UV damage stimulation by ETS transcription factors in skin cancer

项目摘要 暴露于紫外线（UV）光是黑色素瘤和其他皮肤癌的主要病因。UV 光诱导细胞DNA的损伤，主要是环丁烷嘧啶二聚体（CPD）和6-4 光产物（6-4PPs）。对人类皮肤癌DNA的测序显示， 诱导突变，这些突变在基因组中不均匀分布。最近已发现 紫外线诱导的突变在转录因子的DNA结合位点特别丰富。虽然这 已经归因于在这样的结合位点处的较低的修复效率，也可能是初始结合位点的变化导致的。 由于转录因子结合而形成的损伤可刺激突变率。为了验证这个假设，我们 使用我们新开发的CPD-seq方法绘制了整个人类基因组的CPD形成图 UV照射后立即进行。我们的初步数据表明，最初的紫外线损伤形成是 在许多转录因子结合位点（TFBS）显著升高，特别是E26的结合位点 转录因子（TF）的转化特异性（ETS）家族。重要的是，刺激CPD形成， ETS结合与黑色素瘤肿瘤中显著较高的突变率相关，表明 初始DNA损伤形成是人类癌症中突变“景观”的重要贡献者。 这个建议的总体目标是阐明ETS结合刺激UV的机制 损伤的形成，并确定ETS诱导的CPD“热点”是否驱动在 黑色素瘤肿瘤中的单个结合位点。为了研究ETS蛋白结合的机制， 刺激紫外线损伤的形成，我们将描述选定的ETS转录因子对紫外线损伤的影响。 体外损伤形成和修复（目的I）。同时，我们将使用分子动力学模拟来模拟 不同ETS转录因子如何结合DNA使二嘧啶序列易于形成UV 照片产品最后，我们将研究ETS TF结合是否抑制CPD损伤的体外修复。在Aim中 II，我们将开发CPD捕获序列方法来绘制CPD损伤的形成和修复， 测序深度和单核苷酸分辨率在特定的基因组区域的兴趣，包括网站 黑色素瘤和ETS结合位点的复发性启动子突变。同时，我们将绘制ETS结合位点， 使用ChIP-exo方法的人黑素细胞。CPD-捕获-seq数据与UV诱导的 在人类黑色素瘤中发现的突变将使我们能够高分辨率地确定ETS诱导的 CPD“热点”和修复抑制与皮肤癌中的复发性突变相关。这些数据应 为黑色素瘤中一些最常发生的突变的病因学提供了新的见解，这些突变发生在 ETS结合位点。ETS转录因子是已知的癌基因，其调节参与细胞凋亡的许多基因。 分化、迁移、增殖和凋亡;因此，ETS结合位点的复发性突变可能 有助于皮肤癌的致癌作用，并可用于未来的治疗。

PROJECT SUMMARY Exposure to ultraviolet (UV) light is the principal etiological agent for melanoma and other skin cancers. UV light induces damage to the cellular DNA, primarily cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs). Sequencing of DNA from human skin cancers have revealed high levels of UV- induced mutations that are heterogeneously distributed across the genome. It has recently been discovered that UV-induced mutations are particularly enriched at the DNA binding sites of transcription factors. While this has been attributed to less efficient repair at such binding sites, it is also possible that variations in initial damage formation due to transcription factor binding could stimulate mutation rates. To test this hypothesis, we have used our newly developed CPD-seq method to map CPD formation across the human genome immediately following UV irradiation. Our preliminary data indicate that initial UV damage formation is significantly elevated at many transcription factor binding sites (TFBS), particularly binding sites for the E26 transformation-specific (ETS) family of transcription factors (TFs). Importantly, stimulation of CPD formation by ETS binding correlates with significantly higher mutation rates in melanoma tumors, indicating that variations in initial DNA damage formation are an important contributor to the mutational 'landscape' in human cancers. The overall objective of this proposal is to elucidate the mechanism by which ETS binding stimulates UV damage formation and determine whether ETS-induced CPD 'hotspots' drive recurrent mutagenesis at individual binding sites in melanoma tumors. To investigate the mechanism by which ETS protein binding stimulates UV damage formation, we will characterize the effects of selected ETS transcription factors on UV damage formation and repair in vitro (Aim I). In parallel, we will use molecular dynamics simulations to model how DNA binding by different ETS transcription factors predisposes dipyrimidine sequences to form UV photoproducts. Finally, we will examine whether ETS TF binding inhibits repair of CPD lesions in vitro. In Aim II, we will develop the CPD-capture-seq method to map the formation and repair of CPD lesions with high sequencing depth and single nucleotide resolution at specific genomic regions of interest, including sites of recurrent promoter mutations in melanoma and ETS binding sites. In parallel, we will map ETS binding sites in human melanocytes using the ChIP-exo method. Comparison of CPD-capture-seq data with UV-induced mutations identified in human melanomas will allow us determine with high resolution whether ETS-induced CPD 'hotspots' and repair inhibition are associated with recurrent mutations in skin cancer. These data should provide new insights into the etiology of some of the most recurrent mutations in melanoma, which occur at ETS binding sites. ETS transcription factors are known oncogenes that regulate many genes involved in cell differentiation, migration, proliferation, and apoptosis; hence, recurrent mutations at ETS binding sites likely contribute to carcinogenesis in skin cancer and could be exploited in future therapeutics.