HOTTIP-dependent R-loop formation regulates CTCF boundary activity and TAD integrity in leukemia

HOTTIP lncRNA is highly expressed in acute myeloid leukemia (AML) driven by MLL rearrangements or NPM1 mutations to mediate HOXA topologically associated domain (TAD) formation and drive aberrant transcription. However, the mechanism through which HOTTIP accesses CCCTC-binding factor (CTCF) chromatin boundaries and regulates CTCF-mediated genome topology remains unknown. Here, we show that HOTTIP directly interacts with and regulates a fraction of CTCF-binding sites (CBSs) in the AML genome by recruiting CTCF/ cohesin complex and R-loop-associated regulators to form R-loops. HOTTIP-mediated R-loops reinforce the CTCF boundary and facilitate formation of TADs to drive gene transcription. Either deleting CBS or targeting RNase H to eliminate R-loops in the boundary CBS of beta-catenin TAD impaired CTCF boundary activity, inhibited promoter/enhancer interactions, reduced beta-catenin target expression, and mitigated leukemogenesis in xenograft mouse models with aberrant HOTTIP expression. Thus, HOTTIP-mediated R-loop formation directly reinforces CTCF chromatin boundary activity and TAD integrity to drive oncogene transcription and leukemia development.

Automated summary

In acute myeloid leukemia (AML), HOTTIP lncRNA is highly expressed and drives AML development by regulating the formation of HOXA topologically associated domains (TADs) and aberrant transcription. This study reveals that HOTTIP directly interacts with a fraction of CTCF-binding sites (CBSs) in the AML genome and recruits CTCF/cohesin complex and R-loop-associated regulators to form R-loops. HOTTIP-mediated R-loop formation reinforces CTCF chromatin boundary activity and facilitates TAD formation, driving oncogene transcription and leukemia development.