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Abstract

Cell identity plasticity is a normal and essential feature of cells during development, but is also a hallmark of diseases such as cancer. The mechanisms involved in suppressing inappropriate cell fates are poorly understood. Gene repression in terminally-differentiated cells is typically thought to be mediated by passive epigenetic silencing, such as through DNA methylation and repressive histone modifications.

However, recent studies suggest that active repression by cell type-specific transcription factors may play an essential role in maintaining cell identity. MYT1L, a neuron-specific transcriptional repressor, has been shown to maintain neuronal identity by the inhibition of non-neuronal lineages. The observation that deficiencies in MYT1L may lead to diseases such as neurodevelopmental disorders and cancer demonstrates its important role in cell identity maintenance by inhibition of cellular plasticity.

In this thesis, I investigate whether active, lifelong transcriptional repression of alternate lineages – or safeguard repression – is a general mechanism for cell identity stabilisation. Using computational prediction and direct cell reprogramming, I demonstrate that PROX1 safeguards hepatocyte identity by directly suppressing alternate lineage programs, hence demonstrating safeguard repression in a second cell type and germ layer. With single-cell transcriptomics analysis, I find that PROX1 robustly silences donor and alternative cell identities.

I also show that PROX1 directly binds to promoters of key mesodermal transcription factors, including Prrx1 and Pparg, hence decreasing their chromatin accessibility and expression. In addition, repression of PROX1 target genes during hepatocyte reprogramming mimicked effects of Prox1 overexpression. On the other hand, Prox1 deletion or target gene activation permitted inappropriate gene expression.

Finally, in a hepatocellular carcinoma mouse model, Prox1 overexpression prevented tumour initiation, reducing overall tumour load and extending survival substantially. Prox1 depletion in the same model induced cholangiocarcinoma-like morphology and gene expression, demonstrating the role of PROX1 in cell identity maintenance.

These results show that PROX1 is a repressive safeguard of hepatocyte identity. They also support a model whereby continuous silencing of alternate lineage gene expression programs by safeguard repressors prevents cell fate plasticity, thereby maintaining cell identity.