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

摘要： 急性髓系白血病是一种造血干细胞和祖细胞的异质性疾病 （HSPC）与驱动基因突变的顺序获得相关。这些突变通常会导致 干扰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，一种后部HOXA相关的长非编码RNA（lncRNA），重塑CTCF， 拓扑关联域（Topologically Associated Domains，TADs）这种重塑调节同源异型基因相关的 白血病转录程序和促进AML白血病发生，由MLL重排（MLLR+）驱动，或 NMP 1突变（NPM 1C+）。在AML中，HOTTIP调控的最重要的转录基序之一是CTCF结合位点 （CBS），表明HOTTIP在调节CTCF介导的基因组组织和AML中的新功能 发病机制事实上，结合RNA-seq、ChIRP-seq和CTCF ChIP-seq分析显示，HOTTIP 在AML基因组的一个亚组中与CTCF共占据，包括HOXA和WNT/β-连环蛋白靶基因座， 为了他们的激活。然而，HOTTIP lncRNA是否以及如何直接调节CTCF仍然是未知的。 定向基因组组织以促进白血病转录网络和白血病发生。我们的初步 数据表明，HOTTIP能够直接与HOXA和WNT中的关键边界CBS相互作用 通过形成R环结构靶向基因座。我们假设HOTTIP激活介导了异常的R- CBS中形成环以分层CTCF染色质边界，用于重编程AML TAD和白血病 转录网络，导致HSPC扰动和白血病发生。为了验证这一假设，我们将重点 关于改变的CTCF边界和R环形成对AML基因组上HOTTIP活化的影响 调节和基因转录输出。在本提案中，我们将测试HOTTIP激活的重要性- 介导的异常R环形成在调节CTCF边界活性和转录调节中的作用。 急性髓细胞白血病具体来说，我们将确定和表征关键的HOTTIP调节的CTCF染色质边界， AML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HOTTIP介导的异常R环的影响， 在白血病发生和HSPC调节的特定边界上形成。

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.