Molecular mechanisms of differential transcriptional activity of AP-1 factors c-Jun, Fra-1 and ATF-2, and their functional significance in malignant melanoma

近几十年来，已经确定了许多调节黑色素瘤发展和进展的改变途径以及黑色素瘤细胞的高迁移和侵袭潜力，但对疾病发展和进展中定义的分子机制的详细理解仍然不完整。黑色素瘤的发展和进展中的关键步骤是肿瘤促进转录因子（例如活化蛋白-1（AP-1））的失调。AP-1蛋白调节特定靶基因的表达，从而导致肿瘤相关信号通路的失调。因此，AP-1转录因子在包括恶性黑色素瘤在内的各种肿瘤类型中发挥重要作用。细胞中AP-1复合物的一个关键特征是其二聚体组成的异质性。各种研究表明，AP-1二聚体组成的差异导致特异性结合位点的选择发生变化。然而，目前还不清楚AP-1同源二聚体或异源二聚体的直接靶基因引起的功能效应，支持melanomagenesis AP-1家族成员c-Jun是黑色素瘤进展的主要调节因子，并通过调节支持肿瘤细胞增殖和迁移的靶基因发挥作用，从而促进恶性表型的发展。c-Jun、Fra-1和ATF-2的失调是黑色素瘤发生发展的主要事件，但它们之间相互作用的功能相关性和对靶基因表达的分子效应迄今为止还不能详细显示。（c-Jun、Fra-1和ATF-2）对于恶性黑素瘤的发展和进展的作用。ATF-2和Fra-1 ChIP-Seq数据分析将用于分析单个转录因子的DNA结合位点，并鉴定二聚体的潜在不同DNA结合模式，其将在进一步的实验中验证（例如荧光素酶报告基因测定、电泳迁移率变化测定）。此外，将确定所鉴定的AP-1结合基序附近的基序，并确定AP-1辅因子的存在或不存在对调节机制的影响。在不同黑色素瘤细胞系与黑素细胞中通过ChIP-和RNA-Seq确认的靶基因的表达分析（qRT-PCR，蛋白质印迹）中，将揭示可能的失调。将使用功能实验（增殖、迁移、侵袭测定等）研究由c-Jun、Fra-1和/或ATF-2直接调节并且与正常黑素细胞相比在黑色素瘤细胞中显示失调的靶基因。该项目的结果将为阐明AP-1转录因子在黑色素瘤发展中的作用做出重大贡献，也将导致新的治疗靶点和选择的鉴定和开发。

In recent decades, many altered pathways regulating the development and progression of melanoma and the high migratory and invasive potential of melanoma cells have been identified, but a detailed understanding of defined molecular mechanisms in disease development and progression is still incomplete. A crucial step in the development and progression of melanoma is the de-regulation of tumor-promoting transcription factors, for example of the Activating Protein-1 (AP-1). AP-1 proteins regulate the expression of specific target genes, which leads to the de-regulation of tumor-relevant signaling pathways. Thus, AP-1 transcription factors play an important role in various tumor types, including malignant melanoma. A key feature of AP-1 complexes in the cell is their heterogeneity with respect to their dimer composition. Various studies have shown that differences in AP-1 dimer compositions cause a change in the selection of specific binding sites. However, it remains unclear which direct target genes of AP-1 homodimers or heterodimers cause the functional effects that support melanomagenesis.The AP-1 family member c-Jun is a major regulator of melanoma progression and acts by regulating target genes that support tumor cell proliferation and migration, thereby promoting the development of a malignant phenotype. The de-regulation of c-Jun, Fra-1 and ATF-2 are major events in melanoma development, but the functional relevance of their interplay and molecular effects on target gene expression could not be shown in detail so far.In the proposed project, the relevance of interaction of selected AP-1 transcription factors (c-Jun, Fra-1, and ATF-2) for the development and progression of malignant melanoma will be investigated.C-Jun, ATF-2 and Fra-1 ChIP-Seq data analysis will be used to analyze the DNA-binding sites of the individual transcription factors and to identify potentially different DNA-binding modalities of the dimers, which will be validated in further experiments (e.g. Luciferase reporter gene assay, Electrophoretic Mobility Shift Assay). Moreover, motifs near the identified AP-1 binding motifs will be defined and the influence of the presence or absence of AP-1 co-factors on regulatory mechanisms will be determined. In expression analyses (qRT-PCR, Western Blots) of the target genes confirmed by ChIP- and RNA-Seq in different melanoma cell lines versus melanocytes, possible de-regulations will be revealed. Target genes, which are directly regulated by c-Jun, Fra-1, and/or ATF-2 and show a de-regulation in melanoma cells compared to normal melanocytes, will be investigated using functional experiments (proliferation, migration, invasion assays, etc.) for their relevance and function in melanoma.The results of this project will make a significant contribution to elucidate of the role of AP-1 transcription factors in the development of melanoma and would also lead to the identification and development of new therapeutic targets and options.