Regulation of interferon signaling in melanoma by the cohesin complex protein STAG2 via 3D genome organization

总结 哺乳动物基因组在细胞核内以高度组织化的方式折叠。 黏连蛋白复合体通过生成 和维持DNA循环肿瘤抑制因子STAG 2，编码一个核心亚基 在各种癌症中经常发生突变。然而， STAG 2失活对癌细胞中3D基因组组织的影响，特别是长距离 增强子-启动子接触和随后的基因表达控制，仍然很差 明白在我们的初步研究中，我们进行了RNA-seq，ChIP-seq以及 稳定表达STAG 2的诱导型shRNA的黑素瘤细胞中的Hi-C分析。我们有 表明黑色素瘤细胞中STAG 2的缺失导致拓扑学上的扩增， 在STAG 2的结合被转换到其互补结构域的位点处的相关结构域（TADs STAG 1.我们还确定了干扰素调节因子9（IRF 9）作为假定的直接靶点 STAG 2在黑色素瘤细胞中的表达，并证明STAG 2的缺失激活IRF 9， 增强I型干扰素信号传导并增加PD-L1的表达。基于 根据这些初步的发现，我们推测STAG 2的缺失会导致 黑色素瘤中I型干扰素信号通过3D基因组组织的扰动。在aim中 1，我们将描述I型调节的分子机制 STAG 2通过调节3D基因组组织在黑色素瘤中的干扰素应答。我们 还将研究STAG 2中IRF 9位点和其他靶点的染色质相互作用 野生型和STAG 2突变体人黑素瘤样品。在目标2中，我们将系统地 表征STAG 2特异性染色质环，并研究STAG 2 通过实施对黑色素瘤细胞中增强子-启动子相互作用组的灭活 STAG 2-和H3 K27 ac-HiChIP分析。在目标3中，我们将研究STAG 2在以下方面的作用： 调节肿瘤诱导的免疫逃避和测定抗PD-1的敏感性 黑色素瘤免疫检查点阻断。这项研究的结果将非常 可能揭示STAG 2在粘附素复合物中的功能， STAG 2突变型癌症的恶性表型由3D基因组扰动引起。

Summary Mammalian genomes is folded within the nucleus in a highly organized fashion. The cohesin complex participates in the organization of 3D genome through generating and maintaining DNA loops. The tumor suppressor STAG2, which encodes a core subunit of the cohesin complex, is frequently mutated in various cancers. However, the impact of STAG2 inactivation on 3D genome organization in cancer cells, especially the long-range enhancer-promoter contacts and subsequent gene expression control, remains poorly understood. In our preliminary studies, we have carried out RNA-seq, ChIP-seq as well as Hi-C analyses in melanoma cells stably expressing inducible shRNA of STAG2. We have shown that depletion of STAG2 in melanoma cells leads to expansion of topologically associated domains (TADs) at sites where binding of STAG2 is switched to its paralog STAG1. We also identified Interferon Regulatory Factor 9 (IRF9) as a putative direct target of STAG2 in melanoma cells and demonstrated that loss of STAG2 activates IRF9 to enhance type I interferon signaling and increases the expression of PD-L1. Based on these preliminary findings, we hypothesize that loss of STAG2 results in dysregulation of type I interferon signaling in melanoma via perturbation of 3D genome organization. In aim 1, we will characterize the molecular mechanism underlying the regulation of type I interferon response by STAG2 in melanoma via modulating 3D genome organization. We will also examine the chromatin interactions at the IRF9 locus and other targets in STAG2 wildtype and STAG2 mutant human melanoma samples. In aim 2, we will systematically characterize STAG2-specific chromatin loops and investigate the effects of STAG2 inactivation on the enhancer-promoter interactome in melanoma cells by carrying out STAG2- and H3K27ac- HiChIP analyses. In aim 3, we will examine the roles of STAG2 in regulating tumor-induced immune evasion and in determining the sensitivity to anti-PD-1 immune checkpoint blockade in melanoma. Findings from the proposed study will very likely reveal functions of STAG2 within the cohesin complex that contribute to the malignant phenotypes in STAG2 mutant cancer arising from 3D genome perturbation.