Dynamic association of the mammalian insulator protein CTCF with centrosomes and the midbody

CTCF is a highly conserved, ubiquitously expressed DNA-binding protein that has widespread capabilities in gene regulation. CTCF plays important roles in cell growth regulatory processes and epigenetic functions. Ectopic expression of CTCF results in severe cell growth inhibition at multiple points within the cell cycle, indicating that CTCF levels must be stringently monitored. We have investigated the subcellular localization of CTCF in detail. Interestingly, we observe that CTCF shows a dynamic cell cycle-dependent distribution. Immunofluorescent staining reveals that in interphase CTCF is a nuclear protein, which is mainly excluded from the nucleolus. Strikingly, CTCF is associated with the centrosome during mitosis, especially from metaphase to anaphase. At telophase, CTCF dissociates from the centrosome and localizes to the midbody and the reformed nuclei. The association of CTCF with centrosomes and the midbody is further confirmed by biochemical fractionation. Moreover, subcellular fractions of CTCF show cell cycle and organelle-specific posttranslational modifications, suggesting different roles for CTCF at different stages of the cell cycle.     

The CTCF Insulator Protein Is Posttranslationally Modified by SUMO

The CTCF protein is a highly conserved zinc finger protein that is implicated in many aspects of gene regulation and nuclear organization. Its functions include the ability to act as a repressor of genes, including the c-myc oncogene. In this paper, we show that the CTCF protein can be posttranslationally modified by the small ubiquitin-like protein SUMO. CTCF is SUMOylated both in vivo and in vitro, and we identify two major sites of SUMOylation in the protein. The posttranslational modification of CTCF by the SUMO proteins does not affect its ability to bind to DNA in vitro. SUMOylation of CTCF contributes to the repressive function of CTCF on the c-myc P2 promoter. We also found that CTCF and the repressive Polycomb protein, Pc2, are colocalized to nuclear Polycomb bodies. The Pc2 protein may act as a SUMO E3 ligase for CTCF, strongly enhancing its modification by SUMO 2 and 3. These studies expand the repertoire of posttranslational modifications of CTCF and suggest roles for such modifications in its regulation of epigenetic states.     

CTCF regulates the FoxO signaling pathway to affect the progression of prostate cancer

The present research focuses on the influence of CCCTC‐binding factor (CTCF) on prostate cancer (PC) via the regulation of the FoxO signalling pathway. A bioinformatics analysis was conducted to screen out target genes for CTCF in LNCaP cells and to enrich the relevant pathways in LNCaP cells. It was found that the FoxO pathway was enriched according to the ChIP‐seq results of CTCF. The expression of CTCF, pFoxO1a, FoxO1a, pFoxO3a and FoxO3a was tested by RT‐qPCR and Western blot. Inhibition of CTCF could lead to the up‐regulation of the FoxO signalling pathway. The rates of cell proliferation, cell invasion and apoptosis were examined by MTT assay, cell invasion assay and flow cytometry under different interference conditions. Down‐regulation of CTCF could suppress cell proliferation, cell invasion and facilitate cell apoptosis. Lastly, the effect of CTCF on tumour growth was determined in nude mice. Inhibition of CTCF regulated the FoxO signalling pathway, which retarded tumour growth in vivo. In conclusion, CTCF regulates the FoxO signalling pathway to affect the progress of PC.     

The tumor suppressor role of CTCF

CTCF is an evolutionary conserved and ubiquitously expressed protein that binds thousands of sites in the human genome. Ectopic expression of CTCF in various normal and tumoral human cell lines inhibits cell division and clonogenicity, with the consequence to consider CTCF a potential tumor-suppressor factor. In this review article, we focused on the molecular mechanisms engaged by CTCF to modulate the expression of several key-regulators of differentiation, cellular senescence, cell cycle control and progression, whose expression is frequently altered in tumors. Moreover, we discussed common features of CTCF at each tumor-related DNA-binding sequence, such as protein-partners, post-translational modifications, and distinctive epigenetic marks establishment. The investigation of the molecular mechanisms engaged by CTCF to modulate tumor-related genes emphasizes the cell-type dependency of its tumor suppressor role. Indeed, the ability of CTCF to bind their promoters strictly depends by cell-type features as DNA methylation, BORIS-binding and post-translational modifications as PARYlation.     

CTCF-Induced Circular DNA Complexes Observed by Atomic Force Microscopy

The CTCF protein has emerged as a key architectural protein involved in genome organization. Although hypothesized to initiate DNA looping, direct evidence of CTCF-induced DNA loop formation is still missing. Several studies have shown that the 11 zinc finger (11 ZF) domain of CTCF is actively involved in DNA binding. We here use atomic force microscopy to examine the effect of the 11 ZF domain comprising residues 266–579 (11 ZF CTCF) and the 3 ZF domain comprising residues 402–494 (6–8 ZF CTCF) of human CTCF on the DNA morphology. Our results show that both domains alter the DNA architecture from the relaxed morphology observed in control DNA samples to compact circular complexes, meshes, and networks, offering important insights into the multivalent character of the 11 ZF CTCF domain. Atomic force microscopy images reveal quasi-circular DNA/CTCF complexes, which are destabilized upon replacing the 11 ZF CTCF by the 6–8 ZF CTCF domain, highlighting the role of the 11 ZF motif in loop formation. Intriguingly, the formation of circular DNA/CTCF complexes is dominated by non-specific binding, whereby contour length and height profiles suggest a single DNA molecule twice wrapped around the protein.     

Poly(ADP-ribosyl)ation associated changes in CTCF-chromatin binding and gene expression in breast cells

CTCF is an evolutionarily conserved and ubiquitously expressed architectural protein regulating a plethora of cellular functions via different molecular mechanisms. CTCF can undergo a number of post-translational modifications which change its properties and functions. One such modifications linked to cancer is poly(ADP-ribosyl)ation (PARylation). The highly PARylated CTCF form has an apparent molecular mass of 180?kDa (referred to as CTCF180), which can be distinguished from hypo- and non-PARylated CTCF with the apparent molecular mass of 130?kDa (referred to as CTCF130). The existing data accumulated so far have been mainly related to CTCF130. However, the properties of CTCF180 are not well understood despite its abundance in a number of primary tissues. In this study we performed ChIP-seq and RNA-seq analyses in human breast cells 226LDM, which display predominantly CTCF130 when proliferating, but CTCF180 upon cell cycle arrest. We observed that in the arrested cells the majority of sites lost CTCF, whereas fewer sites gained CTCF or remain bound (i.e. common sites). The classical CTCF binding motif was found in the lost and common, but not in the gained sites. The changes in CTCF occupancies in the lost and common sites were associated with increased chromatin densities and altered expression from the neighboring genes. Based on these results we propose a model integrating the CTCF130/180 transition with CTCF-DNA binding and gene expression changes. This study also issues an important cautionary note concerning the design and interpretation of any experiments using cells and tissues where CTCF180 may be present.