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

Keratinocytes undergo a complex differentiation process, coupled with extensive changes in gene expression through which they acquire distinctive features indispensable for cells that form the external body barrier-epidermis. Disturbed epidermal differentiation gives rise to multiple skin diseases. The involvement of epigenetic factors, such as DNA methylation or histone modifications, in the regulation of epidermal gene expression and differentiation has not been fully recognized yet. In this work we performed a CRISPR/Cas9-mediated knockout of SUV39H1, a gene-encoding H3K9 histone methyltransferase, in HaCaT cells that originate from spontaneously immortalized human keratinocytes and examined changes in the expression of selected differentiation-specific genes located in the epidermal differentiation complex (EDC) and other genomic locations by RT-qPCR. The studied genes revealed a diverse differentiation state-dependent or -independent response to a lower level of H3K9 methylation. We also show, by means of chromatin immunoprecipitation, that the expression of genes in the LCE1 subcluster of EDC was regulated by the extent of trimethylation of lysine 9 in histone H3 bound to their promoters. Changes in gene expression were accompanied by changes in HaCaT cell morphology and adhesion.

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

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.

S100A6 activates EGFR and its downstream signaling in HaCaT keratinocytes.

Epidermal growth factor receptor (EGFR) is a central transmitter of mitogenic signals in epithelial cells; enhanced EGFR activity is observed in many tumors of epithelial origin. S100A6 is a small calcium-binding protein, characteristic mainly of epithelial cells and fibroblasts, strongly implicated in cell proliferation and upregulated in tumors. In this study, using biochemical assays along with immunohistochemical and immunocytochemical analysis of organotypic and standard cultures of HaCaT keratinocytes with S100A6 overexpression or knock-down, we have examined the effect of S100A6 on EGFR activity and downstream signaling. We found that HaCaT cells overexpressing S100A6 had enhanced EGFR, phospho EGFR, and phospho extracellular signal-regulated kinase 1/2 (pERK1/2) staining intensity and level coupled to higher signal transducer and activator of transcription 3 (STAT3) activity. Conversely, S100A6 knockdown cells had impaired EGFR signaling that could be enhanced by addition of recombinant S100A6 to the culture media. Altogether the results show that S100A6 may exert its proproliferative effects through activating EGFR.

Comparison of DNA Methylation and Expression Pattern of S100 and Other Epidermal Differentiation Complex Genes in Differentiating Keratinocytes.

Epidermal Differentiation Complex (EDC) is a gene cluster on human chromosome 1 q21, which comprises genes encoding four protein families: S100, S100 fused (SFTP), small proline-rich region (SPRR) and late cornified envelope (LCE) proteins. Contrary to the latter three families, which group proteins important for skin barrier formation, the role of S100 proteins has not been well defined and there are no systematic comparative data concerning their expression in the epidermis. Furthermore, little is known about epigenetic mechanisms controlling changes in S100 and other EDC genes expression in differentiating epidermis. In our study, using real-time PCR, we followed the expression of nine S100 genes at subsequent stages of differentiation of primary human keratinocytes and found that they exhibited different expression patterns. Then, we confronted the expression level in undifferentiated and differentiated keratinocytes with the extent of DNA methylation within their promoter or intragenic regions assessed by bisulfite sequencing. Methylation analysis was also performed for three other EDC genes of known expression pattern (involucrin, loricrin, and NICE-1) and a recently identified evolutionary conserved region with defined enhancer properties. The results indicate that altered EDC genes expression is not accompanied by major changes in DNA methylation.