The role for phase separation in oncogenesis and aberrant chromatin looping formation

项目摘要/摘要 NUP98基因重排(NUP98-r)在急性髓系白血病(AML)等白血病中反复出现。 NUP98-r患者预后差，治疗失败。大多数NUP98-r合作伙伴(&gt；30确定自 患者)是转录因子(Tf；例如HOXA9)的DNA结合域或组蛋白结合基序，例如 植物同源域(PHD)，提示染色质去调控是致癌机制之一。NUP98-R融合 从NUP98中始终如一地保留苯丙氨酸-甘氨酸(FG)重复序列，称为固有无序区(IDR)。 非结构化IDR如何促进肿瘤发生仍是个未知数。我们对急性髓系白血病NUP98-HOXA9的研究 NUP98-TF嵌合体，揭示了NUP98的S IDR对液-液相分离(LLP)的基本要求。 我们还表明IDR和LLP对于NUP98-HOXA9和NUP98-HOXA9和 用于癌基因启动子和增强子之间的长距离染色质环，导致 小鼠侵袭性急性髓系白血病。我们未发表的其他复发性白血病融合的初步研究(即， NUP98-PHD融合和MSI2-HOXA9，一个与白血病相关的嵌合体，由一个较少研究的IDR融合而成 具有HOXA9‘S DNA结合域的核糖核酸结合蛋白)都表明IDR和LLP参与了 致癌作用。因此，我们假设，由于异常的基因融合，一些与白血病相关的肿瘤- TFS和染色质因子获得相分离诱导的IDR来建立LLP，这赋予了嵌合体 基因组靶向能力大大增强；因此，致癌基因表达程序是 在癌基因启动子和增强子之间形成过度激活而异常的染色质环，这 导致侵袭性白血病的形成。对IDR-和阶段-的潜在机制的剖析- 分离介导的癌细胞异常基因组组织和癌基因激活将提供新的 以及关于癌症如何发展的范式转变观点，暗示可能出现新的治疗方法 在未来。为了实现这一目标，我们将进一步说明未研究的IDR(MSI2)在 在体外和细胞内建立LLP(目标1A)，并将使用原代人类造血干/祖细胞 细胞(HSPC)和衍生细胞来确定IDR和LLP在调节基因组靶向中的作用(1B)， 靶基因表达(1C)和不同融合(NUP98-PHD)的体外/体内白血病转化(1D) 和MSI2/NUP98-HOXA9)。LLP诱导的染色质循环是独立于CTCF的，并且代表了以前的 3D染色质组织的潜在机制尚未研究。我们将进一步定义3D染色质结构 疾病相关细胞中不同NUP98-r和MSI2-HOXA9融合引起的改变(Aim 2A)，定义 驱动LLP依赖环(2B)形成/维持的分子机制，并决定 利用新型CRISPR/dCas9-IDR研究LLP DNA环对癌基因持续激活的影响 融合策略(2C)。由于具有相分离能力的分子经常牵涉到广泛的 对于人类癌症和疾病，该项目的意义和总体影响都可能很高。

PROJECT SUMMARY/ABSTRACT Rearrangement of NUP98 gene (NUP98-r) is recurrent in leukemias such as acute myeloid leukemia (AML). Patients with NUP98-r show poor prognosis and therapy failure. Most NUP98-r partners (>30 identified from patients) are a DNA-binding domain of transcription factor (TF; e.g. HOXA9) or a histone-binding motif such as Plant Homeodomain (PHD), suggesting chromatin deregulation as an oncogenic mechanism. NUP98-r fusions invariably retain Phenylalanine-Glycine (FG) repeats, termed intrinsically disordered region (IDR), from NUP98. How unstructured IDR contributes to oncogenesis remains elusive. Our studies of NUP98-HOXA9, an AML NUP98-TF chimera, unveil an essential requirement of NUP98’s IDR for liquid-liquid phase separation (LLPS). We also show that IDR and LLPS are critical for the much-enhanced genome binding by NUP98-HOXA9 and for long-distance chromatin looping between oncogene promoters and enhancers, leading to development of aggressive AML in mice. Our unpublished preliminary studies of other recurrent leukemic fusions (namely, NUP98-PHD fusions and MSI2-HOXA9, a leukemia-related chimera formed by fusing a less-studied IDR of an RNA-binding protein with HOXA9’s DNA-binding domain) all point to involvements of IDR and LLPS for oncogenesis. Thus, we hypothesize that, due to aberrant genic fusions, a number of leukemia-related onco- TFs and chromatin factors acquire a phase-separation-inducing IDR to establish LLPS, which confers chimera a much more enhanced ability in genomic targeting; consequently, an oncogenic gene-expression program is over-activated while aberrant chromatin loops are formed between oncogene promoters and enhancers, which drives formation of aggressive leukemias. Dissection of the mechanisms underlying the IDR- and phase- separation-mediated aberrant genome organization and oncogene activation in cancer cells shall provide new and paradigm-shifting views as for how aggressive cancer develops, implicative of potentially new treatments in future. Towards this goal, we will further characterize the role for the un-studied IDR (that of MSI2) in establishing LLPS in vitro and in cells (Aim 1A) and will use primary human hematopoietic stem/progenitor cells (HSPCs) and derived cells to define roles of IDR and LLPS in regulating genomic targeting (1B), the target gene expression (1C), and leukemic transformation in vitro/vivo (1D) by various fusions (NUP98-PHD and MSI2/NUP98-HOXA9). LLPS-indued chromatin looping is CTCF-independent and represents a previously unstudied mechanism underlying 3D chromatin organization. We will further define the 3D chromatin structure alterations caused by various NUP98-r and MSI2-HOXA9 fusions in disease-relevant cells (Aim 2A), define the molecular mechanisms driving formation/maintenance of LLPS-dependent loops (2B), and determine the impact of LLPS DNA loops on the sustained activation of oncogenes by using a novel CRISPR/dCas9-IDR fusion strategy (2C). As phase-separation-competent molecules are frequently implicated in a wide range of human cancers and diseases, both the significance and overall impact of the project are potentially high.