Deciphering the tumor progression-dependent changes in mitotic bookmarking of triple-negative breast cancer cells

癌症被认为是一种遗传和表观遗传疾病，其结果受环境因素的影响。最近的基因组研究表明，传播肿瘤细胞对外来微环境的适应能力可能依赖于可逆的表观遗传改变和重编程。这些改变通常导致细胞周期控制的丧失和随后的增加，不受控制的增殖。为了维持和传播侵袭性癌症表型，癌细胞的身份在有丝分裂过程中必须以某种方式保持，因为子细胞表现出与母细胞相同的表观遗传改变和基因表达模式。最近的证据强调，这种保存细胞身份是通过有丝分裂书签实现的。研究表明，转录因子（TFs）和表观遗传标记都可以作为细胞身份性状繁殖的有丝分裂书签，在有丝分裂完成后允许快速恢复增强子-启动子环和基因表达模式。但目前还不完全清楚哪些tf参与有丝分裂书签，以及有丝分裂书签模式是否在肿瘤进展过程中发生变化。在这个项目中，我的目标是确定肿瘤相关tf在三阴性乳腺癌（TNBC）肿瘤进展到转移形成过程中癌细胞有丝分裂书签中的作用。此外，我打算确定癌症相关的允许染色质状态是否有利于有丝分裂书签和癌细胞身份通过有丝分裂传播。为了实现这一目标，我将制定一种改进的同步方案，用于在有丝分裂开始时捕获细胞，精确地在G2/M边界和前期中期之间。为了确定特定阶段癌细胞中有丝分裂书签的改变，我将通过对有丝分裂阻滞细胞使用ATAC-seq来描述可接近的染色质区域。通过对这些数据集进行计算分析，我将检索有关TF的DNA足迹的信息，这可能涉及有丝分裂书签。这些发现的相关性将被通过Cut&Run分析鉴定的tf的全基因组DNA结合谱所证实。最后，我将通过使用CRISPR/ cas9介导的基因组编辑来删除鉴定的tf，从功能上验证肿瘤相关tf在有丝分裂书签中的相关性。该项目将有助于了解TNBC细胞中的有丝分裂书签如何在有丝分裂期间受到挑战时允许保存细胞身份，以及哪些tf可能发挥癌症类型的特定作用。

Cancer is considered both a genetic and an epigenetic disease whose outcome is influenced by environmental cues. Recent genomic studies showed that the adaptation capacity of disseminating tumor cells to foreign microenvironments could rely on reversible epigenetic alterations and reprogramming. These alterations often result in a loss of cell cycle control and subsequent increased, uncontrolled proliferation. To maintain and transmit the aggressive cancer phenotype, the cancer cell identity has somehow to be preserved during mitosis, as the daughter cells show the same epigenetic alterations and gene expression patterns as does the mother cell. Recent evidence highlighted that this preservation of cell identity is achieved by mitotic bookmarking. It has been shown that both, transcription factors (TFs) and epigenetic marks can serve as mitotic bookmarks for the propagation of cell identity traits by allowing a rapid restoration of enhancer-promoter loops and gene expression patterns after completion of mitosis. But up to date it is not fully understood which TFs are involved in mitotic bookmarking and whether the pattern of mitotic bookmarking changes during tumor progression. With this project, I aim to define the role of tumor-associated TFs in mitotic bookmarking of cancer cells during tumor progression towards metastasis formation of triple-negative breast cancer (TNBC).Moreover, I intend to determine whether a cancer-associated permissive chromatin state could favor mitotic bookmarking and cancer cell identity propagation through mitosis. In order to achieve this goal, I will work out an improved synchronization protocol for the capture of cells at the onset of mitosis, precisely between the G2/M border and prometaphase. To identify the alterations in mitotic bookmarking in stage-specific cancer cells, I will profile the accessible chromatin regions by employing ATAC-seq on mitotically arrested cells. By performing computational analyses on these datasets, I will retrieve information about DNA footprinting of TF, which might be involved in mitotic bookmarking. The relevance of these finding will be corroborated by profiling genomewide DNA binding of the identified TFs by Cut&Run analysis. Finally, I will functionally validate the relevance of tumor-associated TFs in mitotic bookmarking by employing CRISPR/Cas9-mediated genome editing to delete the identified TFs. This project will help to understand how mitotic bookmarking in TNBC cells allows for the preservation of the cellular identity when challenged during mitosis and which TFs might play a cancer-type specific role.