Ann Dermatol.  2020 Apr;32(2):122-129. 10.5021/ad.2020.32.2.122.

Epigenetic Regulation of Filaggrin Gene Expression in Human Epidermal Keratinocytes

• Lee J
• Jang A
• Seo SJ
• Myung SC

• KMID: 2471338
• DOI: http://doi.org/10.5021/ad.2020.32.2.122

Abstract

BACKGROUND
Loss-of-function mutations in the filaggrin gene (FLG), which encodes an epidermal protein crucial for the formation of a functional skin barrier, have been identified as a major predisposing factor in the etiopathogenesis of atopic dermatitis (AD). Recent reports of relatively low frequencies of FLG-null mutations among specific ethnic groups with AD necessitated analysis of the epigenetic regulation which may control FLG expression without altering its DNA sequence.
OBJECTIVE
The study aimed to identify DNA methylation-dependent regulation of FLG expression.
METHODS
Quantitative polymerase chain reaction was performed to determine the restoration of FLG mRNA expression in normal human epidermal keratinocyte (NHEK) cells after treatment with epigenetic modulating agents. Bisulfite genomic sequencing and pyrosequencing analyses of the FLG promoter region were conducted to identify the citical CpG sites relevant to FLG expression. We performed small-scale pilot study for epidermal tissues obtained from Korean patients with severe AD.
RESULTS
We here show that DNA methylation in the FLG with non-CpG island promoter is responsible for the transcriptional regulation of FLG in undifferentiated NHEK cells. The methylation frequencies in a single CpG site of the FLG promoter were significantly higher in lesional epidermis than those in matched nonlesional epidermis of subjects with severe AD.
CONCLUSION
Our in vitro and clinical studies point to this unique CpG site as a potential DNA methylation marker of FLG, which can be a promising therapeutic target in the complications of filaggrin-related skin barrier dysfunction as well as in AD.

Keyword

Atopic dermatitis; DNA methylation; Keratinocyte

MeSH Terms

Base Sequence
Causality
Dermatitis, Atopic
DNA
DNA Methylation
Epidermis
Epigenomics*
Ethnic Groups
Gene Expression*
Humans*
In Vitro Techniques
Keratinocytes*
Methylation
Pilot Projects
Polymerase Chain Reaction
Promoter Regions, Genetic
RNA, Messenger
Skin
DNA
RNA, Messenger

Figure

• 

  Fig. 1 Epigenetic regulation of filaggrin gene (FLG) expression in undifferentiated normal human epidermal keratinocyte (NHEK) cells. (A) Quantitative polymerase chain reaction was performed to determine the restoration of FLG mRNA expression in undifferentiated NHEK cells after treatment with DNA demethylating agent 2′-deoxy-5-azacytidine (DAC) or histone deacetylase inhibitor trichostatin A (TSA). (B) A promoter reporter assay of the 5′-end region of FLG in HEK293T cells using the pCpGfree-basicLucia reporter plasmid. The synthesized fragment contained four CpG dinucleotide sites (represented by lollipops). (C) Bisulfite sequencing analysis of the FLG promoter harboring CpG 1 through CpG 4 sites in undifferentiated NHEK cells. Circles represent stand-alone CpG sites, and each row represents the DNA sequence of an individual clone. Unmethylated and methylated CpG units are depicted as white and black circles, respectively. The blue asterisk indicates the location of CpG ID in the Illumina HumanMethylation450 BeadChip: CpG 1, cg19855573; CpG 7, cg13447818; CpG 9, cg10500702; CpG 10, cg26390526. ACTB: beta-Actin, RLU: relative light unit, TSS: transcription start site. *p<0.05.

• 

  Fig. 2 Quantitative methylation analysis of the CpG 4 and CpG 2 dinucleotide units in patients with atopic dermatitis (AD) (n=10). (A) Bisulfite pyrosequencing analysis of the CpG 4 (−702 bp) and CpG 2 (−410 bp) sites in the FLG promoter region. (B) Significant association between non-lesional and lesional tissues was found in the methylation frequencies of the CpG 2 unit. The methylation frequencies of the CpG 4 and CpG 2 units were determined using triplicate experiments of bisulphite pyrosequencing. *p<0.05.

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