EWSR1-ATF1 dependent 3D connectivity regulates oncogenic and differentiation programs in Clear Cell Sarcoma [HiChIP-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: 4 HiChIP samples of CCS cell lines including wt cells, and cells transfected with siRNA directed against either EWSR1-ATF1 vs non-targeting siRNA control.

染色体易位产生的嵌合蛋白是癌症中主要的驱动致癌基因。其中，EWSR1与一系列转录因子（Transcription Factors, TFs）的融合可产生具备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融合蛋白与不同肿瘤类型相关，提示许可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，对由EWSR1-ATF1融合基因驱动的透明细胞肉瘤（Clear Cell Sarcoma, CCS）——一种极具侵袭性的人类癌症类型——的表观遗传与三维连接图谱进行了系统解析。研究发现，EWSR1-ATF1呈现出依赖于EWSR1朊病毒样结构域的独特结合模式，尽管野生型ATF1与EWSR1-ATF1蛋白存在物理相互作用，但其结合模式与野生型ATF1截然不同。这种协同作用促使ATF1重新靶向新的远端染色质位点，进而引发染色质激活，并构建出与原发性CCS肿瘤共享的、调控致癌特征与分化特征的三维调控网络。反之，EWSR1-ATF1的敲低会导致三维连接网络发生显著重构，包括出现一批新的调控连接，可控制与神经嵴相关的发育程序。据此，对来自人类皮肤图谱的超过16万个单细胞表达谱进行分析后发现，EWSR1-ATF1表达缺失可诱导肿瘤细胞向更成熟的分化状态转变。综上，本研究揭示了EWSR1-ATF1的协同调控网络，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是这类肿瘤的候选起源细胞。实验设计：本研究包含4组CCS细胞系的HiChIP样本，分别为野生型细胞、转染靶向EWSR1-ATF1的siRNA的细胞，以及转染非靶向性siRNA的阴性对照细胞。