Role of lncRNA mediated R-loops in CTCF boundary function and AML genome organization

摘要： 急性髓系白血病(AML)是一种造血干/祖细胞的异质性疾病 (HSPC)与司机基因突变的顺序获取相关。这些突变通常会导致改变 扰乱HSC自我更新和分化的基因组组织和转录程序。最近， 我们发现，HotTip是一种与HOXA相关的后部长非编码RNA(LncRNA)，它重塑CTCF- 定义的拓扑关联域(TADS)。这种重塑调节与同源异型基因相关的 白血病转录程序，并促进由MLL重排(MLLr)或 NMP1突变(NPM1C)。AML中最重要的HotTip调控转录基序之一是CTCF结合位点 (CBSS)，提示HotIP在调节CTCF介导的基因组组织和AML中具有新的功能 发病机制。实际上，组合RNA-seq、chirp-seq和ctcf芯片-seq分析揭示了HotTip 在包括HOXA和WNT/β-catenin靶基因座在内的急性髓系白血病基因组的一个子集上与ctcf共同占据， 用来激活它们。然而，目前尚不清楚HotTip LncRNA是否以及如何直接调控CTCF- 引导基因组组织以促进白血病转录网络和白血病发生。我们的预赛 数据显示，HotTip能够直接与霍夏和WNT的关键TAD边界CBSS相互作用 靶基因通过形成R-环结构。我们假设热尖的激活介导了异常的R- 在CBSS中形成环以分层CTCF染色质边界以重新编程AML、TADS和白血病 转录网络，导致HSPC扰动和白血病的发生。为了检验这一假设，我们将把重点放在 CTCFTAD边界改变和R环形成对AML基因组热尖端激活的影响 调控和基因转录输出。在这项提案中，我们将测试热提示激活的重要性- 介导的异常R环形成在调控CTCF边界活性和转录调控中的作用 AML。具体地说，我们将识别和表征关键的HotTip调控的CTCF染色质边界 急性髓系白血病基因组。然后我们将定义R环在HotTip介导的CTCF染色质边界中的新作用 定义和基因组组织。最后，我们将评估热尖介导的异常R环的影响 白血病发生中特定TAD边界的形成及其对HSPC的调控。

Abstract: Acute myeloid leukemia (AML) is a heterogeneous disorder of hematopoietic stem and progenitor cells (HSPCs) associated with sequential acquisition of driver gene mutations. These mutations often lead to altered genome organization and transcriptional programs that perturb HSC self-renewal and differentiation. Recently, we discovered that HOTTIP, a posterior HOXA-associated long non-coding RNA (lncRNA), remodels CTCF- defined topologically associated domains (TADs). This remodeling regulates the homeotic gene-associated leukemic transcription program and facilitates AML leukemogenesis, driven by MLL rearrangement (MLLr+) or NMP1 mutation (NPM1C+). One of the top HOTTIP-regulated transcription motifs in AML is CTCF-binding sites (CBSs), suggesting a novel function of HOTTIP in regulating CTCF-mediated genome organization and AML pathogenesis. Indeed, combined RNA-seq, ChIRP-seq, and CTCF ChIP-seq analyses revealed that HOTTIP co-occupied with CTCF in a subset of the AML genome, including HOXA and WNT/β-catenin target gene loci, for their activation. However, it remains unknown whether and how HOTTIP lncRNA directly regulates CTCF- directed genome organization to promote leukemic transcription networks and leukemogenesis. Our preliminary data showed that HOTTIP is capable of directly interacting with key TAD boundary CBSs in the HOXA and WNT target loci via formation of an R-loop structure. We hypothesize that HOTTIP activation mediates aberrant R- loop formation in CBSs to stratify CTCF chromatin boundary for reprograming AML TADs and leukemic transcription networks, leading to HSPC perturbation and leukemogenesis. To test this hypothesis, we will focus on the impact of the altered CTCF TAD boundary and R-loop formation upon HOTTIP activation on AML genome regulation and gene transcription output. In this proposal we will test the importance of the HOTTIP activation- mediated aberrant R-loop formation in modulating the CTCF boundary activity and transcriptional regulation in AML. Specifically, we will identify and characterize critical HOTTIP-regulated CTCF chromatin boundaries in the AML genome. We will then define the novel role of R-loops in HOTTIP-mediated CTCF chromatin boundary definition and genome organization. Finally, we will assess the impact of the HOTTIP-mediated aberrant R-loop formation at specific TAD boundaries on leukemogenesis and HSPC regulation.