PO.MCB07.03 · 分子与细胞生物学
PAX3::FOXO1的动态低复杂度结构域相互作用介导腺泡状横纹肌肉瘤中内源性病理性转录枢纽的形成
Dynamic low-complexity domain interactions of PAX3::FOXO1 mediate endogenous pathological transcriptional hub formation in alveolar rhabdomyosarcoma
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
横纹肌肉瘤（RMS）是一种骨骼肌组织的癌症，是最常见的儿童软组织肉瘤。尽管过去几十年在预后和治疗方面取得了进展，但高危RMS的5年生存率仍不足30%。PAX3::FOXO1（P3F1）是一种融合癌蛋白，见于60%的腺泡状RMS（aRMS，RMS最具侵袭性的亚型）病例，被认为驱动aRMS中的致癌转录。然而，P3F1调控转录的分子机制仍不甚明了，这些转录改变在多大程度上促进肿瘤发生也尚不清楚。在本研究中，我们首先证明外源表达的P3F1表现出其亲本蛋白所不具有的新形态（neomorphic）枢纽形成倾向。我们利用基于CRISPR/Cas9的基因编辑方法，在患者来源的aRMS细胞中以荧光生成型HaloTag对P3F1进行内源标记，从而首次得以在其天然病理环境中可视化P3F1。我们发现内源性P3F1在其靶基因处形成转录枢纽，经光激活定位显微镜（photoactivatable localization microscopy）表征，枢纽平均大小约为117 nm。单颗粒追踪实验显示，与枢纽外的P3F1分子相比，枢纽内的P3F1分子具有更长的染色质结合驻留时间，提示P3F1枢纽在靶基因转录中发挥重要作用。我们发现破坏P3F1的内在无序低复杂度结构域（LCD）会削弱其枢纽形成倾向、靶基因转录以及招募共激活因子p300的能力。利用相分离诱导的相互作用组检测（PhaseID）方法，我们绘制出P3F1枢纽所招募并用以影响致癌转录的独特相互作用蛋白。总之，我们的结果揭示了病理性P3F1的一种新形态枢纽形成行为，并为将靶向P3F1枢纽形成作为aRMS新型治疗手段提供了有力依据。
查看英文原文 English abstract
Rhabdomyosarcoma (RMS), a cancer of skeletal muscle tissue, is the most common pediatric soft tissue sarcoma with 5-year survival rate less than 30% in high-risk RMS, despite advances in prognosis and treatment over the past few decades. PAX3::FOXO1 (P3F1) is a fusion oncoprotein found in 60% cases of alveolar RMS (aRMS), the most aggressive subtype of RMS, and has been thought to drive oncogenic transcription in aRMS. However, the molecular mechanisms by which P3F1 regulates transcription remain poorly understood, and the extent to which these transcriptional changes contribute to tumorigenesis is unclear. In the current study, we first demonstrated exogenously expressed P3F1 exhibited neomorphic hub formation propensity which is not observed for its parental proteins. Using CRISPR/Cas9-based gene editing method, we endogenously labeled P3F1 with a fluorogenic HaloTag in patient-derived aRMS cells, allowing us, for the first time, to visualize P3F1 at its native pathological environment. We discovered that endogenous P3F1 forms transcriptional hubs at its target genes, and the average hub size is ~117 nm characterized by photoactivatable localization microscopy. Single particle tracking experiment showed that P3F1 molecules have longer chromatin bound residence time in the hubs compared to those outside the hubs, suggesting an important role of P3F1 hubs in target gene transcription. We found disrupting the intrinsically disordered low complexity domains (LCDs) of P3F1 impaired its hub formation propensity, target gene transcription and its ability to recruit coactivator p300. Using a phase-separation-induced interactome detection (PhaseID) method, we mapped unique interacting proteins that P3F1 hubs recruit and utilize to influence oncogenic transcription. In summary, our results uncover a neomorphic hub formation behavior of pathological P3F1 and provide a strong rationale of targeting P3F1 hub formation as new therapeutics for aRMS.
利益披露 Disclosure
Y. Zhong, None..
M. Di Martino, None..
S. Chong, None.