EGFR-dependent epigenetic signatures in epidermal differentiation

Cell type specific gene expression is ultimately at the heart of cellular identity. While cell type specific transcription factors are one aspect behind this diversity in gene expression, the "epigenetic" identity of each cell type presents a more basic form of information according to which individual transcriptional programs are being executed. The last decade has brought significant progress in understanding the biochemical mechanisms which are associated with epigenetic gene regulation, yet very little is known about how external signals, which are essential for cell fate decisions, orchestrate the genome-wide changes in chromatin configuration which result in heritable changes of gene expression. Understanding how receptor signaling establishes a cell type defining "epigenome" presents a major challenge for the next decade in the field of chromatin research. The Epidermal Growth Factor Receptor (EGFR) is a critical regulator of skin homeostasis and differentiation. Importantly, EGFR signaling prevents the premature differentiation of epidermal cells in the skin, a phenomenon which can be recapitulated in vitro by culturing EGFR-deficient keratinocytes. Recently, epigenetic mechanisms such as DNA methylation have been demonstrated to regulate epidermal differentiation, similar to EGFR function. One of the questions we will address in this proposal is, therefore, if and how EGFR signaling regulates epigenetic mechanisms controlling the cellular identity of epithelial cells during (epidermal) stem cell self-renewal, differentiation and tissue homeostasis. We will address this by chromatin immunoprecipitation followed by genome wide sequencing in order to generate "epigenomic" maps of normal and EGFR-deficient epidermal cells, thus visualizing EGFR`s impact on chromatin signatures associated with epidermal stem cell homeostasis. In addition, we will analyze the differentiation defect of EGFR-deficient cells in detail, focusing on a link between EGFR function, Dnmt1 expression and DNA methylation.

The last decade has brought significant progress in understanding the biochemical mechanisms which are associated with epigenetic gene regulation, yet very little is known about how external signals, which are essential for the induction of cellular differentiation, orchestrate the genome-wide changes in chromatin configuration which result in heritable changes of gene expression. Understanding how receptor signaling establishes a cell type defining epigenome represents a major challenge for the next decade in the field of chromatin research. We found that the Epidermal Growth Factor Receptor (EGFR) is a critical regulator of skin homeostasis and differentiation. Importantly, EGFR signaling prevents the premature differentiation of epidermal cells in the skin, a phenomenon which can be recapitulated in vitro by culturing and differentiating EGFR-deficient primary keratinocytes. We therefore analyzed whether EGFR is one of the regulators orchestrating chromatin remodeling in the differentiating skin. One of the questions we have addressed is, if and how EGFR signaling regulates epigenetic mechanisms controlling the cellular identity of epithelial cells during stem cell self-renewal, differentiation and tissue homeostasis. We investigated how EGFR signaling regulates epigenetic mechanisms controlling the cellular identity of epithelial cells by Chromatin Immunoprecipitation (ChIP) followed by Next Generation Sequencing (NGS), using keratinocytes as a model system. To complement this analysis, we have addressed the differentiation defect of EGFR-deficient epidermal cells in detail, focusing on a link between EGFR signaling and Dnmt1 expression and function.Major findings:Extensive transcriptome analysis of self-renewing and differentiating Keratinocytes by RNAseq has shown that the loss of EGFR-signaling affects gene expression profiles strongly in the undifferentiated state. These changes are also observed after differentiation with little additional changes. In particular, aberrant Keratin gene expression are detectable along with changes in the epigenetic signature at this multigene cluster. Furthermore EGFR-deficient keratinocytes display a more mesenchymal-like gene expression profile.We have generated transgenic mice overexpressing Dnmt1 in the epidermis. This transgene does not rescue the EGFR-mutant phenotype. In addition, DNA methylation is normal on a genome wide level in EGFR-deficient animals as monitored by whole genome bisulfite sequencing. This indicates that EGFR does not function through aberrant patterning of DNA methylation.