CTCF, Zinc-finger protein, has been identified as a multifunctional transcription factor that regulates gene expression through various mechanisms, including recruitment of other co-activators and binding to promoter regions of target genes. Furthermore, it has been proposed to be an insulator protein that contributes to the establishment of functional three-dimensional chromatin structures. It can disrupt transcription through blocking the connection between an enhancer and a promoter. Previous studies revealed that the onset of various diseases, including breast cancer, could be attributed to the aberrant expression of CTCF itself or one or more of its target genes. In this review, we will describe molecular dysfunction involving CTCF that induces tumorigenesis and summarize the functional roles of CTCF in breast cancer.
The crucial chromatin organizer, CTCF, is involved in transcriptional regulation through various mechanisms such as enhancer-promoter looping formation and insulation. Genome-wide distributions of CTCF have been analyzed by Chromatin Immunoprecipitation Sequencing (ChIP-Seq), additionally, the dynamic roles of CTCF in gene regulation and genomic interactions have been elucidated by chromosome conformation capture (3C) and by Paired-End Tag Sequencing (ChIA-PET). Global CTCF binding sites are normally conserved within tissues; however, specific CTCF binding sites were identified and regulated by epigenetic factors under specific conditions. CTCF binding is strongly associated with DNA methylation status and aberrant CTCF binding to DNA depends on DNA methylation at the IGF2/H19 locus leads to unexpected transcription that can induce Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS), respectively. Since genes regulated by CTCF are related to proliferation and apoptosis, the role of this multifunctional protein has been studied in various cancers, including breast cancer. Breast cancer is a heterogeneous disease associated with the activities of hormone receptors for estrogen (ER), progesterone (PR), and HER2 (human epidermal growth factor receptor 2). Hormone receptor status is an important biomarker for breast cancer and genes regulated by hormones are therefore therapeutic targets for breast cancer. Several studies have investigated that genes regulated by ER are modulated by CTCF through binding to ER target genomic regions, constructing higher-order chromatin structures for enhancer and promoter interactions, thereby limiting the influences of ER when CTCF is occupied with other proteins such as cohesin, ER, or the transcription activator, BRG1 (1–4). Here, we will review recent studies of CTCF function to understand mechanisms that regulate gene expression in breast cancer.
CTCF, a highly conserved protein which contains 11 zinc fingers in eukaryotes, recognizes and binds to specific sequences in the genome. Genome-wide analysis indicated that half of CTCF-binding sites were observed in intergenic regions, while the remaining sites were present in promoters and intragenic regions (3). This global analysis supports findings of its multifunctional properties as a transcription activator/repressor, insulator, recombination modulator, and architect involved in the establishment of three-dimensional chromatin structures (5, 6).
Several studies have shown that CTCF interacts with other co-factors such as homeodomain transcription factors (HOX-TFs) and glucocorticoid receptor (GR) at promoters or enhancers in order to regulate gene expression in primary mesenchymal limb bud cells and hepatic cells (7, 8). Associations between CTCF and HOXA/D family transcription factors (TFs) have been observed in the chicken genome (7). Of the 9 HOX-TFs, 6 HOX-TFs were grouped with each other based on coincident genomic binding patterns confirmed by ChIP-Seq. These binding patterns were also comparable to CTCF binding sites in the genome. It suggests that CTCF plays a role in transcription regulator with HOX-TFs (7). In addition, GR and CTCF co-localization was confirmed at
The influence of CTCF on V(D)J recombination was manifested via modulation of chromatin loop structures (10). CTCF-depletion studies identified the function of CTCF in association with chromatin structure in mouse embryonic stem cells (2). For maintaining stable genomic complex, CTCF and the cohesin complex, consisting of SMC3, SMC1, RAD21, and STAG1 or STAG2, can co-localize (13). Moreover, global analysis of CTCF, SMC3, and RAD1 shift-banding patterns have demonstrated the proximity of protein-DNA binding motif sequences (13).
In a recent study, multi-functional roles of CTCF were demonstrated in tandem situations. The elimination of CTCF confirmed the multifunctional status of the protein as a prominent factor for transcriptional regulation, distinct looping formation, and maintaining chromatin structure with protein complexes such as cohesin in both inter-chromatin and intra-chromatin looping (2).
It has been discovered that aberrant CTCF induces several diseases or disorders, including mental retardation, Wiedemann syndrome, Silver-Russell syndrome, and various cancers (Table 1) (1, 14–19). Germline CTCF missense and frameshift mutations can result in the syndromic intellectual disability, autosomal dominant mental retardation 21 (MRD21);
Missense mutations of the zinc-finger domain of the
CTCF/cohesin-binding sites (CBSs) mutations were investigated in various cancers including gastrointestinal and skin cancers (29, 30). In gastrointestinal cancer, relatively A·T>C·G and A·T>G·C substitutions were preferentially detected at CBSs (19) and these mutations were related with late replication (29). Mutations arising due to differential nucleotide excision repair (NER) across pyrimidine pairs were also identified at specific CBSs in skin cancer (30).
It has been investigated dysfunction of CTCF caused by mutation and aberrant poly(ADP-Ribosyl)ation (PARlation) in breast cancer cells. Missense codon mutation, K344E, in
PARlation of CTCF also contributes to apoptosis regulation and cell growth inhibition through translocation from the nucleoplasm to nucleoli in breast cancer cells (33, 34). The presence of two CTCF isoforms, 130 kDa and 180 kDa, in breast cancer compared with one 180 kDa CTCF formed via PARlation in normal tissues, prompted the suggestion that the 130 kDa CTCF could be used as a breast cancer biomarker (35). A 130 kDa CTCF is negatively correlated with tumor size and tumor stage. Increased cell proliferation was observed when the expression of the 130 kDa CTCF isoform increased in cells. NaB treatment of cells yielded the opposite effect that the formation of the 180 kDa CTCF isoform was observed, there was a reduction of MCF-7 cell proliferation, and the induction of apoptosis was verified (35).
Abnormal expression of CTCF which generated breast cancer cells, was regulated by DNA methylation in genome. Here is a study indicated that how DNA methylation and down-regulated CTCF affects to tumorigenesis gene regulation in breast tumors (24). In tumor suppressor genes and the imprinting regulatory regions, aberrant changes of the DNA methylation status had an impact on decreased CTCF expression levels. Down-regulated CTCF, in particular, was positively correlated with dysregulated methylation pattern at CpGs near genes known to be involved in tumorigenesis, such as
CTCF can affect breast cancer development by regulating target genes, where it can act alone or in combination with other factors to function as a transcription factor, insulator, and/or regulator of chromatin structure (39).
CTCF binding to gene-regulatory regions could be associated with alteration of oncogene expression levels via co-regulatory factors and changes in DNA methylation status (Fig. 1A). A previous study showed alterations of genes could be facilitated by the absence of CTCF binding at target sites in the promoters of cell-proliferation-related genes such as
It has been identified that DNA methylation status involves in CTCF binding affinity at CpG regions containing CTCF binding motif (42, 43). In addition, their binding affinity was also influenced by histone modifications (44). CTCF occupies to the tumor suppressor gene, X-linked inhibitor of the apoptosis (XIAP)–associated factor 1 (XAF), and controls its expression with a methylation and histone modification sensitive manner. Exposure to demethylation modulators such as 5-aza-2′-deoxycytidine (5-A-DC) could affect the observed DNA methylation status of CpG regions in CTCF-regulated target genes. A negative correlation was observed between
Recent studies have also showed the relationship between CTCF binding and miRNAs. The expression levels of miRNAs were also modulated by CTCF binding, DNA methylation, and histone modification (47, 48). In a previous study, it was discovered that increased
CTCF could be involved in regulating the expression levels of CTCF-modulated genes by acting as a transcriptional insulator to induce gene regulation or to block the interaction of enhancers and promoters (12). Another study investigated the binding of CTCF to sites independently regulated by estrogen (50). It was found that estrogen-regulated function of a gene was limited by CTCF-binding sites acting as barriers. Of all the CTCF binding roles, putative insulators were selected at the Trefoil factor (
One well-known CTCF function is to modulate chromatin structure as a chromatin boundary factor involved in the regulation of transcription. CTCF constructs chromatin architecture with co-factors, such as the cohesion complex, which includes RAD21, SMC1, SMC3, and BRG1 (4, 13, 51–53). Tang and colleagues revealed that the chromatin topological domain could be altered by CTCF binding to specific DNA motif sequences (51). Cell-line specific mutations in CTCF binding motifs resulted in the formation of different chromatin loops. Although BRG1 and CTCF co-localization have not been extensively investigated, it is known that this phenomenon is rare as a function of the entire genome. BRG1 and CTCF interactions could preserve sturdy, topologically-associated domains (TAD) or boundaries (4). BRG1 is a transcriptional co-regulated of the ATP-dependent transcriptional remodeling complex, SWI/SNF. In recent studies, it was shown BRG1 could function to develop cancer cells in a different manner via hormone regulation in breast cancers (54, 55). In previous studies, it was shown BRG1 overexpression had positive correlation with poor prognosis in breast cancer cells (55, 56). Deficient BRG1 was revealed it affected to inhibit cell proliferation through cell cycle arrest, especially G1 phase, caused by decreased cyclin D1 and cyclin E, and increased p27 expression (55). Recently, BRG1 was investigated and the role was confirmed that BRG1 regulation was essential to preserve the topologically-associated domains (TAD) correlated with CTCF binding in the normal MCF-10A and MEF breast cell lines (4). Loss of BRG1 was used to confirm that BRG1 was a causative factor in week formation of a TAD boundary and a reduction in nucleosomes surrounding the CTCF binding site. The strength of the BRG1 and CTCF interaction was enhanced when the event occurred within a range of 1 kb from one another. Dysregulated BRG1 expression may be associated with a disrupted chromatin structure, especially regulatory regions involved in breast cancer cell proliferation. Consequently, it was suggested that related genes could be differentially expressed and that the net result could affect the rate of cell proliferation (4). Co-localization of CTCF with relevant cofactors could stabilize substantial chromatin structures. The direct effect of aberrant chromatin structure and attenuated chromatin looping domains impacted by altered binding of CTCF in breast cancer is the subject of ongoing investigation. It could be expected to affect transcriptional regulation eg, significant alterations in gene expression could lead to tumorigenesis.
Multifunctional CTCF is also associated with the formation of alternative splicing variants. The function of CTCF binding to induce RNA Polymerase II (Pol II) elongation and regulate alternative splicing at exon 5 of
A previous study (59) certified the correlation of the presence of CTCF and ER in breast cancer cell lines. CTCF and ER binding patterns differed in many cell lines, including breast cancers stratified into different subtypes. Cell-line specific colocalization of CTCF and ER binding to target regions were observed in the breast cancer cell line, MCF-7 (59). Most of the ER and CTCF co-localization sites were within 20 kb from estrogen-regulated genes. Therefore, one explanation is that estrogen-mediated gene expression levels could be regulated by ER and CTCF. Fiorito and colleagues studied CTCF-binding enrichment is modulated by ER stimulation to impede the expanded loops of ER regulated enhancers and promoters of genes in MCF-7 (3). Investigators confirmed that the alteration of chromatin looping depended on the timing of an estrogen treatment. CTCF binding sites were increased at intergenic regions with increased duration of the estrogen stimulation time. When estrogen-induced transcripts were highly expressed at specific time points, the CTCF binding density also increased in a proportional manner. ER has an important role in intra-chromosomal interactions to regulate ER target genes. ER-ER chromatin looping was investigated using Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET). CTCF binding was found to co-localize with ER binding in ER-ER loops. CTCF occupancy was observed in more than half of the ER looping regions. A 24% increase in ER binding was detected by Chromosome conformation capture (3C)-PCR in CTCF-depleted cells. For example, P2Y purinoceptor 2 (
In breast cancer cells, dysregulated gene expression clustered specifically in apoptosis-associated genes, may be a consequence of aberrant CTCF gene regulation. An association was confirmed NaB treatment could affect to apoptosis through CTCF regulation (60). NaB treatment led to increased levels of the 130 kDa CTCF isoform, cell proliferation, reduced apoptosis and CTCF translocation in MCF-7 cells (35). As previously mentioned, CTCF binding was sensitive to DNA methylation status at target binding sites. Indeed, CTCF was not able to occupy methylated regions (40, 44, 45, 47, 48, 50). Therefore, DNA-methylation-related drugs such as 5-A-DC, could be considered as potential targeted therapies in breast cancers. In a previous study, Sulforaphane (SFN) was shown to have an effect on the down-regulation of human telomerase reverse transcriptase (
However, CTCF is known to be an important factor in the prevention or induction of tumorigenesis, either by oncogenes or tumor suppressor genes. Unlike CTCF targeted therapies, Brother of the Regulator of Imprinted Sites (BORIS) has been studied as a more common target of cancer treatment. BORIS, also called CTCF-like protein (CTCFL), shares a conserved 11 zinc finger domains with CTCF and it is a paralogue with related CTCF-binding motif sequences (62). It has been reported that BORIS and CTCF have a complementary effect (62). Up-regulated CTCF helps protect cells from apoptosis (40, 60). Conversely, induced
CTCF is a dynamic multifunctional protein that regulates transcription via its role as a transcription factor, insulator, and organizer of higher-order chromatin structures or loops. CTCF mutations could alter the protein or its binding sites, resulting in diverse diseases such as different cancers. Missense codon mutations and lowered poly(ADP-Ribosyl)ation of CTCF have been observed to alter CTCF binding to target sites in breast cancer. These events have led to aberrant gene regulation through inhibition of enhancer activity. Although several studies have confirmed a role for CTCF dysfunction in breast cancer, the mechanisms underlying this involvement at specific loci associated with tumorigenesis have yet to be elucidated. The role of CTCF in regulating chromatin loop formation at specific loci could not be confirmed by siRNA disruption of CTCF expression. Continued studies should reveal the potential role of CTCF as therapeutic target for breast cancer, since numerous genes regulated by CTCF are associated with proliferation and apoptosis in breast cancer cells.
This work was supported by fund from Sookmyung Women’s University (1-1603-2052) and the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2017R1C1B2002806).
Diseases caused by CTCF variations
|Mental retardation, autosomal dominant 21 (MRD21)||Mutation in the CTCF gene||Frameshift mutation; c375dupT, c.1186dupA||(14, 16)|
|Beckwith-wiedemann syndrome (BWS)||Mutation or deletion of imprinted genes||Igf2 expression, H19 inhibition||(15)|
|Silver-russell syndrome (SRS)||Igf2 inhibition, H19 expression|
|Cancer||Testicular cancer||Methylation in CTCF-binding sites||Igf2 inhibition, H19 inhibition||(20)|
|Colorectal cancer||Hypermethylation of CpG sites in IGF2/H19||Loss of imprinting of the IGF2||(21)|
|Bladder cancer||Hypomethylation of CTCF-binding site||Loss of imprinting of the H19||(22)|
|Ovarian cancer||Increase DNA methylation and reduce insulator protein CTCF||H19 inhibition||(23)|
|Endometrial cancer||CTCF mutation||Missense mutation, R377C||(30)|
|Prostate cancer||ZF mutation in the CTCF||ZF3 H345R mutation, CAC→CGC||(31)|
|Wilms’ tumor||ZF3 R339W mutation, CGG→TGG
|Breast cancer||ZF3 K344E mutation, AAA→GAA||(33, 34)|
|Gastrointestinal cancer||CTCF binding site mutations||Mutation at CTCF/cohesion binding site||(19)|
|Skin cancer||CTCF binding site mutations||Asymmetric mutation style with CTCF||(36)|