CTCF-binding element regulates ESC differentiation via orchestrating long-range chromatin interaction between enhancers and HoxA

J Biol Chem. 2021 Jan-Jun:296:100413. doi: 10.1016/j.jbc.2021.100413. Epub 2021 Feb 11.


Proper expression of Homeobox A cluster genes (HoxA) is essential for embryonic stem cell (ESC) differentiation and individual development. However, mechanisms controlling precise spatiotemporal expression of HoxA during early ESC differentiation remain poorly understood. Herein, we identified a functional CTCF-binding element (CBE+47) closest to the 3'-end of HoxA within the same topologically associated domain (TAD) in ESC. CRISPR-Cas9-mediated deletion of CBE+47 significantly upregulated HoxA expression and enhanced early ESC differentiation induced by retinoic acid (RA) relative to wild-type cells. Mechanistic analysis by chromosome conformation capture assay (Capture-C) revealed that CBE+47 deletion decreased interactions between adjacent enhancers, enabling formation of a relatively loose enhancer-enhancer interaction complex (EEIC), which overall increased interactions between that EEIC and central regions of HoxA chromatin. These findings indicate that CBE+47 organizes chromatin interactions between its adjacent enhancers and HoxA. Furthermore, deletion of those adjacent enhancers synergistically inhibited HoxA activation, suggesting that these enhancers serve as an EEIC required for RA-induced HoxA activation. Collectively, these results provide new insight into RA-induced HoxA expression during early ESC differentiation, also highlight precise regulatory roles of the CTCF-binding element in orchestrating high-order chromatin structure.

Keywords: CTCF-binding element; HoxA; differentiation; embryonic stem cells; enhancer; enhancer–enhancer interaction complex; long-range chromatin interaction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CCCTC-Binding Factor / metabolism*
  • CCCTC-Binding Factor / physiology
  • Cell Differentiation
  • Cell Line
  • Chromatin / genetics
  • Chromatin / metabolism
  • Chromatin Assembly and Disassembly
  • Embryonic Stem Cells / metabolism*
  • Embryonic Stem Cells / physiology
  • Enhancer Elements, Genetic / genetics
  • Gene Expression / genetics
  • Gene Expression Regulation / genetics
  • Genes, Homeobox / genetics
  • Homeodomain Proteins / genetics
  • Homeodomain Proteins / metabolism*
  • Homeodomain Proteins / physiology
  • Mice
  • Transcriptional Activation
  • Tretinoin / pharmacology


  • CCCTC-Binding Factor
  • Chromatin
  • Ctcf protein, mouse
  • Homeodomain Proteins
  • HoxA protein
  • Tretinoin