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

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. Author summary Our tissues are subject to chronic physiological and environmental damage, yet little is known about how healthy human cells normally respond to stress. We examined the effect of damage on cells obtained from breast tissue of disease-free women. Unexpectedly, we identified a well-known protein regulator of chromosomal function, CTCF, as a robust target of stress signals. In normal mammary cells, a pool of CTCF is localized to large depots within the nucleus that regulate RNA processing. Upon cellular damage, CTCF rapidly disappears from nuclear depots by stress-inducible protein degradation while genome occupancy by CTCF is relatively unaffected. We observe the same phenomenon in neuronal progenitors differentiated from human pluripotent stem cells. We propose that in specific cell types, stress-sensitive forms of CTCF exist that have a unique function in RNA metabolism potentially by fine-tuning gene expression near nuclear speckles, which may maintain cells in a progenitor or adaptive state. Upon stress, this particular CTCF function is rapidly disabled, which may change the identity of cells most vulnerable to disease in order to safeguard them from becoming dysfunctional. Persistently stressed cells have lost this CTCF function, which may facilitate the genesis of damage-induced cancer initiation and neuro-degeneration.

Automated summary

The study reveals that CTCF is highly sensitive to diverse forms of stress in human primary cells, leading to changes in its localization and interactions with RNA. The stress-sensitive pool of CTCF protein can be restored through proteasome inhibition. This stress response in CTCF is observed during neuronal differentiation, highlighting its potential role in cell differentiation regulation.