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

Mol Cell. 2022 Feb 17;82(4):833-851.e11. doi: 10.1016/j.molcel.2022.01.014.

Abstract

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 β-catenin TAD impaired CTCF boundary activity, inhibited promoter/enhancer interactions, reduced β-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.

Keywords: AML leukemogenesis; CTCF chromatin boundary; HOTTIP lncRNA; R-loops; TAD formation; canonical Wnt transcription; cohesin complex; enhancer/promoter interactions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CCCTC-Binding Factor / genetics
  • CCCTC-Binding Factor / metabolism*
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism
  • Cell Line, Tumor
  • Chromatin / genetics
  • Chromatin / metabolism*
  • Chromosomal Proteins, Non-Histone / genetics
  • Chromosomal Proteins, Non-Histone / metabolism
  • Cohesins
  • Gene Expression Regulation, Leukemic
  • HEK293 Cells
  • Humans
  • Leukemia, Myeloid, Acute / genetics
  • Leukemia, Myeloid, Acute / metabolism*
  • Leukemia, Myeloid, Acute / pathology
  • Mice, Transgenic
  • R-Loop Structures*
  • RNA, Long Noncoding / genetics
  • RNA, Long Noncoding / metabolism*
  • Structure-Activity Relationship
  • Transcription, Genetic
  • Transcriptional Activation
  • beta Catenin / genetics
  • beta Catenin / metabolism*

Substances

  • CCCTC-Binding Factor
  • CTCF protein, human
  • CTNNB1 protein, human
  • Cell Cycle Proteins
  • Chromatin
  • Chromosomal Proteins, Non-Histone
  • RNA, Long Noncoding
  • beta Catenin
  • long noncoding RNA HOTTIP, human