The coordinates and numbers of insertions of transposons reflect the locations where the factor binds and the proportion of time the factor is bound to the locus. Transposon integration by targeted transposases has been used to identify genomic regions in several contexts. BAP1 mutations have been found in other aggressive cancers, including skin-derived melanomas, mesotheliomas, and renal cell carcinomas, suggesting a general role for BAP1 as a suppressor of metastasis in cancer. Loss of BAP1 causes UM cells to assume a rounded, epithelioid morphology, to deposit distinctive extracellular matrix materials, and to grow well under clonogenic conditions, and BAP1-depleted UM cells display increased diapedesis through endothelial monolayers in a cell-culture model of transendothelial migration, which may reflect their ability to metastasize. Over 95% of class 2 UMs show complete loss of expression of BAP1 protein, with inactivating somatic mutations in the BAP1 gene in 80% of these tumors. Class 2 UMs have a dismal prognosis they are high-grade, aggressive and nearly always metastasize. Class 1 UMs have a favorable prognosis the cancers are low-grade, indolent, and rarely metastasize. UMs can be divided into two classes by molecular and genetic analysis of the tumor. Melanomas arising from the pigmented layers (uvea) of the eye are highly aggressive cancers: almost half of patients with uveal melanoma (UM) die from metastatic disease, even after the primary tumor is completely removed by surgical excision of the eye, because we are unable to prevent or treat metastatic spread of the cancer. The HCFC1 subunit is a transcriptional co-activator that can bind transcription factors such as E2F, YY1, and Ets-related transcription factors however, its role in targeting BAP1 to chromatin has not been fully elucidated. This complex, a component of the polycomb pathway, removes mono-ubiquitin from histone H2A. The BAP1 polypeptide is the catalytic subunit of the polycomb-repressive deubiquitinase complex, which requires either ASXL1 or ASXL2, and which can include HCFC1, OGT and other factors. This technique has generated a new and expanded list of BAP1 targets in UM that provides important insight into metastasis pathways and identifies novel potential therapeutic targets.īAP1 is a histone deubiquitinase that remodels chromatin to regulate gene expression. The calling card methodology works equally well for chromatin regulatory factors that do not interact directly with DNA as for transcription factors. Further, a subset of the BAP1 genomic target genes was able to discriminate aggressive tumors in published gene expression data from primary UM tumors. Sequence consensus analysis of BAP1-bound sites showed enrichment of motifs specific for YY1, NRF1 and Ets transcription factors, which have been shown to interact with BAP1 in other cell types. BAP1-bound genomic loci showed narrow distributions of insertions and were near transcription start sites, consistent with recruitment of BAP1 to these sites by specific DNA-binding proteins. We found a strong correlation between multiple calling-card transposon insertions targeted by BAP1-PBase and BAP1-responsive expression of adjacent genes. Sets of significant genes were analyzed for common pathways, transcription factor binding sites, and ability to identify molecular tumor classes. We also examined RNA expression in the same OCM-1A UM cells after BAP1 depletion to identify BAP1 binding sites associated with BAP1-responsive genes. The insertion of transposons near BAP1 binding sites in UM cells were identified by genomic sequencing. The transposase piggyBac (PBase) was fused to BAP1 and expressed in OCM-1A UM cells. This system was developed to identify the genomic loci visited by transcription factors that bind directly to DNA our study is the first use of the system with a chromatin-remodeling factor that binds to histones but does not interact directly with DNA. We have used the “Calling Card System” of transposase-directed transposon insertion mapping to identify the genomic targets of BAP1 in uveal melanoma (UM). BAP1 is a histone deubiquitinase that acts as a tumor and metastasis suppressor associated with disease progression in human cancer.
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