Impact of BAP1 inactivation on the formation of the class specific methylation pattern in uveal melanoma

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Uveal melanoma (UM) is an eye cancer which can be divided in two major classes based on the tumours` gene expression pattern (GEP), chromosome 3 copy number or gene mutation pattern. UM with loss of an entire chromosome 3 (monosomy 3, M3) frequently metastasize whereas UM with disomy 3 (D3) are associated with a favourable prognosis. In almost all UM with monosomy 3 BAP1 (BRCA1 associated protein 1), a tumour suppressor gene located on 3p21, shows inactivating somatic mutations on the remaining chromosome 3. Due to its chromatin modifying function BAP1 can be regarded as epigenetic modifier.Recently we have performed whole genome bisulfite sequencing (WGBS) on DNA from 4 primary UM and were able to define a set of regions that are differentially methylated (DMRs) between both UM classes. These DMRs can be used to classify UM based on their DNA methylation pattern. Here we want to characterize the UM class specific methylation patterns of M3 and D3 tumours in more detail. In addition, to identify recurrent methylation changes that arise in the course of tumourigenesis, we will analyse the melanocytic precursor cells (melanocytes from the uveal tract) by WGBS and compare their methylome with that of the primary tumours. To examine the regulatory effects of the differentially methylated regions we will determine the transcriptome of the primary UM by RNA sequencing and correlate the DMR methylation data with the gene expression levels of neighbouring genes. As we found 25% of all identified DMRs located between two different transcription start sites (TSS) of the same gene we will test if methylation of these DMRs might be associated with differential TSS usage. The main focus of this project addresses the role of BAP1 in the epigenetic reprograming of a cell during tumour development and progression. We will evaluate if BAP1 inactivation in UM cell lines with D3 is sufficient to induce a M3 or M3 like methylation pattern. And we will examine if vice versa restoring BAP1 wild-type expression in M3 cell lines is sufficient to restore the D3 methylation pattern in the genome edited cells. In order to perform genome editing of BAP1 in UM cell lines will utilize CRISPR/Cas technology. To monitor the epigenetic class switch in the cells subjected to BAP1 editing we will perform targeted methylation analysis of a set of class specific methylated DMRs by deep amplicon sequencing which allows for precise quantitative detection of methylation patterns.If the results of the present application provide sufficient evidence for a causal role of BAP1 inactivation in epigenetic reprogramming of UM cells it is our long term goal to further dissect the mechanisms of epigenetic reprogramming.