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

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.

CTCF as a regulator of alternative splicing: new tricks for an old player

“…( B ) CTCF regulation of DNA methylation via activation of PARP1 and PARylation ( 60 , 61 ), which are also involved in other AS-related regulatory activities ( 66–69 ). ( C ) Splicing factor recruitment might also take part in CTCF-mediated AS regulation through direct interaction with RNA binding proteins ( 42 , 58 , 70 ) or other transcription factors involved in AS such as PARP1 ( 66 ), MeCP2 ( 38 ), YB-1 ( 108 , 109 ) and HP1α ( 46 , 124 , 127 ). SF3B1 and SRSF3 are examples of RNA-binding proteins.…”

“…This could be tested by examining the effect of CTCF on PARP1-mediated recruitment of SF3B1 and subsequently pre-mRNA splicing ( 66 ). Proteomics analysis of CTCF-interacting partners in MCF10A cells identified RNA-binding proteins including snRNPs, heterogeneous nuclear ribonucleoproteins (hnRNPs) and serine-arginine proteins ( 70 ), which are essential components of the splicing machinery ( 51 ). In addition, CTCF can transcriptionally regulate or directly interact with multiple splicing factors.…”

Pericentromeric noncoding RNA changes DNA binding of CTCF and inflammatory gene expression in senescence and cancer

“…In the previous study, Zirkel et al revealed that, upon senescence entry, the high-mobility group B protein (HMGB2) nuclear depletion provokes the alteration of CTCF distribution and CTCF spatial clustering ( 18 ). Moreover, Lehman et al showed that stressors, such as acute oxidative stress, cause CTCF reduction from nuclear speckles and changes in CTCF RNA interaction ( 17 ). These reports support our findings that pericentromeric satellite RNA up-regulated during cellular senescence directly binds to CTCF and disturbs CTCF function.…”

“…Chromatin organization and global gene expression are coordinately maintained by the CCCTC-binding factor (CTCF), a zinc-finger (ZF) nucleic acidbinding protein, and the cohesin complex; together, these factors orchestrate higher-order chromatin conformation through the formation of intrachromosomal and interchromosomal loops (14)(15)(16). Given that the nuclear localization and RNA-binding capacity of CTCF dynamically change due to cellular stress (17) and the alteration of CTCF distribution and/or followed by chromatin reorganization occur during cellular senescence…”

“…Chromatin organization and global gene expression are coordinately maintained by the CCCTC-binding factor (CTCF), a zinc-finger (ZF) nucleic acid–binding protein, and the cohesin complex; together, these factors orchestrate higher-order chromatin conformation through the formation of intrachromosomal and interchromosomal loops ( 14 – 16 ). Given that the nuclear localization and RNA-binding capacity of CTCF dynamically change due to cellular stress ( 17 ) and the alteration of CTCF distribution and/or followed by chromatin reorganization occur during cellular senescence ( 13 , 18 ), postulating that CTCF distribution is associated with SASP gene expression in senescent cells is reasonable. However, the molecular mechanism underlying the connection between CTCF regulation and its association with SASP gene expression remains elusive.…”