EWSR1-ATF1 dependent 3D connectivity regulates oncogenic and differentiation programs in Clear Cell Sarcoma [RNA-seq]

Chimeric proteins resulting from chromosomal translocations play a major role as driver oncogenes in cancer. Among them, fusions between EWSR1 and a set of transcription factors (TFs) generate oncogenes with powerful chromatin regulatory activities, capable of establishing complex gene expression programs. However, specific EWSR1 fusion proteins have been implicated in distinct tumor types, suggesting an enhancement of their functional properties by permissive precursor cells. Here we combined functional epigenomics with nuclear topology mapping to define the epigenetic and 3D connectivity landscape of Clear Cell Sarcoma (CCS), one of the most aggressive forms of human cancer, driven by the EWSR1-ATF1 fusion gene. We find that EWSR1-ATF1 display a distinctive binding pattern that depends on the EWSR1 prion-like domain, and is divergent from wild type ATF1, despite the physical interaction between wt and EWSR1-ATF1 proteins. This cooperativity promotes ATF1 retargeting to new distal sites, leading to chromatin activation and the establishment of a 3D network that controls oncogenic and differentiation signatures shared with primary CCS tumors. Conversely, EWSR1-ATF1 depletion results in a marked reconfiguration of 3D connectivity, including the emergence of a new set of connections controlling neural crest-related developmental programs. Accordingly, interrogation of more than 160,000 single cell expression profiles from the human skin atlas reveals loss of EWSR1-ATF1 expression to induce a more differentiated tumor cell state. Taken together, our study uncovers the cooperativity network of EWSR1-ATF1, delineates the molecular underpinnings of its epigenetic function in CCS, and points to precursor cells along the Schwann cell-melanocytic axis as a candidate origin for these tumors. Overall design: 18 RNAseq samples on CCS cell lines, CCS and AFH Primary tumor samples and MSC primary cells. The study also includes perturbed systems; cells transfected with siRNA directed against EWSR1-ATF1, or cells infected with an EWSR1-ATF1 overexpression construct, and their respective controls. DTC1 samples include three biological, and one technical, replicate experiments. >>> Raw files unavailable for Samples GSM5455379 GSM5455380 GSM5455382 GSM5455383 because they were generated using old archival samples, which have IRB limitations in terms of data deposition. These raw files are on Zenodo (http://doi.org/10.5281/zenodo.5112706). <<<染色体易位产生的嵌合蛋白，在癌症中作为驱动致癌基因发挥关键作用。其中，EWSR1与一系列转录因子（Transcription Factors, TFs）的融合，可产生具有强大染色质调控活性的致癌基因，能够构建复杂的基因表达程序。然而，特定的EWSR1融合蛋白与不同的肿瘤类型相关，这提示允许性前体细胞可增强其功能特性。本研究将功能表观基因组学与细胞核拓扑结构绘图相结合，以明确透明细胞肉瘤（Clear Cell Sarcoma, CCS）——一种由EWSR1-ATF1融合基因驱动的高侵袭性人类癌症——的表观遗传与三维（3D）连接图谱。研究发现，EWSR1-ATF1呈现独特的结合模式，该模式依赖于EWSR1的朊蛋白样结构域，且与野生型ATF1（wild type, wt）存在差异，尽管野生型ATF1与EWSR1-ATF1蛋白之间存在物理相互作用。这种协同作用促使ATF1重新靶向至新的远端位点，进而引发染色质激活，并构建出一套三维网络，该网络可调控与原发性CCS肿瘤共有的致癌特征与分化特征。反之，EWSR1-ATF1的敲除会导致三维连接网络发生显著重构，包括出现一套新的连接通路，可调控与神经嵴相关的发育程序。据此，通过分析来自人类皮肤图谱的超过16万个单细胞表达谱，研究发现EWSR1-ATF1表达的缺失可诱导肿瘤细胞进入更分化的状态。综上，本研究揭示了EWSR1-ATF1的协同网络，阐明了其在CCS中发挥表观遗传功能的分子基础，并指出施万细胞-黑素细胞轴相关的前体细胞可作为这类肿瘤的候选起源细胞。实验设计：本研究包含18份CCS细胞系、CCS与AFH原发性肿瘤样本以及间充质干细胞（Mesenchymal Stem Cell, MSC）原代细胞的RNA测序（RNA-seq）样本。此外，研究还涉及扰动系统实验：包括转染靶向EWSR1-ATF1的小干扰RNA（siRNA）的细胞，以及转染空载体的对照细胞；还有过表达EWSR1-ATF1的细胞及其相应的对照细胞。DTC1样本包含3次生物学重复与1次技术重复实验。>>> 样本GSM5455379、GSM5455380、GSM5455382、GSM5455383的原始文件无法获取，原因是这些样本来自老旧存档样本，受伦理审查委员会（Institutional Review Board, IRB）关于数据提交的相关限制。这些原始文件已上传至Zenodo平台（http://doi.org/10.5281/zenodo.5112706）。<<<