CTCF Function Research Articles

Expression and Effect of CTCF in Pediatric Acute Lymphoblastic Leukemia

AbstractAbstract 4298Acute lymphoblastic leukemia (ALL) is the most frequent-occurring malignant neoplasm in children, but the pathogenesis of the disease remains unclear. In a microarray assay using samples from 100 Chinese children with ALL, CTCF was found to be up-regulated. DNA-binding nuclear protein CTCF (encoded by CTCF gene) is a highly conserved zinc finger protein involved in multiple cellular processes including transcriptional activation/repression, insulation, imprinting and × chromosome inactivation. It has been shown to be associated with cell apoptosis and differentiation in tumors; however, the biological function of CTCF in pediatric ALL is presently unknown.To investigate the expression features of CTCF in pediatric ALL cells, matched newly diagnosis (ND), complete remission (CR) and relapse (RE) bone marrow samples from 24 patients were collected. Ten ND-CR paired samples (n=20) were selected to detect the mRNA levels of CTCF by Q-PCR. Besides, the protein levels of CTCF at different stages of disease progression were measured by western blot in all patients (20 ND-CR paired samples, n=40; 4 ND-CR-RE matched samples, n=12). To further explore the role of CTCF in the pathogenesis of leukemia, the potential effect of CTCF on the cell apoptosis in lymphoblastic cells was investigated by flow cytometry.We identified significant up-regulation of CTCF in the ND samples. Importantly, the expression of CTCF returned to normal level after CR, but rebounded in the RE samples. Knock-down of CTCF resulted in nearly 3–fold and 15–fold increases in early and late apoptosis of leukemic cells respectively, which indicated that CTCF is an anti-apoptotic factor and plays an anti-apoptotic role in lymphoblastic cells.Our results indicate that CTCF may represent a promising indicator of disease progression as well as reflecting the ongoing therapeutic effects of treatment. Furthermore, CTCF serves as an anti-apoptotic factor and potentially contributes to leukemogenesis in pediatric ALL patients. Disclosures:Zhang:Beijing Health System High-level Technical Personel Plan: Research Funding. Zheng:Beijing Health System High-level Technical Personel Plan: Research Funding.

CTCF function is modulated by neighboring DNA binding factors

The zinc-finger protein CTCF was originally identified in the context of gene silencing and gene repression (Baniahmad et al. 1990; Lobanenkov et al. 1990). CTCF was later shown to be involved in several transcriptional mechanisms such as gene activation (Vostrov et al. 2002) and enhancer blocking (Filippova et al. 2001; Hark et al. 2000; Kanduri et al. 2000; Lutz et al. 2003; Szabó et al. 2000; Tanimoto et al. 2003; Phillips and Corces 2009; Bell et al. 1999; Zlatanova and Caiafa 2009a, 2009b). Insulators block the action of enhancers when positioned between enhancer and promoter. CTCF was found to be required in almost all cases of enhancer blocking tested in vertebrates. This CTCF-mediated enhancer blocking is in many instances conferred by constitutive CTCF action. For some examples however, a modulation of the enhancer blocking activity was documented (Lutz et al. 2003; Weth et al. 2010). One mechanism is achieved by regulation of binding to DNA. It was shown that CTCF is not able to bind to those binding-sites containing methylated CpG sequences. At the imprinting control region (ICR) of the Igf2/H19 locus the binding-site for CTCF on the paternal allele is methylated. This prevents DNA-binding of CTCF, resulting in the loss of enhancer blocking (Bell and Felsenfeld 2000; Chao et al. 2002; Filippova et al. 2001; Hark et al. 2000; Kanduri et al. 2000, 2002; Szabó et al. 2000; Takai et al. 2001). Not only can DNA methylation interfere with CTCF binding to DNA, it was also shown in one report that RNA transcription through the CTCF binding site results in CTCF eviction (Lefevre et al. 2008). In contrast to these cases most of the DNA sites are not differentially bound by CTCF. Even CTCF interaction with its cofactor cohesin does not seem to differ in different cell types (Schmidt et al. 2010). These results indicate that regulation of CTCF activity might be achieved by neighboring factors bound to DNA. In fact, whole genome analyses of CTCF binding sites identified several classes of neighboring sequences (Dickson et al. 2010; Boyle et al. 2010; Essien et al. 2009). Therefore, in this review we will summarize those results for which a combined action of CTCF with factors bound adjacently was found. These neighboring factors include the RNA polymerases I, II and III, another zinc finger factor VEZF1 and the factors YY1, SMAD, TR and Oct4. Each of these seems to influence, modulate or determine the function of CTCF. Thereby, at least some of the pleiotropic effects of CTCF can be explained.

CTCF-mediated functional chromatin interactome in pluripotent cells.

Mammalian genomes are viewed as functional organizations that orchestrate spatial and temporal gene regulation. CTCF, the most characterized insulator-binding protein, has been implicated as a key genome organizer. Yet, little is known about CTCF-associated higher order chromatin structures at a global scale. Here, we applied Chromatin Interaction Analysis by Paired-End-Tag sequencing to elucidate the CTCF-chromatin interactome in pluripotent cells. From this analysis, 1,480 cis and 336 trans interacting loci were identified with high reproducibility and precision. Associating these chromatin interaction loci with their underlying epigenetic states, promoter activities, enhancer binding and nuclear lamina occupancy, we uncovered five distinct chromatin domains that suggest potential new models of CTCF function in chromatin organization and transcriptional control. Specifically, CTCF interactions demarcate chromatin-nuclear membrane attachments and influence proper gene expression through extensive crosstalk between promoters and regulatory elements. This highly complex nuclear organization offers insights towards the unifying principles governing genome plasticity and function.

Complete Biallelic Insulation at the H19/Igf2 Imprinting Control Region Position Results in Fetal Growth Retardation and Perinatal Lethality

BackgroundThe H19/Igf2 imprinting control region (ICR) functions as an insulator exclusively in the unmethylated maternal allele, where enhancer-blocking by CTCF protein prevents the interaction between the Igf2 promoter and the distant enhancers. DNA methylation inhibits CTCF binding in the paternal ICR allele. Two copies of the chicken β-globin insulator (ChβGI)2 are capable of substituting for the enhancer blocking function of the ICR. Insulation, however, now also occurs upon paternal inheritance, because unlike the H19 ICR, the (ChβGI)2 does not become methylated in fetal male germ cells. The (ChβGI)2 is a composite insulator, exhibiting enhancer blocking by CTCF and chromatin barrier functions by USF1 and VEZF1. We asked the question whether these barrier proteins protected the (ChβGI)2 sequences from methylation in the male germ line.Methodology/Principal FindingsWe genetically dissected the ChβGI in the mouse by deleting the binding sites USF1 and VEZF1. The methylation of the mutant versus normal (ChβGI)2 significantly increased from 11% to 32% in perinatal male germ cells, suggesting that the barrier proteins did have a role in protecting the (ChβGI)2 from methylation in the male germ line. Contrary to the H19 ICR, however, the mutant (mChβGI)2 lacked the potential to attain full de novo methylation in the germ line and to maintain methylation in the paternal allele in the soma, where it consequently functioned as a biallelic insulator. Unexpectedly, a stricter enhancer blocking was achieved by CTCF alone than by a combination of the CTCF, USF1 and VEZF1 sites, illustrated by undetectable Igf2 expression upon paternal transmission.Conclusions/SignificanceIn this in vivo model, hypomethylation at the ICR position together with fetal growth retardation mimicked the human Silver-Russell syndrome. Importantly, late fetal/perinatal death occurred arguing that strict biallelic insulation at the H19/Igf2 ICR position is not tolerated in development.

Regulation of the apoptotic genes in breast cancer cells by the transcription factor CTCF

CTCF is a highly conserved and ubiquitous transcription factor with versatile functions. We previously demonstrated that elevated protein levels of CTCF in breast cancer cells were associated with the specific anti-apoptotic function of CTCF. We used proteomics and microarray approaches to identify regulatory targets of CTCF specific for breast cancer cells. Among the CTCF identified targets were proteins involved in the control of apoptosis. A proapoptotic protein, Bax, negatively regulated by CTCF, was chosen for further investigation. Repression of the human Bax gene at the transcriptional level by CTCF in breast cancer cells was confirmed by real-time PCR. Two CTCF binding sites within the Bax promoter were identified by electrophoretic mobility shift assay and footprinting. In reporter assays, the Bax-luciferase reporter construct, containing CTCF-binding sites, was negatively regulated by CTCF. In vivo, CTCF occupied its binding sites in breast cancer cells and tissues, as confirmed by chromatin immunoprecipitation assay. Our findings suggest a possible mechanism of the specific CTCF anti-apoptotic function in breast cancer cells whereby CTCF is bound to the Bax promoter, resulting in repression of Bax and inhibition of apoptosis; depletion of CTCF leads to activation of Bax and apoptotic death. CTCF binding sites in the Bax promoter are unmethylated in all cells and tissues inspected. Therefore, specific CTCF interaction with the Bax promoter in breast cancer cells, and the functional outcome, may depend on a combination of epigenetic factors characteristic for these cells. Interestingly, CTCF appears to be a negative regulator of other proapoptotic genes (for example, Fas, Apaf-1, TP531NP1). Conversely, stimulating effects of CTCF on the anti-apoptotic genes (Bcl-2, Bag-3) have been observed. Taken together, these findings suggest that specific mechanisms have evolved in breast cancer cells to protect them from apoptosis; regulation of apoptotic genes by CTCF appears to be one of the resistance strategies.

BORIS (CTCFL) Is Not Expressed in Most Human Breast Cell Lines and High Grade Breast Carcinomas

BORIS (CTCFL) is the only known paralog of the versatile regulatory protein CTCF, a multifunctional DNA binding protein that mediates distinct gene regulatory functions involved in cell growth, differentiation, and apoptosis. Unlike CTCF, the expression of BORIS is normally restricted to specific cells in testes (the only cells where CTCF is not expressed), where it may play a role in reprogramming the methylation pattern of male germ line DNA. Frequent amplification of the 20q13.2 region, which contains the BORIS gene, and expression of BORIS transcripts in diverse human tumors and cell lines have led to the hypothesis that aberrant expression of BORIS may play a role in tumorigenesis by interfering with CTCF functions. However, recent studies using more quantitative methods indicate low frequency of BORIS expression in melanoma, ovarian, prostate, and bladder carcinomas. To investigate the relationship between chromosome 20q13 amplification and BORIS mRNA levels within breast cancer cell lines and tissues, we developed a quantitative RT-PCR assay to measure the levels of BORIS mRNA. Endpoint RT-PCR assays were also used to investigate the possible expression of alternatively spliced variants. Using multiple primer sets and controls, we found that neither mature BORIS transcripts nor spliced variants are commonly expressed at detectable levels in malignant breast cells or tissues, although endogenous BORIS transcripts can be induced in MCF-7 cells following 5-aza-2′-deoxycytidine treatment. In conclusion, in most breast cancer cells, endogenous BORIS is unlikely to be expressed at sufficient levels to interfere with CTCF functions. Thus it is improbable that aberrant BORIS expression plays a role in most human breast cancers.

Mutational analysis of the poly(ADP-ribosyl)ation sites of the transcription factor CTCF provides an insight into the mechanism of its regulation by poly(ADP-ribosyl)ation.

Poly(ADP-ribosyl)ation of the conserved multifunctional transcription factor CTCF was previously identified as important to maintain CTCF insulator and chromatin barrier functions. However, the molecular mechanism of this regulation and also the necessity of this modification for other CTCF functions remain unknown. In this study, we identified potential sites of poly(ADP-ribosyl)ation within the N-terminal domain of CTCF and generated a mutant deficient in poly(ADP-ribosyl)ation. Using this CTCF mutant, we demonstrated the requirement of poly(ADP-ribosyl)ation for optimal CTCF function in transcriptional activation of the p19ARF promoter and inhibition of cell proliferation. By using a newly generated isogenic insulator reporter cell line, the CTCF insulator function at the mouse Igf2-H19 imprinting control region (ICR) was found to be compromised by the CTCF mutation. The association and simultaneous presence of PARP-1 and CTCF at the ICR, confirmed by single and serial chromatin immunoprecipitation assays, were found to be independent of CTCF poly(ADP-ribosyl)ation. These results suggest a model of CTCF regulation by poly(ADP-ribosyl)ation whereby CTCF and PARP-1 form functional complexes at sites along the DNA, producing a dynamic reversible modification of CTCF. By using bioinformatics tools, numerous sites of CTCF and PARP-1 colocalization were demonstrated, suggesting that such regulation of CTCF may take place at the genome level.

Vertebrate Protein CTCF and its Multiple Roles in a Large-Scale Regulation of Genome Activity

The CTCF transcription factor is an 11 zinc fingers multifunctional protein that uses different zinc finger combinations to recognize and bind different sites within DNA. CTCF is thought to participate in various gene regulatory networks including transcription activation and repression, formation of independently functioning chromatin domains and regulation of imprinting. Sequencing of human and other genomes opened up a possibility to ascertain the genomic distribution of CTCF binding sites and to identify CTCF-dependent cis-regulatory elements, including insulators. In the review, we summarized recent data on genomic distribution of CTCF binding sites in the human and other genomes within a framework of the loop domain hypothesis of large-scale regulation of the genome activity. We also tried to formulate possible lines of studies on a variety of CTCF functions which probably depend on its ability to specifically bind DNA, interact with other proteins and form di- and multimers. These three fundamental properties allow CTCF to serve as a transcription factor, an insulator and a constitutive dispersed genome-wide demarcation tool able to recruit various factors that emerge in response to diverse external and internal signals, and thus to exert its signal-specific function(s).

Cohesins form chromosomal cis-interactions at the developmentally regulated IFNG locus.

Cohesin-mediated sister chromatid cohesion is essential for chromosome segregation and post-replicative DNA repair1,2. In addition, evidence from model organisms3-6 and from human genetics7 suggests that cohesin plays a role in the control of gene expression8,9. This non-canonical role has recently been rationalized by the findings that mammalian cohesin complexes are recruited to a subset of DNase I hypersensitive sites (HSS) and to conserved non-coding sequences by the DNA binding protein CTCF10-13. CTCF functions at insulators (which control interactions between enhancers and promoters) and at boundary elements (which demarcate regions of distinct chromatin structure)14, and cohesin contributes to CTCF’s enhancer blocking activity10,11. The underlying mechanisms remain unknown, and the full spectrum of cohesin functions remains to be elucidated. Here we show that cohesin forms the topological and mechanistic basis for cell type-specific long-range chromosomal interactions in cis at the developmentally regulated cytokine locus IFNG. Hence, cohesin’s ability to constrain chromosome topology is utilized not only for the purpose of sister chromatid cohesion1,2, but also to dynamically define the spatial conformation of specific loci. This novel aspect of cohesin function is likely of importance to normal development3-6 and to disease7.

CTCF and its protein partners: divide and rule?

CTCF is a ubiquitous transcription factor that is involved in numerous, seemingly unrelated functions. These functions include, but are not limited to, positive or negative regulation of transcription, enhancer-blocking activities at developmentally regulated gene clusters and at imprinted loci, and X-chromosome inactivation. Here, we review recent data acquired with state-of-the-art technologies that illuminate possible mechanisms behind the diversity of CTCF functions. CTCF interacts with numerous protein partners, including cohesin, nucleophosmin, PARP1, Yy1 and RNA polymerase II. We propose that CTCF interacts with one or two different partners according to the biological context, applying the Roman principle of governance, 'divide and rule' (divide et impera).

Active promoters and insulators are marked by the centrosomal protein 190

For the compact Drosophila genome, several factors mediating insulator function, such as su(Hw) and dCTCF, have been identified. Recent analyses showed that both these insulator-binding factors are functionally dependent on the same cofactor, CP190. Here we analysed genome-wide binding of CP190 and dCTCF. CP190 binding was detected at CTCF, su(Hw) and GAF sites and unexpectedly at the transcriptional start sites of actively transcribed genes. Both insulator and transcription start site CP190-binding elements are strictly marked by a depletion of histone H3 and, therefore, a loss of nucleosome occupancy. In addition, CP190/dCTCF double occupancy was seen at the borders of many H3K27me3 'islands'. As before, these sites were also depleted of H3. Loss of either dCTCF or CP190 causes an increase of H3 and H3K27 trimethylation at these sites. Thus, for both types of cis-regulatory elements, domain borders and promoters, the chromatin structure is dependent on CP190.

How cohesin and CTCF cooperate in regulating gene expression

Cohesin is a DNA-binding protein complex that is essential for sister chromatid cohesion and facilitates the repair of damaged DNA. In addition, cohesin has important roles in regulating gene expression, but the molecular mechanisms of this function are poorly understood. Recent experiments have revealed that cohesin binds to the same sites in mammalian genomes as the zinc finger transcription factor CTCF. At a few loci CTCF has been shown to function as an enhancer-blocking transcriptional insulator, and recent observations indicate that this function depends on cohesin. Here we review what is known about the roles of cohesin and CTCF in regulating gene expression in mammalian cells, and we discuss how cohesin might mediate the insulator function of CTCF.

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.

The Insulator Binding Protein CTCF Positions 20 Nucleosomes around Its Binding Sites across the Human Genome

Chromatin structure plays an important role in modulating the accessibility of genomic DNA to regulatory proteins in eukaryotic cells. We performed an integrative analysis on dozens of recent datasets generated by deep-sequencing and high-density tiling arrays, and we discovered an array of well-positioned nucleosomes flanking sites occupied by the insulator binding protein CTCF across the human genome. These nucleosomes are highly enriched for the histone variant H2A.Z and 11 histone modifications. The distances between the center positions of the neighboring nucleosomes are largely invariant, and we estimate them to be 185 bp on average. Surprisingly, subsets of nucleosomes that are enriched in different histone modifications vary greatly in the lengths of DNA protected from micrococcal nuclease cleavage (106–164 bp). The nucleosomes enriched in those histone modifications previously implicated to be correlated with active transcription tend to contain less protected DNA, indicating that these modifications are correlated with greater DNA accessibility. Another striking result obtained from our analysis is that nucleosomes flanking CTCF sites are much better positioned than those downstream of transcription start sites, the only genomic feature previously known to position nucleosomes genome-wide. This nucleosome-positioning phenomenon is not observed for other transcriptional factors for which we had genome-wide binding data. We suggest that binding of CTCF provides an anchor point for positioning nucleosomes, and chromatin remodeling is an important component of CTCF function.

Cohesin branches out

The cohesin complex — best known for its role in cell division — does not rest between divisions, and instead participates in regulating gene expression. How it does this is only now becoming clear. A study of the distribution of cohesin binding sites in the human genome shows that they co-locate with sites for the transcription insulator protein CTCF. Cohesin is well known as a protein that acts as a 'chromosome glue', promoting sister chromatid cohesion during chromosome segregation. This new finding has uncovered a second and independent role for cohesin, facilitating CTCF function by acting as a transcription insulator. The discovery could be of medical relevance as a pointer to pathways that could be involved in 'cohesinopathies' such as Roberts syndrome and Cornelia de Lange syndrome.

CTCF Regulates Growth and Erythroid Differentiation of Human Myeloid Leukemia Cells

CTCF is a transcription factor and a candidate tumor suppressor that contains a DNA-binding domain composed of 11 zinc fingers. We reported previously that CTCF is differentially regulated during differentiation of human myeloid leukemia cells. In this study we aimed to investigate the role of CTCF in myeloid cell differentiation. A human cell line, K562, that can be chemically induced to differentiate into various hematopoietic lineages was chosen as a model system for this study. Several K562 cell lines with constitutive and conditional expression of CTCF have been generated. By using these model systems we demonstrated that: (i) ectopic expression of CTCF in K562 cells led to growth retardation and promotion of differentiation into the erythroid lineage; (ii) CTCF knock-down significantly inhibited differentiation of K562 cells into erythroid lineage; (iii) differentiation of K562 into the megakaryocytic lineage was not significantly affected; and (iv) down-regulation of MYC has been identified as one of the mechanisms by which CTCF promotes erythroid differentiation. Taken together our results demonstrate that CTCF is involved in the control of myeloid cell growth and differentiation.

Regulation of the transcription factor, CTCF, by phosphorylation with protein kinase CK2

CTCF is a transcription factor involved in various aspects of gene regulation. We previously reported that CTCF function is modulated by protein kinase CK2. In this report we investigate further the role of CK2 in regulating the transcriptional properties of CTCF. We demonstrate that coexpression of CTCF with CK2 switches function of CTCF from repressor to activator. The non-phosphorylatable mutant increases repression by CTCF and potentiates the growth-suppressive ability of the protein, whereas the phospho-mimetic mutant behaves in the opposite fashion. Mutation of the individual serines reveals that Serine 612 is a critical residue in regulation of CTCF by CK2.

Imprinting regulation of the murine Meg1/Grb10 and human GRB10 genes; roles of brain-specific promoters and mouse-specific CTCF-binding sites.

The imprinted mouse gene Meg1/Grb10 is expres sed from maternal alleles in almost all tissues and organs, except in the brain, where it is expressed biallelically, and the paternal allele is expressed preferentially in adulthood. In contrast, the human GRB10 gene shows equal biallelic expression in almost all tissues and organs, while it is almost always expressed paternally in the fetal brain. To elucidate the molecular mechanisms of the complex imprinting patterns among the different tissues and organs of humans and mice, we analyzed in detail both the genomic structures and tissue-specific expression profiles of these species. Experiments using 5'-RACE and RT-PCR demonstrated the existence in both humans and mice of novel brain- specific promoters, in which only the paternal allele was active. The promoters were located in the primary differentially methylated regions. Interest ingly, CTCF-binding sites were found only in the mouse promoter region where CTCF showed DNA methylation-sensitive binding activity. Thus, the insulator function of CTCF might cause reciprocal maternal expression of the Meg1/Grb10 gene from another upstream promoter in the mouse, whereas the human upstream promoter is active in both parental alleles due to the lack of the corresponding insulator sequence in this region.

The vertebrate protein CTCF functions as an insulator in Saccharomyces cerevisiae.

Insulators are elements that shelter genes from the effects of silencers or enhancers. CTCF is the only vertebrate protein that has a recognized role in transcriptional insulation, but how it exerts its effect is unknown. In an attempt to better understand how CTCF functions, we have used an insulation assay in Saccharomyces cerevisiae. We show that CTCF acts as an insulator in yeast, where it can efficiently block the spreading of repressive telomeric chromatin. We identify two domains of the protein that are responsible for this activity: a short and very potent N-terminal domain, as well as the C-terminus of the protein.

Molecular cloning and expression of the chromatin insulator protein CTCF in Xenopus laevis

The zinc finger protein CTCF has been shown to mediate multiple functions connected to gene repression. Transcriptional inhibition as well as enhancer blocking and chromatin insulation are documented for CTCF in men, mice and chickens. Additionally, hCTCF has been linked to epigenetics and disease. In line with these basic cellular functions, CTCF has been found to be expressed in every cell type and adult tissue tested and has thus been deemed an ubiquitous protein. Here, we report the identification of the CTCF homologue from Xenopus and the analysis of the spatio-temporal expression of xCTCF during embryogenesis. Within the DNA binding domain, xCTCF is virtually identical to other identified vertebrate CTCF proteins. Homology also extends to other conserved regions that are important for CTCF function. Although xCTCF mRNA is present during all stages of early Xenopus development, a remarkable increase in expression is observed in neuronal tissues. Early in development, xCTCF is highly expressed in the neural plate and later in the neural tube and developing brain. By tailbud stage, elevated expression is also seen in the developing sensory organs of the head. This is the first detailed description of the expression pattern of a vertebrate insulator protein during embryogenesis.