Mutational synergy coordinately remodels chromatin accessibility, enhancer landscape and 3-Dimensional DNA topology to alter gene expression during leukemia induction (ChIP-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. Overall design: Chromatin states were profiled by performing ChIP-seq on histone modifications (including H3K4me1, H3K4me3 and H3K27ac) in WT and mutant HSPCs, as well as in WT neutrophils. Two biological replicates were performed for each cellular state.

转录调控异常是急性髓系白血病（acute myeloid leukemia, AML）的核心特征，但突变如何协同重塑表观遗传景观并重构三维（3-D）DNA拓扑结构的机制仍未阐明。本研究构建并利用了携带AML最常见突变——Flt3-ITD与Npm1c的小鼠等位基因系列模型，该模型覆盖了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单一种突变的单突变体，single mutant, SM）以及恶性状态（双突变体，double mutant, DM）。研究人员对野生型及突变小鼠的造血干祖细胞（hematopoietic stem and progenitor cells, HSPCs）开展了多维度组学分析：包括基因表达检测（RNA测序，RNA-seq）、染色质激活状态解析（针对H3K4me1、H3K4me3、H3K27ac的染色质免疫共沉淀测序，ChIP-seq）、染色质可及性检测（ATAC测序，ATAC-seq），以及启动子锚定的三维染色质互作分析（启动子捕获Hi-C，promoter capture HiC, pCHiC）；并整合上述多组学分析结果，阐明AML发生过程中的转录、表观遗传及DNA拓扑结构演化规律。本研究成果可识别远距离顺式调控回路以及更为庞大精细的基因调控网络，并通过扰动网络核心成员验证了此类网络的生物学重要性。总体实验设计：针对野生型及突变型造血干祖细胞、野生型中性粒细胞，通过组蛋白修饰染色质免疫共沉淀测序（涵盖H3K4me1、H3K4me3及H3K27ac）分析染色质状态；每种细胞状态均设置2个生物学重复。