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

葡萄膜黑色素瘤（UM）是一种眼癌，可根据肿瘤的基因表达模式（GEP）、3号染色体拷贝数或基因突变模式分为两大类。 丢失整个3号染色体（3号单体性，M3）的UM经常转移，而丢失3号二体性（D3）的UM与良好的预后相关。在几乎所有具有单体3的UM中，位于3 p21上的肿瘤抑制基因BAP 1（BRCA 1相关蛋白1）在剩余的3号染色体上显示失活体细胞突变。由于其染色质修饰功能，BAP 1可被视为表观遗传修饰剂。最近，我们对4种原发性UM的DNA进行了全基因组亚硫酸氢盐测序（WGBS），并能够定义一组两种UM类别之间的差异甲基化区域（DMR）。这些DMR可用于基于其DNA甲基化模式对UM进行分类。在这里，我们想更详细地描述M3和D3肿瘤的UM类特异性甲基化模式。此外，为了鉴定肿瘤发生过程中出现的复发性甲基化变化，我们将通过WGBS分析黑素细胞前体细胞（来自葡萄膜的黑素细胞），并将其甲基化组与原发性肿瘤的甲基化组进行比较。为了检查差异甲基化区域的调节作用，我们将通过RNA测序确定主要UM的转录组，并将DMR甲基化数据与相邻基因的基因表达水平相关联。由于我们发现所有鉴定的DMR中有25%位于同一基因的两个不同转录起始位点（TSS）之间，因此我们将测试这些DMR的甲基化是否可能与差异TSS使用相关。该项目的主要重点是BAP 1在肿瘤发展和进展过程中细胞表观遗传重编程中的作用。我们将评估在具有D3的UM细胞系中BAP 1失活是否足以诱导M3或M3样甲基化模式。我们将检查反之亦然，在M3细胞系中恢复BAP 1野生型表达是否足以恢复基因组编辑细胞中的D3甲基化模式。为了在UM细胞系中进行BAP 1的基因组编辑，将利用CRISPR/Cas技术。为了监测经历BAP 1编辑的细胞中的表观遗传类别转换，我们将通过深度扩增子测序对一组类别特异性甲基化DMR进行靶向甲基化分析，这允许精确定量检测甲基化模式。如果本申请的结果为BAP 1失活在UM细胞的表观遗传重编程中的因果作用提供足够的证据，那么我们的长期目标是进一步研究BAP 1失活在UM细胞的表观遗传重编程中的因果作用。剖析表观遗传重编程的机制。

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.