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Repurposed nuclear speckle and mitosis components induce CTCF clustering to sustain the senescence splicing program [ChIPmentation]

ABSTRACT: Senescence —the end-point of the replicative life span of normal cells— is characterized by a complex sequence of molecular and biochemical events. One of these is the dramatic reorganization of CTCF into large senescence-induced CTCF clusters (SICCs). However, the molecular determinants, genomic consequences, and functional purpose of SICCs remained unknown. Here, we combine super-resolution imaging, 3D genomics, and functional assays with computational modelling to address all aspects of SICC emergence. First, we used modelling and data from the overexpression of nuclear factors lost upon senescence entry to explain how changes in the nuclear environment allow for CTCF clustering. Second, we discovered that cells entering senescence repurpose SRRM2 —a key component of nuclear speckles— and BANF1 —a protein known to act as ‘molecular glue’ for chromosomes during mitosis— to cluster CTCF on the surface of speckles, while rewiring higher-order genome architecture. Last, we could show that SICCs are required for the faithful execution of the senescence splicing program as their disruption by SRRM2 or BANF1 depletion reverts splicing choices. Together, the senescence-specific clustering of CTCF on nuclear speckles is a new paradigm of a change in the biochemical environment of the nucleus being translated into its architectural reorganization to allow for the deployment of a splicing program driving homeostatic deregulation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE269858 | GEO | 2024/06/17

REPOSITORIES: GEO

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Project description: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 analysis, mass spectrometry, RNA immunoprecipitation-sequence analysis, 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, the majority of CTCF protein forms non-genomic complexes that localize to Serine/arginine-rich splicing factor (SC-35)-containing nuclear speckles, exclusive of its canonical association with chromatin. Upon stress, non-genomic 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. CTCF complexes that associate with genomic DNA are resistant to stress-induced degradation and CTCF-DNA binding is largely unchanged. Restoration of cellular 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 (hPSC). 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 non-canonical CTCF complexes serve a largely non-genomic role in RNA processing, potentially to maintain cells in a particular differentiation state, that is dynamically regulated by environmental signals. The non-canonical, 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.

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Repurposed nuclear speckle and mitosis components induce CTCF clustering to sustain the senescence splicing program [ChIPmentation]

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