Abstract
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.
Highlights
Exposure of an organism or tissue to physiological stress results in an orchestrated cellular response that induces profound changes in gene expression, RNA processing, and protein synthesis that drive cell fate
We propose that in specific cell types, stress-sensitive forms of CCCTC-binding factor (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
We extended this analysis by examining CTCF protein levels during a time course of the human mammary epithelial cells (HMECs) to variant” HMECs (vHMECs) transition from days 1–70 (Fig 1B)
Summary
Exposure of an organism or tissue to physiological stress results in an orchestrated cellular response that induces profound changes in gene expression, RNA processing, and protein synthesis that drive cell fate. This coordinated strategy accelerates adaptive processes necessary for individual cells within a population to survive diverse and unanticipated forms of stress [1]. Much of our knowledge about the human cellular stress response comes from studies in which human cancer cell lines were exposed to genotoxic agents While valuable, these conditions do not adequately reflect how our normal healthy cells respond to physiological stressors that they are routinely subject to in vivo (e.g., hypoxia, inflammation, oxidative damage). Beyond the scope of our initial aim, our data unexpectedly revealed that the multi-functional nuclear protein CCCTC-binding factor (CTCF) is an exquisitely sensitive target of diverse forms of cellular stress
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