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

项目总结/摘要 NUP 98基因重排（NUP 98-r）在白血病如急性髓性白血病（AML）中是复发性的。 NUP 98-r患者预后差，治疗失败。大多数NUP 98-r合作伙伴（从 患者）是转录因子（TF;例如HOXA 9）的DNA结合结构域或组蛋白结合基序， 植物同源结构域（PHD），提示染色质失调是一种致癌机制。NUP 98-r融合体 总是保留来自NUP 98称为固有无序区（IDR）的苯丙氨酸-甘氨酸（FG）重复序列。 非结构化的IDR如何促进肿瘤发生仍然是难以捉摸的。NUP 98-HOXA 9是一种急性髓细胞白血病， NUP 98-TF嵌合体揭示了NUP 98 IDR对液-液相分离（LLPS）的基本要求。 我们还表明IDR和LLPS对于NUP 98-HOXA 9和H 0XA 9的增强的基因组结合是至关重要的。 癌基因启动子和增强子之间的长距离染色质环，导致 小鼠的侵袭性AML。我们对其他复发性白血病融合的未发表的初步研究（即， NUP 98-PHD融合体和MSI 2-HOXA 9，一种白血病相关嵌合体，通过融合一种研究较少的白血病细胞的IDR而形成。 RNA结合蛋白与HOXA 9的DNA结合结构域）都指出IDR和LLPS参与 肿瘤发生因此，我们假设，由于异常的基因融合，一些白血病相关的肿瘤- TF和染色质因子获得相分离诱导IDR以建立LLPS，其赋予嵌合体 基因组靶向能力大大增强;因此， 过度激活，同时在癌基因启动子和增强子之间形成异常染色质环， 导致恶性白血病的形成剖析IDR的机制-和阶段- 分离介导的异常基因组组织和癌基因激活在癌细胞中将提供新的 以及关于侵袭性癌症如何发展的范式转变观点，暗示着潜在的新治疗方法 日后为了实现这一目标，我们将进一步描述未研究的IDR（MSI 2）的作用， 在体外和细胞中建立LLPS（目标1A），并将使用原代人造血干/祖细胞 细胞（HSPC）和衍生细胞，以确定IDR和LLPS在调节基因组靶向中的作用（1B）， 靶基因表达（1C）和通过各种融合（NUP 98-PHD）体外/体内白血病转化（1D 和MSI 2/NUP 98-HOXA 9）。LLPS诱导的染色质成环是CTCF独立的，代表了先前的 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 在疾病相关细胞中由各种NUP 98-r和MSI 2-HOXA 9融合引起的改变（Aim 2A），定义了 驱动LLPS依赖性环形成/维持的分子机制（2B），并确定 通过使用新型CRISPR/dCas 9-IDR，LLPS 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涉及广泛的生物学领域， 人类癌症和疾病，该项目的重要性和总体影响可能很高。

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 Our unpublished preliminary studies of other recurrent leukemic fusions (namely, NUP98-PHD fusions and MSI2-HOXA9, a leukemia-related chimera formed by fusing a less-studied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.