Dynamic regulation of CTCF stability and sub-nuclear localization in response to stress

PLoS Genet. 2021 Jan 7;17(1):e1009277. doi: 10.1371/journal.pgen.1009277. eCollection 2021 Jan.


The nuclear protein CCCTC-binding factor (CTCF) has diverse roles in chromatin architecture and gene regulation. Functionally, CTCF associates with thousands of genomic sites and interacts with proteins, such as cohesin, or non-coding RNAs to facilitate specific transcriptional programming. In this study, we examined CTCF during the cellular stress response in human primary cells using immune-blotting, quantitative real time-PCR, chromatin immunoprecipitation-sequence (ChIP-seq) analysis, mass spectrometry, RNA immunoprecipitation-sequence analysis (RIP-seq), and Airyscan confocal microscopy. Unexpectedly, we found that CTCF is exquisitely sensitive to diverse forms of stress in normal patient-derived human mammary epithelial cells (HMECs). In HMECs, a subset of CTCF protein forms complexes that localize to Serine/arginine-rich splicing factor (SC-35)-containing nuclear speckles. Upon stress, this species of CTCF protein is rapidly downregulated by changes in protein stability, resulting in loss of CTCF from SC-35 nuclear speckles and changes in CTCF-RNA interactions. Our ChIP-seq analysis indicated that CTCF binding to genomic DNA is largely unchanged. Restoration of the stress-sensitive pool of CTCF protein abundance and re-localization to nuclear speckles can be achieved by inhibition of proteasome-mediated degradation. Surprisingly, we observed the same characteristics of the stress response during neuronal differentiation of human pluripotent stem cells (hPSCs). CTCF forms stress-sensitive complexes that localize to SC-35 nuclear speckles during a specific stage of neuronal commitment/development but not in differentiated neurons. We speculate that these particular CTCF complexes serve a role in RNA processing that may be intimately linked with specific genes in the vicinity of nuclear speckles, potentially to maintain cells in a certain differentiation state, that is dynamically regulated by environmental signals. The stress-regulated activity of CTCF is uncoupled in persistently stressed, epigenetically re-programmed "variant" HMECs and certain cancer cell lines. These results reveal new insights into CTCF function in cell differentiation and the stress-response with implications for oxidative damage-induced cancer initiation and neuro-degenerative diseases.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • CCCTC-Binding Factor / genetics*
  • Cell Differentiation
  • Cell Line, Tumor
  • Chromatin
  • Chromosomes
  • DNA-Binding Proteins / genetics*
  • Epigenesis, Genetic / genetics
  • Gene Expression Regulation
  • Genomics
  • Humans
  • Mammary Glands, Human / cytology
  • Mammary Glands, Human / metabolism
  • Neoplasms / genetics*
  • Neoplasms / pathology
  • Neurodegenerative Diseases / genetics*
  • Neurodegenerative Diseases / pathology
  • Neurons / metabolism
  • Neurons / pathology
  • Oxidative Stress / genetics
  • Pluripotent Stem Cells / metabolism
  • Pluripotent Stem Cells / pathology
  • Protein Binding
  • RNA Processing, Post-Transcriptional / genetics
  • Serine-Arginine Splicing Factors / genetics*
  • Stress, Physiological / genetics


  • CCCTC-Binding Factor
  • CTCF protein, human
  • CTCFL protein, human
  • Chromatin
  • DNA-Binding Proteins
  • Serine-Arginine Splicing Factors