The Effect of Single CpG Demethylation on the Pattern of DNA-Protein Binding

Abstract

Epidermal differentiation is a complex process and its regulation may involve epigenetic factors. Analysis of DNA methylation in 20 selected regions within the epidermal differentiation complex (EDC) gene cluster by targeted next-generation sequencing (NGS) detected no or only minor changes in methylation, mostly slight demethylation, occurring during the course of keratinocyte differentiation. However, a single CpG pair within the exon of the PGLYRP3 gene underwent a pronounced demethylation concomitant with an increase in PGLYRP3 expression. We have employed a DNA-affinity precipitation assay (DAPA) and mass spectrometry to examine changes in the composition of proteins that bind to DNA containing either methylated or unmethylated CpG. We found that the unmethylated probe attracted mostly RNA binding proteins, including splicing factors, which suggests that demethylation of this particular CpG may facilitate PGLYRP3 transcription and/or pre-mRNA splicing.

Epigenetic Regulation of Epidermal Differentiation

“…Results of the latter analysis revealed a stable methylation landscape and a lack of correlation between the methylation status and gene expression level. This finding was in agreement with earlier studies focused on genes of the Epidermal Differentiation Complex (EDC), which maintained their methylation status throughout the differentiation of primary keratinocytes despite large changes in expression [ 26 , 27 ]. This may indicate that the impact of DNA methylation on epidermal differentiation is exerted at the early stage, probably deciding on the balance between proliferation/quiescence of progenitor cells, with a lesser effect on gene expression in keratinocytes already poised for differentiation.…”

Beta-defensins and analogs in Helicobacter pylori infections: mRNA expression levels, DNA methylation, and antibacterial activity

“…In fact, its single demethylation site corresponds to its expression in CD4+ T cells [60]. Finally, prominent demethylation of a single cytosine residue situated in the 5th exon of the PGLYRP3 gene is associated with an increment of mRNA level of PGLYRP3 [61]. This information supports our hypothesis that a single demethylation in one specific CpG dinucleotide could reactivate the gene.…”

Effect of SUV39H1 Histone Methyltransferase Knockout on Expression of Differentiation-Associated Genes in HaCaT Keratinocytes

“…To obtain undifferentiated HaCaT cells the medium was exchanged for DMEM without calcium (Life Technologies-Thermo Fisher Scientific, Waltham, MA, USA) supplemented with calcium-free 10% FBS as described in [21], and the cells were cultured in these conditions for at least 14 days. Primary human keratinocytes, NHEK (PromoCell, Heidelberg, Germany), were cultured in Keratinocyte Growth Medium 2 (PromoCell, Heidelberg, Germany) with growth supplement as described in [16]. To induce differentiation of NHEKs and of undifferentiated HaCaT cells the respective culture media were supplemented with 1.8 mM CaCl 2 (final concentration) and the cells were cultured for 72 h.…”

“…Epigenetic regulation through DNA methylation, histone modifications, and miRNA activity may also be involved in this process [13]. Initial studies on DNA methylation of gene promoters revealed clear differences between undifferentiated and differentiated keratinocytes [14], although many gene promoters and putative regulatory regions within EDC were shown to maintain their methylation status during differentiation [15,16]. Lack of major changes in DNA methylation within EDC and elsewhere in the genome was recently confirmed by a genome-wide methylation analysis of in vitro differentiating keratinocytes [17].…”