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

摘要 哺乳动物的基因组以高度有序的方式在细胞核内折叠。 粘附素复合体通过生成参与3D基因组的组织 以及维持DNA环路。肿瘤抑制基因STAG2，编码核心亚单位 在各种癌症中，粘附素复合体的基因经常发生突变。然而， STAG2对癌细胞3D基因组组织的失活作用，尤其是长程作用 增强子-启动子接触和随后的基因表达控制仍然很差 明白了。在我们的初步研究中，我们已经进行了RNA-SEQ、CHIP-SEQ以及 稳定表达STAG2可诱导shRNA的黑色素瘤细胞的HI-C分析。我们有 研究表明，黑色素瘤细胞中STAG2的缺失会导致拓扑结构的扩张 STAG2结合转换为其平行对数的位点上的相关结构域(TADS) STAG1。我们还确定干扰素调节因子9(IRF9)是一个假定的直接靶标 STAG2在黑色素瘤细胞中的表达，并证明了STAG2的缺失激活了IRF9以 增强I型干扰素信号，增加PD-L1的表达。基于 这些初步的发现，我们假设STAG2的缺失导致了信号转导通路的失调。 黑色素瘤中的I型干扰素信号通过3D基因组组织的扰动。在AIM 1，我们将描述I型调控的分子机制 STAG2通过调节3D基因组结构在黑色素瘤中的干扰素反应。我们 我还将研究IRF9基因座和STAG2中其他靶点的染色质相互作用 野生型和STAG2突变的人类黑色素瘤样本。在目标2中，我们将系统地 鉴定STAG2特异的染色质环并研究STAG2的作用 对黑色素瘤细胞增强子-启动子相互作用组失活的研究 STAG2-和H3K27ac-HiChIP分析。在目标3中，我们将研究STAG2在 调节肿瘤诱导的免疫逃避和确定抗PD-1的敏感性 黑色素瘤的免疫检查点阻断。拟议的研究结果将非常 可能揭示了STAG2在粘连蛋白复合体中的功能，这些功能有助于 3D基因组扰动引起的STAG2突变癌的恶性表型。

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.