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

• Published in: PLoS genetics Vol. 17; no. 1; p. e1009277
• Main Authors: Lehman, Bettina J., Lopez-Diaz, Fernando J., Santisakultarm, Thom P., Fang, Linjing, Shokhirev, Maxim N., Diffenderfer, Kenneth E., Manor, Uri, Emerson, Beverly M.
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 07.01.2021; Public Library of Science (PLoS)
• Subjects: Binding Sites; Biology and Life Sciences; CCCTC-Binding Factor - genetics; Cell Differentiation; Cell Line, Tumor; Cell nuclei; Chromatin; Chromosomes; Control; DNA-Binding Proteins - genetics; Epigenesis, Genetic - genetics; Gene Expression Regulation; Genetic aspects; Genetic regulation; Genomics; Humans; Mammary Glands, Human - cytology; Mammary Glands, Human - metabolism; Medicine and Health Sciences; Methods; Neoplasms - genetics; Neoplasms - pathology; Neurodegenerative Diseases - genetics; Neurodegenerative Diseases - pathology; Neurons - metabolism; Neurons - pathology; Oxidative Stress - genetics; Physiological aspects; Pluripotent Stem Cells - metabolism; Pluripotent Stem Cells - pathology; Protein Binding; RNA Processing, Post-Transcriptional - genetics; Serine-Arginine Splicing Factors - genetics; Stress (Physiology); Stress, Physiological - genetics; Transcription factors
• ISSN: 1553-7404; 1553-7390; 1553-7404
• DOI: 10.1371/journal.pgen.1009277

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.