Elucidating the Effect of DNA Methylation on the Regulation of Human Neonatal Fc Receptor (FcRn)

Abstract

ObjectivesThe neonatal Fc receptor (FcRn) plays a crucial role in maintaining homeostatic blood concentrations of immunoglobulin G and albumin in humans, as well as extending serum half‐lives of therapeutic monoclonal antibodies and albumin conjugates. A series of pH specific binding and dissociation events allow FcRn to recycle or transcytose ligands, affording protection from catabolism and transport across cellular barriers. The interaction of FcRn with biological therapeutics makes it an integral component of observed pharmacokinetic and pharmacodynamic properties. The epigenetic regulation of FcRn, and its contribution to differential FcRn expression in humans, remains unknown. The objective of this study was to determine the location and extent of DNA methylation in the promoter region of the FcRn alpha‐chain encoding gene, FCGRT, in human tissue specimens. We also sought to identify the effect of the DNA demethylating agent 5‐azacytidine on the expression of FCGRT using in vitro cell culture models.MethodsGenomic DNA was extracted from human tissue specimens from heart (n = 10), liver (n = 10), and lung (n = 5). DNA methylation analysis was conducted using the EpiTYPER massARRAY system (Agena), with PCR primers designed to encompass the theoretical promoter region of FCGRT, as well as the 1st exon and 3′‐untranslated region. The effect of 5‐azacytidine on HepG2 cells was evaluated following 72 hours of treatment (1 μM) with daily renewal of drug. Expression of FCGRT was analyzed by quantitative real‐time PCR.ResultsA total of 53 CpG sites were analyzed in the 5′‐untransated region of FCGRT (−1355bp to ATG). The average CpG methylation in this region was 27.0%, 19.5%, and 15.6% for liver, heart and lung tissues respectively. In all tissues analyzed a methylated region was observed immediately prior to the transcription start site (−1355bp to −707bp, relative to ATG). Within this region the highest degree of DNA methylation (mean ± SD) was observed with liver specimens (58.7% ± 12.91), followed by heart (41.2% ± 9.07) and lung (29.9% ± 6.59). On average, CpG sites located between the transcription start site and translation start site were demethylated in all specimens (−693bp to ATG). The highest degree of inter‐individual variability in methylation was observed with CpG sites located between −1135bp and −878bp (relative to ATG) in all tissues. HepG2 cells treated with 5‐azacytidine displayed a 64.4 ± 16.6% increase in FCGRT expression after 72 hours relative to a vehicle treated control (mean ± SD, P = 0.0002, Two‐tailed Students t‐test).ConclusionsThis study successfully identified the DNA methylation profile of the theoretical promoter region of FCGRT. In all tissue specimens DNA methylation followed a specific pattern, with highest methylation occurring at CpG sites immediately prior to the transcription start site. This methylated region of the promoter also encompassed a cluster of CpG sites which displayed the highest degree of methylation variability, in all tissues. Treatment of HepG2 cells with the demethylating agent 5‐azacytidine resulted in an increase in FCGRT expression, indicating DNA methylation may influence the expression of FcRn in humans.Support or Funding InformationThis work was funded by the National Institutes of Health (GM073646, HD076055), and Mae Stone Goode Trust.