Dynamic 3D chromatin remodeling in acute leukemia - Resubmission - 1

摘要 染色质构象捕捉技术的最新进展彻底改变了我们对染色质构象的理解。 并以前所未有的详细程度提供了新颖的见解。几项研究已经 在DNA-DNA接触图中识别出与生物相关的结构，例如A/B隔间，从拓扑上- 关联结构域(TADS)，隔离邻域，并阐明了染色质结构在 基因调控和细胞特性的维持。我们实验室和其他实验室的几项最新研究表明 研究表明，异常的TAD激活或重新连接启动子-增强子的相互作用可以促进癌症的生长。 然而，还没有研究在全基因组范围内解决染色质组织的破坏。 或这种干扰如何改变启动子-增强子格局导致耐药和 旧病复发。使用原发急性白血病患者样本，我们第一次确定了复发的TAD 白血病中涉及关键癌基因(如NOTCH1、MYC)及其靶点的干扰。例如，我们知道- 发现了MYC基因座中3D染色质拓扑结构的反复中断，这是一种以前没有特征性的非特征性 编码CTCF结合区，使MYC与下游增强子隔绝。这种破坏使染色质 MYC癌基因与下游增强子之间的相互作用导致MYC表达增加。 西翁。基于我们的初步结果，我们建议研究3D染色体景观重组，如 癌症发生、发展和复发的新机制及发现新的非编码调控 导致白血病的因素(增强子3D枢纽及其TAD边界)。为此，我们将首先介绍和 分析大量白血病患者在诊断和复发时使用Hi-C和H3K27ac HiChIP的情况 识别反复复发的特定3D重组事件。我们将把计算方法与或- 发现转录因子和表观遗传修饰物使其出现的同源CRISPR策略 通过增强子-启动子染色质环的重新连接而产生的耐药性。然后，我们将重点介绍增强型 Hubs：我们和其他人已经证明，与目标启动子和其他启动子密切相关的增强子 增强子(即增强子中心)是基因表达的强大调节器，它们的干扰可能会影响 多基因调控。基于这些发现，我们将测试这种增强子中枢及其3D 拓扑可以通过激活体外和体内的致癌基因来驱动耐药。我们建议的研究 不仅将阐明3D结构在白血病诊断和复发中的作用，而且还将推动 我们对治疗抗药性的理解，并开发新的方法来克服它。

ABSTRACT Recent advances in chromatin conformation capture techniques have revolutionized our understanding of chro- matin organization and have provided novel insights at an unprecedented level of detail. Several studies have identified biologically-relevant structures in DNA-DNA contact maps, such as A/B compartments, topologically- associating domains (TADs), insulated neighborhoods, and have elucidated the role of chromatin architecture in gene regulation and maintenance of cell identity. A handful of very recent studies from our lab and others have shown that aberrant TAD activation or “rewiring” promoter-enhancer interactions can promote cancer growth. However, no study has yet addressed the disruptions of chromatin organization on a genome-wide scale in cancer patients or how such disruptions modify the promoter-enhancer landscape leading to drug resistance and relapse. Using primary acute leukemia patient samples, we have, for the first time, identified recurrent TAD disruptions in leukemia involving key oncogenes (e.g. NOTCH1, MYC) and their targets. For example, we iden- tified a recurrent disruption of 3D chromatin topology in the MYC locus at a previously uncharacterized non- coding CTCF-bound region that insulates MYC from downstream enhancers. This disruption enables chromatin interactions between the MYC oncogene and the downstream enhancers leading to an increase in MYC expres- sion. Based on our preliminary results, we propose to investigate 3D chromosomal landscape reorganization as a new mechanism of cancer initiation, progression and relapse, and to discover novel non-coding regulatory elements (enhancer 3D hubs and their TAD boundaries) that drive leukemia. To this end, we will first profile and analyze a large cohort of leukemia patients using Hi-C and H3K27ac HiChIP both at diagnosis and at relapse to identify recurrent relapse-specific 3D reorganization events. We will combine computational methods with or- thogonal CRISPR strategies to discover transcription factors and epigenetic modifiers that enable the emergence of drug resistance via the rewiring of enhancer-promoter chromatin looping. We will then focus on enhancer hubs: we and others have shown that enhancers that are densely connected with target promoters and other enhancers (i.e. enhancer hubs) are robust regulators of gene expression and their disruption can impact the regulation of multiple genes. Based on these findings, we will test whether such enhancer hubs and their 3D topology can be drivers of drug resistance by activating oncogenic loci in vitro and in vivo. Our proposed study will not only elucidate the role of 3D architecture in leukemia at diagnosis and relapse, but it will also advance our understanding of resistance to therapy and develop new approaches to overcome it.