HOTTIP reinforces CTCF-defined TAD boundaries by forming R-loops to drive Wnt/b-catenin target gene expression in AML leukemogenesis

HOTTIP lncRNA is highly expressed in acute myeloid leukemia (AML) patients carrying MLL rearrangement and NPM1c+ mutations, and positively correlates with poor patient survival by facilitating formation of leukemic-specific topologically associated domains (TADs) to drive leukemic transcription program. However, the direct mechanism through which HOTTIP accesses and regulates leukemic genome topology remains unknown. Here, we showed that HOTTIP directly interacts and regulates a fraction of CTCF binding sites (CBSs) including key WNT/b-catenin target loci in the AML genome by forming R-loop structure that reinforces the CTCF boundary, WNT/b-catenin TADs and gene transcription. Importantly, either deleting CBSs or targeting RNaseH to eliminate R-loop structure in boundaries of the Wnt/b-catenin TADs resulted in inhibited Wnt target promoter interactomes and Wnt target expression, and mitigation of leukemogenesis of PDX mouse models with aberrant HOTTIP expression. Thus, HOTTIP reinforces CTCF-defined TAD boundaries by forming R-loops to drive Wnt/b-catenin aberration and pathogenesis of MLL-rearranged/NPM1c+ AML. Overall design: We have finished the RNA-SEQ, ATAC-seq, ChIP-seq, DRIP-seq, DRIPc-seq, NG-Capture-C and single cell RNA-seq and scATAC-seq for WT and HOTTIP-KO MOLM13 leukemia cells and Hottip Transgenic mice cells. LK cells (Lin- Kit+) and LSK cells (Lin- Sca1+ Kit+) were sorted by FACS from the bone marrow of WT and Hottip transgenic mice for RNA-seq, ATAC-seq and single cell-RNA seq and scATAC-seq assay