Mutational synergy coordinately remodels chromatin accessibility, enhancer landscape and 3-Dimensional DNA topology to alter gene expression during leukemia induction (RNA-seq)

Altered transcription is a cardinal feature of acute myeloid leukemia (AML), however, exactly how mutations synergise to remodel the epigenetic landscape and rewire 3-Dimensional (3-D) DNA topology is unknown. We have utilized an allelic series of mice carrying the most common mutations in AML, namely Flt3-ITD and Npm1c. These model different “transition states” (normal: wild-type (WT); pre-malignant: single mutant (SM) with either Flt3-ITD or Npm1c; malignant: double mutant (DM)) during AML induction. We have analyzed hematopoietic stem and progenitor cells (HSPCs) from WT and mutant mice for gene expression (RNA-seq), chromatin activation states (ChIP-seq for H3K4me1, H3K4me3, H3K27ac), chromatin accessibility (ATAC-seq), and promoter-anchored 3-D chromatin interaction (promoter capture HiC, pCHiC) and have integrated these analyses to determine the transcriptional, epigenetic and DNA-topological evolution of AML. These findings allow the identification of long-range cis-regulatory circuits, as well as larger and more detailed gene-regulatory networks, whose importance we demonstrate through perturbation of network members. Global gene expression was profiled using RNA-seq in WT and mutant HSPCs, as well as in WT neutrophils. Two biological replicates were performed for each cellular state.

转录失调是急性髓系白血病（acute myeloid leukemia, AML）的核心特征，但目前尚未明确突变如何协同重塑表观遗传景观并重构三维（3-D）DNA拓扑结构。本研究采用携带AML最常见突变Flt3-ITD与Npm1c的等位基因系列小鼠，该模型涵盖了AML发生进程中的三类过渡状态：正常状态（野生型，wild-type, WT）、癌前状态（仅携带Flt3-ITD或Npm1c任一突变的单突变体，single mutant, SM）以及恶性状态（双突变体，double mutant, DM）。我们对野生型及突变型小鼠的造血干祖细胞（hematopoietic stem and progenitor cells, HSPCs）开展了多组学分析，涵盖基因表达检测（RNA测序，RNA-seq）、染色质激活状态分析（针对H3K4me1、H3K4me3、H3K27ac的染色质免疫沉淀测序，ChIP-seq）、染色质可及性分析（转座酶可及性测序，ATAC-seq），以及启动子锚定的三维染色质相互作用检测（启动子捕获Hi-C，pCHiC），并整合多组学数据解析AML发生过程中的转录、表观遗传及DNA拓扑结构演化规律。本研究的发现可用于识别远距离顺式调控环路以及更为庞大精细的基因调控网络，并通过扰动该网络中的成员验证了这些网络的重要性。此外，我们通过RNA-seq对野生型及突变型造血干祖细胞、野生型中性粒细胞开展了全基因表达谱分析，每一种细胞状态均设置2次生物学重复。