The role of epigenetic-mediated regulation of gene expression in the terminal differentiation of keratinocytes has been studied both in vitro and in vivo, with regulators of methylation such as the polycomb repressor complex 2 (PRC2) and DNA Methyltransferases having a role in the coordination of gene expression during squamous differentiation. In particular, the catalytic subunit of PRC2, EZH2, has been shown to be the predominant driver of PRC2 activity. With respect to normal epidermal epithelium, in vitro studies suggest that changes in H3K27me3 mediated by the demethylase JMJD3 are also an important driver of squamous differentiation. However, in vivo studies suggest a more complex role of H3K27me3 and its epigenetic modifiers, where mice that lack H3K27me3 have an intact epidermis. In contrast, the role of PRC2 and EZH2 in differentiation-defective squamous cell carcinoma cells is a little clearer. For example, we have previously shown that abberant differentiation in malignant keratinocytes is attributable to epigenetic defects mediated by pathological overexpression of EZH2 (Gannon, 2013, Clinical Cancer Research, 19;428). However, it remains unclear what the role of H3K27 methylation itself may be in squamous differentiation in normal keratinocytes.
In the present work, we have examined the potential involvement of H3K27 and an additional non-PRC2-mediated repressive histone modification, H3K9me3, in squamous differentiating keratinocytes. We have observed that inhibition of EZH2 can induce differentiation in normal and malignant keratinocytes. However, H3K27me3 or H3K9me3 have not yet been characterized during confluence-induced differentiation. We used ChIP-seq to analyse H3K27me3 and H3K9me3 in proliferative and confluence differentiated keratinocytes in order to 1) characterize the gene-specific changes in epigenetic modifications upon squamous differentiation and 2) characterize novel differentiation associated genes.
Our results suggest that confluence-induced squamous differentiation is associated with a reduction in EZH2 levels in the absence of global changes in H3K27me3 or altered localization of H3K27me3 on differentiation genes. Our results also highlight a potential for H3K9me3 in the regulation of novel squamous differentiation genes.