Hottip-Mediated R-Loops Regulate CTCF TAD Boundary to Control WNT/b-Catenin Pathway in AML Genome

lncRNA, transcribed from genomic DNA, can form a DNA: RNA hybridization structure to access regulatory elements. HOX loci associated long noncoding RNA (lncRNA), HOTTIP, acts as an epigenetic regulator to define oncogenic HOXA topologically associated domain (TAD) and drive HOXA associated leukemic transcription program. Activation of HOTTIP promoted AML pathogenesis by perturbing leukemic transcription program and hematopoietic/progenitor stem cells (HS/PCs) function. However, the mechanism by which HOTTIP accesses and regulates genomic DNA transcriptional output remains unknown.

Here, Combined RNA-seq, ChIP-seq, NG-Capture-C and Hi-C integrated analysis demonstrated that HOTTIP dependent stratification/formation of TADs at WNT/b-catenin loci is critical for b-catenin (CTNNB1) target gene transcription in AML cells. We further showed that HOTTIP specifically interacts with CTCF-occupied TAD boundary CTCF binding sites (CBSs) at b-catenin and its target loci (e.g. MYC and MECOM) by formation of R-loops. To test whether HOTTIP-mediated R-loops contribute to boundary maintanence and TAD stablization, we created CRISPR/dCas9-RNaseH-mediated disruption of R-loop structure In MOLM13 cells by which the Ribonuclease H (RNase H) was targeted to the HOTTIP/CTCF co-occupied CTNNB1 or MYC boundary CBSs to specifically target RNA: DNA hybridization region. Our DNA-RNA immunoprecipitation (DRIP)-qPCR data indicated that CRISPR/dCas9-RNaseH-mediated sgRNAs disrupted the R-loop structure in MYC and CTNNB1 defined TAD boundary regions. Furthermore, HOTTIP CHIRP-qPCR and CTCF CHIP-qPCR suggested that loss of R-loop structure in MYC or CTNNB1 TAD boundary CBSs decreased both CTCF and HOTTIP binding in these CBSs. Disruption of R-loops also affects the long-range interaction within CTNNB1 or MYC TADs that are important for WNT/b-catenin signaling network and AML leukemogenesis.

In conclusion, our finding demonstrated that HOTTIP forming DNA∙RNA hybridized R-loops mediate AML genome organization at specific gene loci and control leukemic transcription program. Thus, HOTTIP lncRNA mediated R-loop structure will be considered as a novel molecular mechanism in AML leukemogensis.