Chromatin and stem cell adaptation in wound healing and skin cancer

In numerous adult epithelia, specific pools of stem cells continually renew multiple cell types throughout life. While maintaining their lineage identity, stem cells and their differentiated progeny tightly crosstalk to ensure proper tissue homeostasis. The skin is a physical, chemical and immunological barrier that protect us from the outside environment. Following an injury, distinct cell lineages at various stages of differentiation collaborate to restore skin integrity through the wound healing process, in which they acquire an unexpected plasticity. Indeed, when epithelial cells are recruited to repair an injury, they progressively lose their initial identity and are reprogrammed to acquire the lineage of the repaired epithelial niche. Recent studies highlight the existence of shared molecular mechanisms in wound healing and skin cancer, suggesting possible detrimental consequences of cell plasticity (FIGURE 1).

Moreover, the precise transcriptional and chromatin factors that govern this process remain unidentified, and the connection between plasticity and cancer is still incompletely understood.

In the lab (www.donatilab.org), we integrate state-of-the-art molecular and cellular biology techniques in vitro and in vivo to decipher the regulatory mechanisms of cell plasticity in wound healing and its consequences on cell fate, such as cancer.  The first approach that we set up in our lab employs in vitro genetic screenings, such as shRNA pooled screenings. The technology allows to target, in an unbiased way, thousands of transcriptional and chromatin factors. Pooled lentiviral screenings (with shRNAs or CRISPR-Cas9) are powerful tools to elucidate phenotype drivers, gene-gene interactions and determine critical nodes in biological pathways and processes. The combination of functional genomics in primary cell cultures with histological and ‘omics approaches in in vivo models represents a powerful strategy for the study of a complex process as cell plasticity. Therefore, in the lab we use in vivo lineage tracing of different epidermal lineages in longitudinal experiments such as at different phases of wound healing. Lineage tracing allows to follow the progeny of a selected stem cell population; it represents an essential tool for studying stem cell properties in adult mammalian tissues and it is particularly helpful to study cell fate after plasticity acquisition and its long-term consequences. Our research employs a combination of in vitro and in vivo approaches, complemented by high-throughput methods that analyze the transcriptional and epigenetic profiles of cells, both at the bulk and single-cell levels (FIGURE 2).

In particular, to study the transcriptome we employ single cell or bulk RNA-sequencing and a more recent technique, named Spatial Transcriptomic, that add the spatial resolution component to the transcript quantification. Additionally, chromatin profile and protein-DNA binding are assessed through chromatin immunoprecipitation coupled with sequencing (Chip-seq) and chromatin accessibility assay (ATAC-seq). Finally, the lab comprises a team of computational biologists that enable the interpretation and the integration of the data coming from high-throughput techniques. To validate our finding in human skin diseases, we have access to fresh and stored skin samples from our network of national and international of collaborators including several dermatologists.

Recent findings. We recently identified and functionally characterized a so-called distal memory, a cell adaptation of specific epidermal stem cells. Cellular adaptation refers to the capacity of cells to respond to diverse stimuli and adverse environmental changes. While adaptive programs have been widely described in adaptive and innate immune cells, it has recently emerged that epithelial cells in vivo can also acquire a memory. We established a two consecutive skin injuries in vivo model, combining lineage tracing with single cell omics to understand the spatial extent of wound memory and the full spectrum of the adaptive responses of multiple epithelial stem cells in skin. We demonstrated that away from injured site, after a first injury a specific epithelial stem cell population gives rise to long term wound-memory progenitors residing in their own niche of origin. Notably these progenitors have not taken part in the first wound healing but become pre-activated through priming. Mechanistically, we demonstrated that a transcriptional de-repression, linked to the reduction of a H2AK119 ubiquitination (a chromatin marker of transcriptional repression), is functional for memory onset.  We also analyzed the the consequences of distal memory. We found that not only this wound memory exacerbated tumorigenesis, but which onset occurs earlier within these primed cells and follow their spatial distribution. Therefore, we showed that sub-organ scale adaptation of an injury relies on spatially organized and memory-dedicated progenitors, characterized by a chromatin actionable cell state, that establishes an epigenetic field cancerization and predisposes to tumour onset ( FIGURE 3).