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，许多调节黑色素瘤发生和发展的通路以及黑色素瘤细胞的高迁移和侵袭潜力已经被确定，但对疾病发生和发展的分子机制的详细理解仍然不完整。黑色素瘤发生和发展的一个关键步骤是肿瘤促进转录因子（如激活蛋白-1 (AP-1)）的去调控。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去调控。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它们的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，目前尚不清楚AP-1同二聚体或异二聚体的哪些直接靶基因导致支持黑色素瘤形成的功能影响。AP-1家族成员c-Jun是黑色素瘤进展的主要调节因子，通过调节支持肿瘤细胞增殖和迁移的靶基因起作用，从而促进恶性表型的发展。c-Jun、Fra-1和ATF-2的去调控是黑色素瘤发生过程中的重要事件，但其相互作用的功能相关性和分子效应对靶基因表达的影响目前尚未得到详细的研究。本项目将研究AP-1转录因子（c-Jun、fr -1和ATF-2）相互作用与恶性黑色素瘤发生和发展的相关性。c-Jun、ATF-2和fr -1 ChIP-Seq数据分析将用于分析单个转录因子的dna结合位点，并确定二聚体的潜在不同dna结合方式，这些结果将在进一步的实验中得到验证（例如荧光素酶报告基因测定）。电泳迁移率转移试验)。此外，将确定已确定的AP-1结合基序附近的基序，并确定AP-1辅助因子的存在或不存在对调控机制的影响。ChIP-和RNA-Seq证实的靶基因在不同黑色素瘤细胞系和黑色素细胞中的表达分析（qRT-PCR, Western Blots）将揭示可能的去调控。靶基因由c-Jun、Fra-1和/或ATF-2直接调控，与正常黑色素细胞相比，在黑色素瘤细胞中表现出去调控，将通过功能实验（增殖、迁移、侵袭试验等）来研究它们在黑色素瘤中的相关性和功能。该项目的结果将对阐明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确定和发展。

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.