):8674.
doi: 10.1038/s41598-019-45203-1.

Contribution of DNA methylation to the expression of FCGRT in human liver and myocardium

R B Cejas  1 , D C Ferguson  1 , A Quiñones-Lombraña  1 , J E Bard  2 , J G Blanco  3
Affiliations

Contribution of DNA methylation to the expression of FCGRT in human liver and myocardium

R B Cejas et al. Sci Rep. .

Abstract

FcRn mediates recycling and transcytosis of IgG and albumin in various cell types. The MHC-class-I-like protein of the FcRn heterodimer is encoded by FCGRT. Few determinants of variable FCGRT expression in humans have been identified so far. In this study, we investigated the presence of DNA methylation in regulatory regions of FCGRT in samples of human liver and myocardium tissue, and we examined the impact of FCGRT methylation on FcRn expression in model cell lines. Quantitative DNA methylation analysis of the FCGRT locus revealed differentially methylated regions in DNA from liver and myocardium. Methylation status in individual CpG sites correlated with FCGRT mRNA expression. Data from model cell lines suggest that differential methylation in the -1058 to -587 bp regulatory region of FCGRT contributes to FcRn expression. Chromatin immunoprecipitation assays indicate that CpG site methylation impacts the binding of the methylation sensitive transcription factors Zbtb7a and Sp1. This study provides a foundation to further define the contribution of epigenetic factors during the control of FcRn expression and IgG traffic in human tissues.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Quantitative DNA methylation analysis of the FCGRT locus in human tissues. Each point represents DNA methylation ratios ± SD (n = 2–10) at individual CpG sites in liver (upper panel) and myocardium (lower panel). Left panels represent the 5′UTR and partial genic regions of FCGRT. Right panels represent the 3′UTR of FCGRT. Nucleotide positions are relative to the A+1TG (left panel) and T+1GA codons (right panel), respectively. Blue-shaded regions represent CpG islands. The dotted line represents the −744 bp reference site for “distal” and “proximal” 5′UTR regions, and the dashed line represents the transcription start site for FCGRT transcript NM_004107.4 (−222 bp).
Figure 2
Figure 2
DNA methylation status and FCGRT expression correlation analysis in human liver and myocardium. (a) Relative FCGRT mRNA fold expression in samples from human liver (n = 10) and myocardium (n = 10). Each point represents the mean from two separate measurements performed in triplicates. (b) Linear regression analysis of methylation at individual CpG sites in DNA samples from liver (◦) and myocardium (▫) versus FCGRT mRNA relative fold expression. Detection of the −707 bp CpG site failed in 4 liver samples (◦), and detection of CpG sites −1017, −903 and −842 bp was successful in 9 out of 10 myocardial samples (▫). (c) Linear regression analysis of DNA methylation ratios versus FCGRT mRNA relative expression in liver-derived cell lines (n = 16. Data from the Cancer Cell Line Encyclopedia database). Data show mean methylation ratios in the −1021 to −628 bp region of FCGRT. *P < 0.05, **P < 0.01, Spearman rank-order correlation.
Figure 3
Figure 3
FCGRT expression and DNA methylation status in AC16, Hep G2, and SNU-475 cells. (a) Basal FCGRT mRNA relative fold expression. Each point represents individual measurements. Horizontal bars show the mean ± SD from three determinations performed in quintuplicates. *** P < 0.001, ns = not significant, 1-way ANOVA (Turkey’s test). (b) Fluorescence microcopy analysis of basal FcRn expression (yellow). Nuclei were stained with DAPI (blue). Scale bar: 10 µm. (c) Quantitation of FcRn relative expression. Each point represents an individual measurement. Horizontal bars show the mean ± SD (arbitrary units, AU) from a representative experiment. *** P < 0.001, ** P < 0.01 ANOVA (Kruskal-Wallis test). (d) Bisulfite analysis of CpG sites methylation status in the −1058 to −587 bp region of FCGRT. Black circles represent methylated CpG sites (≥1 positive clone), and clear circles represent demethylated CpG sites (no positive clones).
Figure 4
Figure 4
Impact of Aza treatment on FCGRT expression. (a) FCGRT mRNA expression in AC16, Hep G2, and SNU-475 cells after Aza treatment (5 µM, 72 h). Plots depict fold differences in FCGRT mRNA expression levels relative to controls (0 µM Aza). Each point represents individual measurements. Horizontal bars show the mean ± SD from three experiments performed in quintuplicates. *** P < 0.001, ns = not significant, Student’s t test. (b) Cellular FcRn protein expression (yellow) before and after Aza treatment (5 µM, 72 h. Representative images). Nuclei were stained with DAPI (blue). Scale bar: 10 µm. (c) Quantitation of FcRn relative fold expression after Aza treatments. Each point represents individual measurements. Horizontal bars show the mean ± SD from two independent experiments (8–10 fields per condition). *** P < 0.001, ns = not significant, Student’s t test.
Figure 5
Figure 5
Effect of Aza treatment on FCGRT DNA methylation. Methylation levels at individual CpG sites in the −1058 to −587 bp region of FCGRT after Aza treatment (5 µM, 72 h). Circles represent individual CpG sites. Mean methylation level (%) at individual CpG sites were determined by bisulfite sequencing.
Figure 6
Figure 6
Role of DNA methylation on the binding of transcription factors to FCGRT. (a) Bioinformatics analysis of transcription factors potentially targeting the FCGRT locus (−1793 bp to A+1TG region). The −1058 bp to A+1TG region is shown in orange. Regions amplified by primer sets 1, 2, and 3 are indicated in magenta, cyan and green, respectively. CpG sites are represented as circles. (b) Chromatin immunoprecipitation (ChIP) assays evaluating the binding of Sp1, GABPα, and Zbtb7a, to the FCGRT locus in AC16 cells with or without Aza treatment (5 µM, 72 h). Panels show representative agarose gel electrophoresis analysis of PCR amplification products obtained with primers sets 1–3 using input samples and immunoprecipitated DNA samples. IgG controls are also shown (right panels, top, and bottom). Images were cropped from pictures taken from 3 individual agarose gels (full length pictures are included in supplementary material file 1, Figure S3). (c) Quantitative PCR analysis of ChIP samples assessing transcription factor enrichment in FCGRT regions amplified by primer sets 1 and 2. Values are expressed relative to values from IgG control antibody. Data show the mean ± SD from two independent qPCR experiments (3–4 replicates). *** P < 0.0001, 1-way ANOVA (Turkey’s test).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Praetor A, Hunziker W. beta(2)-Microglobulin is important for cell surface expression and pH-dependent IgG binding of human FcRn. J Cell Sci. 2002;115:2389–2397. - PubMed
    1. Dickinson BL, et al. Bidirectional FcRn-dependent IgG transport in a polarized human intestinal epithelial cell line. J Clin Invest. 1999;104:903–911. doi: 10.1172/JCI6968. - DOI - PMC - PubMed
    1. Antohe, F., Radulescu, L., Gafencu, A., Ghetie, V. & Simionescu, M. Expression of functionally active FcRn and the differentiated bidirectional transport of IgG in human placental endothelial cells. Hum Immunol62, 93–105, doi:S0198885900002445 (2001). - PubMed
    1. Ober RJ, Martinez C, Vaccaro C, Zhou J, Ward ES. Visualizing the site and dynamics of IgG salvage by the MHC class I-related receptor, FcRn. J Immunol. 2004;172:2021–2029. doi: 10.4049/jimmunol.172.4.2021. - DOI - PubMed
    1. Anderson Clark L., Chaudhury Chaity, Kim Jonghan, Bronson C.L., Wani Manzoor A., Mohanty Sudhasri. Perspective – FcRn transports albumin: relevance to immunology and medicine. Trends in Immunology. 2006;27(7):343–348. doi: 10.1016/j.it.2006.05.004. - DOI - PubMed

Publication types

MeSH terms