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

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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.