Journal articles on the topic 'CTCF protein'
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1
Voutsadakis, Ioannis. "Molecular Lesions of Insulator CTCF and Its Paralogue CTCFL (BORIS) in Cancer: An Analysis from Published Genomic Studies." High-Throughput 7, no. 4 (October 1, 2018): 30. http://dx.doi.org/10.3390/ht7040030.
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CTCF (CCCTC-binding factor) is a transcription regulator with hundreds of binding sites in the human genome. It has a main function as an insulator protein, defining together with cohesins the boundaries of areas of the genome called topologically associating domains (TADs). TADs contain regulatory elements such as enhancers which function as regulators of the transcription of genes inside the boundaries of the TAD while they are restricted from regulating genes outside these boundaries. This paper will examine the most common genetic lesions of CTCF as well as its related protein CTCFL (CTCF-like also called BORIS) in cancer using publicly available data from published genomic studies. Cancer types where abnormalities in the two genes are more common will be examined for possible associations with underlying repair defects or other prevalent genetic lesions. The putative functional effects in CTCF and CTCFL lesions will also be explored.
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MacPherson, Melissa J., Linda G. Beatty, Wenjing Zhou, Minjie Du, and Paul D. Sadowski. "The CTCF Insulator Protein Is Posttranslationally Modified by SUMO." Molecular and Cellular Biology 29, no. 3 (November 24, 2008): 714–25. http://dx.doi.org/10.1128/mcb.00825-08.
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ABSTRACT 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.
3
Pugacheva, Elena M., Naoki Kubo, Dmitri Loukinov, Md Tajmul, Sungyun Kang, Alexander L. Kovalchuk, Alexander V. Strunnikov, Gabriel E. Zentner, Bing Ren, and Victor V. Lobanenkov. "CTCF mediates chromatin looping via N-terminal domain-dependent cohesin retention." Proceedings of the National Academy of Sciences 117, no. 4 (January 14, 2020): 2020–31. http://dx.doi.org/10.1073/pnas.1911708117.
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The DNA-binding protein CCCTC-binding factor (CTCF) and the cohesin complex function together to shape chromatin architecture in mammalian cells, but the molecular details of this process remain unclear. Here, we demonstrate that a 79-aa region within the CTCF N terminus is essential for cohesin positioning at CTCF binding sites and chromatin loop formation. However, the N terminus of CTCF fused to artificial zinc fingers was not sufficient to redirect cohesin to non-CTCF binding sites, indicating a lack of an autonomously functioning domain in CTCF responsible for cohesin positioning. BORIS (CTCFL), a germline-specific paralog of CTCF, was unable to anchor cohesin to CTCF DNA binding sites. Furthermore, CTCF–BORIS chimeric constructs provided evidence that, besides the N terminus of CTCF, the first two CTCF zinc fingers, and likely the 3D geometry of CTCF–DNA complexes, are also involved in cohesin retention. Based on this knowledge, we were able to convert BORIS into CTCF with respect to cohesin positioning, thus providing additional molecular details of the ability of CTCF to retain cohesin. Taken together, our data provide insight into the process by which DNA-bound CTCF constrains cohesin movement to shape spatiotemporal genome organization.
4
Klenova, E. M., R. H. Nicolas, H. F. Paterson, A. F. Carne, C. M. Heath, G. H. Goodwin, P. E. Neiman, and V. V. Lobanenkov. "CTCF, a conserved nuclear factor required for optimal transcriptional activity of the chicken c-myc gene, is an 11-Zn-finger protein differentially expressed in multiple forms." Molecular and Cellular Biology 13, no. 12 (December 1993): 7612–24. http://dx.doi.org/10.1128/mcb.13.12.7612-7624.1993.
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A novel sequence-specific DNA-binding protein, CTCF, which interacts with the chicken c-myc gene promoter, has been identified and partially characterized (V. V. Lobanenkov, R. H. Nicolas, V. V. Adler, H. Paterson, E. M. Klenova, A. V. Polotskaja, and G. H. Goodwin, Oncogene 5:1743-1753, 1990). In order to test directly whether binding of CTCF to one specific DNA region of the c-myc promoter is important for chicken c-myc transcription, we have determined which nucleotides within this GC-rich region are responsible for recognition of overlapping sites by CTCF and Sp1-like proteins. Using missing-contact analysis of all four nucleotides in both DNA strands and homogeneous CTCF protein purified by sequence-specific chromatography, we have identified three sets of nucleotides which contact either CTCF or two Sp1-like proteins binding within the same DNA region. Specific mutations of 3 of 15 purines required for CTCF binding were designed to eliminate binding of CTCF without altering the binding of other proteins. Electrophoretic mobility shift assay of nuclear extracts showed that the mutant DNA sequence did not bind CTCF but did bind two Sp1-like proteins. When introduced into a 3.3-kbp-long 5'-flanking noncoding c-myc sequence fused to a reporter CAT gene, the same mutation of the CTCF binding site resulted in 10- and 3-fold reductions, respectively, of transcription in two different (erythroid and myeloid) stably transfected chicken cell lines. Isolation and analysis of the CTCF cDNA encoding an 82-kDa form of CTCF protein shows that DNA-binding domain of CTCF is composed of 11 Zn fingers: 10 are of C2H2 class, and 1 is of C2HC class. CTCF was found to be abundant and conserved in cells of vertebrate species. We detected six major nuclear forms of CTCF protein differentially expressed in different chicken cell lines and tissues. We conclude that isoforms of 11-Zn-finger factor CTCF which are present in chicken hematopoietic HD3 and BM2 cells can act as a positive regulator of the chicken c-myc gene transcription. Possible functions of other CTCF forms are discussed.
5
Klenova, E. M., R. H. Nicolas, H. F. Paterson, A. F. Carne, C. M. Heath, G. H. Goodwin, P. E. Neiman, and V. V. Lobanenkov. "CTCF, a conserved nuclear factor required for optimal transcriptional activity of the chicken c-myc gene, is an 11-Zn-finger protein differentially expressed in multiple forms." Molecular and Cellular Biology 13, no. 12 (December 1993): 7612–24. http://dx.doi.org/10.1128/mcb.13.12.7612.
Full textAbstract:
A novel sequence-specific DNA-binding protein, CTCF, which interacts with the chicken c-myc gene promoter, has been identified and partially characterized (V. V. Lobanenkov, R. H. Nicolas, V. V. Adler, H. Paterson, E. M. Klenova, A. V. Polotskaja, and G. H. Goodwin, Oncogene 5:1743-1753, 1990). In order to test directly whether binding of CTCF to one specific DNA region of the c-myc promoter is important for chicken c-myc transcription, we have determined which nucleotides within this GC-rich region are responsible for recognition of overlapping sites by CTCF and Sp1-like proteins. Using missing-contact analysis of all four nucleotides in both DNA strands and homogeneous CTCF protein purified by sequence-specific chromatography, we have identified three sets of nucleotides which contact either CTCF or two Sp1-like proteins binding within the same DNA region. Specific mutations of 3 of 15 purines required for CTCF binding were designed to eliminate binding of CTCF without altering the binding of other proteins. Electrophoretic mobility shift assay of nuclear extracts showed that the mutant DNA sequence did not bind CTCF but did bind two Sp1-like proteins. When introduced into a 3.3-kbp-long 5'-flanking noncoding c-myc sequence fused to a reporter CAT gene, the same mutation of the CTCF binding site resulted in 10- and 3-fold reductions, respectively, of transcription in two different (erythroid and myeloid) stably transfected chicken cell lines. Isolation and analysis of the CTCF cDNA encoding an 82-kDa form of CTCF protein shows that DNA-binding domain of CTCF is composed of 11 Zn fingers: 10 are of C2H2 class, and 1 is of C2HC class. CTCF was found to be abundant and conserved in cells of vertebrate species. We detected six major nuclear forms of CTCF protein differentially expressed in different chicken cell lines and tissues. We conclude that isoforms of 11-Zn-finger factor CTCF which are present in chicken hematopoietic HD3 and BM2 cells can act as a positive regulator of the chicken c-myc gene transcription. Possible functions of other CTCF forms are discussed.
6
Wang, Jie, Yumei Wang, and Luo Lu. "De-SUMOylation of CCCTC Binding Factor (CTCF) in Hypoxic Stress-induced Human Corneal Epithelial Cells." Journal of Biological Chemistry 287, no. 15 (February 21, 2012): 12469–79. http://dx.doi.org/10.1074/jbc.m111.286641.
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Epigenetic factor CCCTC binding factor (CTCF) plays important roles in the genetic control of cell fate. Previous studies found that CTCF is positively and negatively regulated at the transcriptional level by epidermal growth factor (EGF) and ultraviolet (UV) stimulation, respectively. However, it is unknown whether other stresses modify the CTCF protein. Here, we report that regulation of CTCF by de-SUMOylation is dependent upon hypoxic and oxidative stresses. We found that stimulation of human corneal epithelial cells with hypoxic stress suppressed a high molecular mass form of CTCF (150 kDa), but not a lower molecular weight form of CTCF (130 kDa). Further investigation revealed that the hypoxic stress-suppressed 150-kDa CTCF was a small ubiquitin-related modifier (SUMO)ylated form of the protein. Hypoxic stress-induced de-SUMOylation of human CTCF was associated with lysine 74 and 689 residues, but not to the phosphorylation of CTCF. Overexpression of SENP1 induced de-SUMOylation of CTCF. However, knockdown of SENP1 could not rescue hypoxic stress-induced CTCF de-SUMOylation. Overexpression of SUMO1 and SUMO2 increased SUMOylation of CTCF and partially blocked hypoxic stress-induced CTCF de-SUMOylation, suggesting that free cellular SUMO proteins play roles in regulating hypoxia-induced CTCF de-SUMOylation. In addition, hypoxic stress significantly inhibited PAX6 mRNA and protein expressions by suppression of PAX6-P0 promoter activity. The result was further supported by data showing that knockdown of CTCF significantly enhanced expression of PAX6 and abolished hypoxia-induced suppression of PAX6. Thus, we conclude that hypoxic stress induces de-SUMOylation of CTCF to functionally regulate CTCF activity.
7
Lehman, Bettina J., Fernando J. Lopez-Diaz, Thom P. Santisakultarm, Linjing Fang, Maxim N. Shokhirev, Kenneth E. Diffenderfer, Uri Manor, and Beverly M. Emerson. "Dynamic regulation of CTCF stability and sub-nuclear localization in response to stress." PLOS Genetics 17, no. 1 (January 7, 2021): e1009277. http://dx.doi.org/10.1371/journal.pgen.1009277.
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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.
8
Daruliza Kernain and Shaharum Shamsuddin. "Interaction between Two Transcriptional Factors CTCF and YB-1 – Truncated domains in Brain Cancer Cell line." International Journal of Research in Pharmaceutical Sciences 10, no. 4 (October 16, 2019): 3332–38. http://dx.doi.org/10.26452/ijrps.v10i4.1642.
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CTCF is a protein involved in the regulation of transcription, insulator function, and the X-chromosome inactivation. It is an 11 ZF transcriptional factor which is highly conserved between the species. Identification of proteins interacting with CTCF can help to elucidate the function in the cell. Previously reported studies had identified numerous CTCF protein interacting partners, and one of the interacting partners chosen in this study is YB-1. Brain cancer cell –RGBM was selected as a model to study the interaction between CTCF and YB-1. Firstly, proteins were transformed and expressed in the bacterial expression system, and these proteins were chosen to further map the interaction via pull-down assay. Results showed CTCF-ZF was the only domain able to binds to YB-1 CSD. Other truncated areas did not show any interaction hence demonstrating the interaction between these two proteins took place at the ZF for CTCF and CSD for YB-1. Next, the significant of the interaction was further characterized using the mammalian two-hybrid system. Results show strong interaction when both we co-transfected into RGBM cells. Thus, this study shows a significant binding between CTCF/YB-1 interaction in the brain cell line.
9
Chernukhin, Igor, Shaharum Shamsuddin, Sung Yun Kang, Rosita Bergström, Yoo-Wook Kwon, WenQiang Yu, Joanne Whitehead, et al. "CTCF Interacts with and Recruits the Largest Subunit of RNA Polymerase II to CTCF Target Sites Genome-Wide." Molecular and Cellular Biology 27, no. 5 (January 8, 2007): 1631–48. http://dx.doi.org/10.1128/mcb.01993-06.
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ABSTRACT CTCF is a transcription factor with highly versatile functions ranging from gene activation and repression to the regulation of insulator function and imprinting. Although many of these functions rely on CTCF-DNA interactions, it is an emerging realization that CTCF-dependent molecular processes involve CTCF interactions with other proteins. In this study, we report the association of a subpopulation of CTCF with the RNA polymerase II (Pol II) protein complex. We identified the largest subunit of Pol II (LS Pol II) as a protein significantly colocalizing with CTCF in the nucleus and specifically interacting with CTCF in vivo and in vitro. The role of CTCF as a link between DNA and LS Pol II has been reinforced by the observation that the association of LS Pol II with CTCF target sites in vivo depends on intact CTCF binding sequences. “Serial” chromatin immunoprecipitation (ChIP) analysis revealed that both CTCF and LS Pol II were present at the β-globin insulator in proliferating HD3 cells but not in differentiated globin synthesizing HD3 cells. Further, a single wild-type CTCF target site (N-Myc-CTCF), but not the mutant site deficient for CTCF binding, was sufficient to activate the transcription from the promoterless reporter gene in stably transfected cells. Finally, a ChIP-on-ChIP hybridization assay using microarrays of a library of CTCF target sites revealed that many intergenic CTCF target sequences interacted with both CTCF and LS Pol II. We discuss the possible implications of our observations with respect to plausible mechanisms of transcriptional regulation via a CTCF-mediated direct link of LS Pol II to the DNA.
10
Maksimenko, Oksana G., Dariya V. Fursenko, Elena V. Belova, and Pavel G. Georgiev. "CTCF As an Example of DNA-Binding Transcription Factors Containing Clusters of C2H2-Type Zinc Fingers." Acta Naturae 13, no. 1 (March 15, 2021): 31–46. http://dx.doi.org/10.32607/actanaturae.11206.
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In mammals, most of the boundaries of topologically associating domains and all well-studied insulators are rich in binding sites for the CTCF protein. According to existing experimental data, CTCF is a key factor in the organization of the architecture of mammalian chromosomes. A characteristic feature of the CTCF is that the central part of the protein contains a cluster consisting of eleven domains of C2H2-type zinc fingers, five of which specifically bind to a long DNA sequence conserved in most animals. The class of transcription factors that carry a cluster of C2H2-type zinc fingers consisting of five or more domains (C2H2 proteins) is widely represented in all groups of animals. The functions of most C2H2 proteins still remain unknown. This review presents data on the structure and possible functions of these proteins, using the example of the vertebrate CTCF protein and several well- characterized C2H2 proteins in Drosophila and mammals.
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Burcin, M., R. Arnold, M. Lutz, B. Kaiser, D. Runge, F. Lottspeich, G. N. Filippova, V. V. Lobanenkov, and R. Renkawitz. "Negative protein 1, which is required for function of the chicken lysozyme gene silencer in conjunction with hormone receptors, is identical to the multivalent zinc finger repressor CTCF." Molecular and Cellular Biology 17, no. 3 (March 1997): 1281–88. http://dx.doi.org/10.1128/mcb.17.3.1281.
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The transcriptional repressor negative protein 1 (NeP1) binds specifically to the F1 element of the chicken lysozyme gene silencer and mediates synergistic repression by v-ERBA, thyroid hormone receptor, or retinoic acid receptor. Another protein, CCCTC-binding factor (CTCF), specifically binds to 50-bp-long sequences that contain repetitive CCCTC elements in the vicinity of vertebrate c-myc genes. Previously cloned chicken, mouse, and human CTCF cDNAs encode a highly conserved 11-Zn-finger protein. Here, NeP1 was purified and DNA bases critical for NeP1-F1 interaction were determined. NeP1 is found to bind a 50-bp stretch of nucleotides without any obvious sequence similarity to known CTCF binding sequences. Despite this remarkable difference, these two proteins are identical. They have the same molecular weight, and NeP1 contains peptide sequences which are identical to sequences in CTCF. Moreover, NeP1 and CTCF specifically recognize each other's binding DNA sequence and induce identical conformational alterations in the F1 DNA. Therefore, we propose to replace the name NeP1 with CTCF. To analyze the puzzling sequence divergence in CTCF binding sites, we studied the DNA binding of 12 CTCF deletions with serially truncated Zn fingers. While fingers 4 to 11 are indispensable for CTCF binding to the human c-myc P2 promoter site A, a completely different combination of fingers, namely, 1 to 8 or 5 to 11, was sufficient to bind the lysozyme silencer site F1. Thus, CTCF is a true multivalent factor with multiple repressive functions and multiple sequence specificities.
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Renda, Mario, Ilaria Baglivo, Bonnie Burgess-Beusse, Sabrina Esposito, Roberto Fattorusso, Gary Felsenfeld, and Paolo V. Pedone. "Critical DNA Binding Interactions of the Insulator Protein CTCF." Journal of Biological Chemistry 282, no. 46 (September 7, 2007): 33336–45. http://dx.doi.org/10.1074/jbc.m706213200.
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The DNA-binding protein CTCF (CCCTC binding factor) mediates enhancer blocking insulation at sites throughout the genome and plays an important role in regulating allele-specific expression at the Igf2/H19 locus and at other imprinted loci. Evidence is also accumulating that CTCF is involved in large scale organization of genomic chromatin. Although CTCF has 11 zinc fingers, we show here that only 4 of these are essential to strong binding and that they recognize a core 12-bp DNA sequence common to most CTCF sites. By deleting individual fingers and mutating individual sites, we determined the orientation of binding. Furthermore, we were able to identify the specific finger and its point of DNA interaction that are responsible for the loss of CTCF binding when CpG residues are methylated in the imprinted Igf2/H19 locus. This single interaction appears to be critical for allele-specific binding and insulation by CTCF.
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Peña-Hernández, Rodrigo, Maud Marques, Khalid Hilmi, Teijun Zhao, Amine Saad, Moulay A. Alaoui-Jamali, Sonia V. del Rincon, et al. "Genome-wide targeting of the epigenetic regulatory protein CTCF to gene promoters by the transcription factor TFII-I." Proceedings of the National Academy of Sciences 112, no. 7 (February 2, 2015): E677—E686. http://dx.doi.org/10.1073/pnas.1416674112.
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CCCTC-binding factor (CTCF) is a key regulator of nuclear chromatin structure and gene regulation. The impact of CTCF on transcriptional output is highly varied, ranging from repression to transcriptional pausing and transactivation. The multifunctional nature of CTCF may be directed solely through remodeling chromatin architecture. However, another hypothesis is that the multifunctional nature of CTCF is mediated, in part, through differential association with protein partners having unique functions. Consistent with this hypothesis, our mass spectrometry analyses of CTCF interacting partners reveal a previously undefined association with the transcription factor general transcription factor II-I (TFII-I). Biochemical fractionation of CTCF indicates that a distinct CTCF complex incorporating TFII-I is assembled on DNA. Unexpectedly, we found that the interaction between CTCF and TFII-I is essential for directing CTCF to the promoter proximal regulatory regions of target genes across the genome, particularly at genes involved in metabolism. At genes coregulated by CTCF and TFII-I, we find knockdown of TFII-I results in diminished CTCF binding, lack of cyclin-dependent kinase 8 (CDK8) recruitment, and an attenuation of RNA polymerase II phosphorylation at serine 5. Phenotypically, knockdown of TFII-I alters the cellular response to metabolic stress. Our data indicate that TFII-I directs CTCF binding to target genes, and in turn the two proteins cooperate to recruit CDK8 and enhance transcription initiation.
14
Kotova, E. S., S. B. Akopov, D. A. Didych, N. V. Petrova, O. V. Iarovaia, S. V. Razin, and L. G. Nikolaev. "Binding of Protein Factor CTCF within Chicken Genome Alpha-Globin Locus." Acta Naturae 8, no. 1 (March 15, 2016): 90–97. http://dx.doi.org/10.32607/20758251-2016-8-1-90-97.
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A systematic search for DNA fragments containing potential CTCF transcription factor binding sites in the chicken alpha-globin domain and its flanking regions was performed by means of the two-dimension electrophoretic mobility shift assay. For the alpha-globin domain fragments selected, the occupancy by the CTCF in erythroid and lymphoid chicken cells was tested by chromatin immunoprecipitation. Only one of 13 DNA fragments capable of CTCF binding in vitro was efficiently bound to this protein in vivo in erythroid cells, and somewhat less efficiently - in lymphoid cells. So, binding of CTCF to the DNA fragment in vitro in most cases does not mean that this fragment will be occupied by CTCF in the cell nucleus. Yet, CTCF binding in vivo, as a rule, is accompanied by the binding of the protein to this DNA region in vitro. During the erythroid differentiation, no significant changes in CTCF binding to the DNA fragments studied were detected.
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Xiao, Tiaojiang, Xin Li, and Gary Felsenfeld. "The Myc-associated zinc finger protein (MAZ) works together with CTCF to control cohesin positioning and genome organization." Proceedings of the National Academy of Sciences 118, no. 7 (February 8, 2021): e2023127118. http://dx.doi.org/10.1073/pnas.2023127118.
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The Myc-associated zinc finger protein (MAZ) is often found at genomic binding sites adjacent to CTCF, a protein which affects large-scale genome organization through its interaction with cohesin. We show here that, like CTCF, MAZ physically interacts with a cohesin subunit and can arrest cohesin sliding independently of CTCF. It also shares with CTCF the ability to independently pause the elongating form of RNA polymerase II, and consequently affects RNA alternative splicing. CTCF/MAZ double sites are more effective at sequestering cohesin than sites occupied only by CTCF. Furthermore, depletion of CTCF results in preferential loss of CTCF from sites not occupied by MAZ. In an assay for insulation activity like that used for CTCF, binding of MAZ to sites between an enhancer and promoter results in down-regulation of reporter gene expression, supporting a role for MAZ as an insulator protein. Hi-C analysis of the effect of MAZ depletion on genome organization shows that local interactions within topologically associated domains (TADs) are disrupted, as well as contacts that establish the boundaries of individual TADs. We conclude that MAZ augments the action of CTCF in organizing the genome, but also shares properties with CTCF that allow it to act independently.
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Valletta, Mariangela, Rosita Russo, Ilaria Baglivo, Veronica Russo, Sara Ragucci, Annamaria Sandomenico, Emanuela Iaccarino, et al. "Exploring the Interaction between the SWI/SNF Chromatin Remodeling Complex and the Zinc Finger Factor CTCF." International Journal of Molecular Sciences 21, no. 23 (November 25, 2020): 8950. http://dx.doi.org/10.3390/ijms21238950.
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The transcription factor CCCTC-binding factor (CTCF) modulates pleiotropic functions mostly related to gene expression regulation. The role of CTCF in large scale genome organization is also well established. A unifying model to explain relationships among many CTCF-mediated activities involves direct or indirect interactions with numerous protein cofactors recruited to specific binding sites. The co-association of CTCF with other architectural proteins such as cohesin, chromodomain helicases, and BRG1, further supports the interplay between master regulators of mammalian genome folding. Here, we report a comprehensive LC-MS/MS mapping of the components of the switch/sucrose nonfermentable (SWI/SNF) chromatin remodeling complex co-associated with CTCF including subunits belonging to the core, signature, and ATPase modules. We further show that the localization patterns of representative SWI/SNF members significantly overlap with CTCF sites on transcriptionally active chromatin regions. Moreover, we provide evidence of a direct binding of the BRK-BRG1 domain to the zinc finger motifs 4–8 of CTCF, thus, suggesting that these domains mediate the interaction of CTCF with the SWI/SNF complex. These findings provide an updated view of the cooperative nature between CTCF and the SWI/SNF ATP-dependent chromatin remodeling complexes, an important step for understanding how these architectural proteins collaborate to shape the genome.
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Del Moral-Morales, Aylin, Marisol Salgado-Albarrán, Yesennia Sánchez-Pérez, Nina Kerstin Wenke, Jan Baumbach, and Ernesto Soto-Reyes. "CTCF and Its Multi-Partner Network for Chromatin Regulation." Cells 12, no. 10 (May 10, 2023): 1357. http://dx.doi.org/10.3390/cells12101357.
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Architectural proteins are essential epigenetic regulators that play a critical role in organizing chromatin and controlling gene expression. CTCF (CCCTC-binding factor) is a key architectural protein responsible for maintaining the intricate 3D structure of chromatin. Because of its multivalent properties and plasticity to bind various sequences, CTCF is similar to a Swiss knife for genome organization. Despite the importance of this protein, its mechanisms of action are not fully elucidated. It has been hypothesized that its versatility is achieved through interaction with multiple partners, forming a complex network that regulates chromatin folding within the nucleus. In this review, we delve into CTCF’s interactions with other molecules involved in epigenetic processes, particularly histone and DNA demethylases, as well as several long non-coding RNAs (lncRNAs) that are able to recruit CTCF. Our review highlights the importance of CTCF partners to shed light on chromatin regulation and pave the way for future exploration of the mechanisms that enable the finely-tuned role of CTCF as a master regulator of chromatin.
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Kang, Hyojeung, and Paul M. Lieberman. "Cell Cycle Control of Kaposi's Sarcoma-Associated Herpesvirus Latency Transcription by CTCF-Cohesin Interactions." Journal of Virology 83, no. 12 (April 15, 2009): 6199–210. http://dx.doi.org/10.1128/jvi.00052-09.
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ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) latency is characterized by the highly regulated transcription of a few viral genes essential for genome maintenance and host cell survival. A major latency control region has been identified upstream of the divergent promoters for the multicistronic transcripts encoding LANA (ORF73), vCyclin (ORF72), and vFLIP (ORF71) and for the complementary strand transcript encoding K14 and vGPCR (ORF74). Previous studies have shown that this major latency control region is occupied by the cellular chromatin boundary factor CTCF and chromosome structural maintenance proteins SMC1, SMC3, and RAD21, which comprise the cohesin complex. Deletion of the CTCF-cohesin binding site caused an inhibition of cell growth and viral genome instability. We now show that the KSHV genes regulated by CTCF-cohesin are under cell cycle control and that mutation of the CTCF binding sites abolished cell cycle-regulated transcription. Cohesin subunits assembled at the CTCF binding sites and bound CTCF proteins in a cell cycle-dependent manner. Subcellular distribution of CTCF and colocalization with cohesins also varied across the cell cycle. Ectopic expression of Rad21 repressed CTCF-regulated transcription of KSHV lytic genes, and a Rad21-CTCF chimeric protein converted CTCF into an efficient transcriptional repressor of KSHV genes normally activated in the G2 phase. We conclude that cohesins interact with CTCF in mid-S phase and repress CTCF-regulated genes in a cell cycle-dependent manner. We propose that the CTCF-cohesin complex plays a critical role in regulating the cell cycle control of viral gene expression during latency and that failure to maintain cell cycle control of latent transcripts inhibits host cell proliferation and survival.
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Zhu, Lin, Han Zhang, Xiao Liu, Chaohao Du, Shanshan Zhu, Wei Zhang, Xiaoxi Zhao, Zhigang Li, Shilai Bao, and Huyong Zheng. "Expression and Effect of CTCF in Pediatric Acute Lymphoblastic Leukemia." Blood 120, no. 21 (November 16, 2012): 4298. http://dx.doi.org/10.1182/blood.v120.21.4298.4298.
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Abstract Abstract 4298 Acute 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.
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Kapalova, Martina, Juraj Kokavec, Nikola Curik, Pavel Burda, Arthur I. Skoultchi, and Tomas Stopka. "Smarca5 Regulates Ctcf Recruitment to Chromatin, Including to Regulatory Loci Involved In Control of Globin Gene Expression In Erythroleukemia." Blood 116, no. 21 (November 19, 2010): 5159. http://dx.doi.org/10.1182/blood.v116.21.5159.5159.
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Abstract Abstract 5159 Transcription factor Ctcf (CCCTC-binding factor) represents a major regulatory component of epigenetic regulation by recognizing its unmethyled DNA binding sites, resulting in changes in expression of neighboring genes. Ctcf plays an important role in transgenerational genetic imprinting. Very little is known about its role in hematologic malignancies. Ctcf has been described to promote differentiation of human erythroleukemia K562 cells (Torano 2005). We studied Ctcf in mouse erythroleukemia (MEL) cells and found it is expressed at both the mRNA and protein levels. Using chromatin immunoprecipitation (ChIP), we found that Ctcf is recruited to the H19/Igf2 imprinting control region (ICR) and also to the promoters of the alpha globin genes (Hba-a1, Hba-a2) as well as the beta globin locus control region (LCR) in MEL cells. To determine the mechanism by which Ctcf interacts with chromatin, we tested its interaction with chromatin remodeling proteins that associate with these DNA targets, including the well known Imitation Switch (ISWI class) ATPase Smarca5 (Snf2h). Using coimmunopreciptiation and ChIP experiments we found that Smarca5 and Ctcf interact on DNA. Next, we used MEL cells expressing an inducible Smarca5 shRNA. Doxycycline induction of Smarca5 shRNA led to a 5-fold decrease in Smarca5 mRNA and protein levels within 48hrs. ChIP experiments demonstrated that depletion of Smarca5 was accompanied by loss of Ctcf from the aforementioned loci indicating Ctcf requires Smarca5 for its association with chromatin. Furthermore, this was followed by significantly decreased levels H19 RNA. Our data provide evidence that Smarca5 regulates Ctcf recruitment to chromatin, including to regulatory loci involved in controlling globin gene expression. (Grants # IGA 10310-3, MSMT 2B06077, GAUK 251070 45410, SVV-2010-254260507, NIH R01CA154239). Disclosures: No relevant conflicts of interest to declare.
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Zhang, He, Beibei Niu, Ji-Fan Hu, Shengfang Ge, Haibo Wang, Tao Li, Jianqun Ling, Brandon N. Steelman, Guanxiang Qian, and Andrew R. Hoffman. "Interruption of intrachromosomal looping by CCCTC binding factor decoy proteins abrogates genomic imprinting of human insulin-like growth factor II." Journal of Cell Biology 193, no. 3 (May 2, 2011): 475–87. http://dx.doi.org/10.1083/jcb.201101021.
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Monoallelic expression of IGF2 is regulated by CCCTC binding factor (CTCF) binding to the imprinting control region (ICR) on the maternal allele, with subsequent formation of an intrachromosomal loop to the promoter region. The N-terminal domain of CTCF interacts with SUZ12, part of the polycomb repressive complex-2 (PRC2), to silence the maternal allele. We synthesized decoy CTCF proteins, fusing the CTCF deoxyribonucleic acid–binding zinc finger domain to CpG methyltransferase Sss1 or to enhanced green fluorescent protein. In normal human fibroblasts and breast cancer MCF7 cell lines, the CTCF decoy proteins bound to the unmethylated ICR and to the IGF2 promoter region but did not interact with SUZ12. EZH2, another part of PRC2, was unable to methylate histone H3-K27 in the IGF2 promoter region, resulting in reactivation of the imprinted allele. The intrachromosomal loop between the maternal ICR and the IGF2 promoters was not observed when IGF2 imprinting was lost. CTCF epigenetically governs allelic gene expression of IGF2 by orchestrating chromatin loop structures involving PRC2.
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Washington, Shannan D., Farhana Musarrat, Monica K. Ertel, Gregory L. Backes, and Donna M. Neumann. "CTCF Binding Sites in the Herpes Simplex Virus 1 Genome Display Site-Specific CTCF Occupation, Protein Recruitment, and Insulator Function." Journal of Virology 92, no. 8 (February 7, 2018): e00156-18. http://dx.doi.org/10.1128/jvi.00156-18.
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ABSTRACTThere are seven conserved CTCF binding domains in the herpes simplex virus 1 (HSV-1) genome. These binding sites individually flank the latency-associated transcript (LAT) and the immediate early (IE) gene regions, suggesting that CTCF insulators differentially control transcriptional domains in HSV-1 latency. In this work, we show that two CTCF binding motifs in HSV-1 display enhancer blocking in a cell-type-specific manner. We found that CTCF binding to the latent HSV-1 genome was LAT dependent and that the quantity of bound CTCF was site specific. Following reactivation, CTCF eviction was dynamic, suggesting that each CTCF site was independently regulated. We explored whether CTCF sites recruit the polycomb-repressive complex 2 (PRC2) to establish repressive domains through a CTCF-Suz12 interaction and found that Suz12 colocalized to the CTCF insulators flanking the ICP0 and ICP4 regions and, conversely, was removed at early times postreactivation. Collectively, these data support the idea that CTCF sites in HSV-1 are independently regulated and may contribute to lytic-latent HSV-1 control in a site-specific manner.IMPORTANCEThe role of chromatin insulators in DNA viruses is an area of interest. It has been shown in several beta- and gammaherpesviruses that insulators likely control the lytic transcriptional profile through protein recruitment and through the formation of three-dimensional (3D) chromatin loops. The ability of insulators to regulate alphaherpesviruses has been understudied to date. The alphaherpesvirus HSV-1 has seven conserved insulator binding motifs that flank regions of the genome known to contribute to the establishment of latency. Our work presented here contributes to the understanding of how insulators control transcription of HSV-1.
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Cummings, Christopher T., and M. Jordan Rowley. "Implications of Dosage Deficiencies in CTCF and Cohesin on Genome Organization, Gene Expression, and Human Neurodevelopment." Genes 13, no. 4 (March 25, 2022): 583. http://dx.doi.org/10.3390/genes13040583.
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Properly organizing DNA within the nucleus is critical to ensure normal downstream nuclear functions. CTCF and cohesin act as major architectural proteins, working in concert to generate thousands of high-intensity chromatin loops. Due to their central role in loop formation, a massive research effort has been dedicated to investigating the mechanism by which CTCF and cohesin create these loops. Recent results lead to questioning the direct impact of CTCF loops on gene expression. Additionally, results of controlled depletion experiments in cell lines has indicated that genome architecture may be somewhat resistant to incomplete deficiencies in CTCF or cohesin. However, heterozygous human genetic deficiencies in CTCF and cohesin have illustrated the importance of their dosage in genome architecture, cellular processes, animal behavior, and disease phenotypes. Thus, the importance of considering CTCF or cohesin levels is especially made clear by these heterozygous germline variants that characterize genetic syndromes, which are increasingly recognized in clinical practice. Defined primarily by developmental delay and intellectual disability, the phenotypes of CTCF and cohesin deficiency illustrate the importance of architectural proteins particularly in neurodevelopment. We discuss the distinct roles of CTCF and cohesin in forming chromatin loops, highlight the major role that dosage of each protein plays in the amplitude of observed effects on gene expression, and contrast these results to heterozygous mutation phenotypes in murine models and clinical patients. Insights highlighted by this comparison have implications for future research into these newly emerging genetic syndromes.
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Fursenko, D. V., P. G. Georgiev, and A. N. Bonchuk. "Study of the N-Terminal Domain Homodimerization in Human Proteins with Zinc Finger Clusters." Doklady Biochemistry and Biophysics 499, no. 1 (July 2021): 257–59. http://dx.doi.org/10.1134/s1607672921040050.
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Abstract CTCF belongs to a large family of transcription factors with clusters of C2H2-type zinc finger domains (C2H2 proteins) and is a main architectural protein in mammals. Human CTCF has a homodimerizing unstructured domain at the N-terminus which is involved in long-distance interactions. To test the presence of similar N-terminal domains in other human C2H2 proteins, a yeast two-hybrid system was used. In total, the ability of unstructured N-terminal domains to homodimerize was investigated for six human C2H2 proteins with an expression profile similar to CTCF. The data indicate the lack of the homodimerization ability of these domains. On the other hand, three C2H2 proteins containing the structured domain DUF3669 at the N-terminus demonstrated homo- and heterodimerization activity.
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Hsu, Sarah C., Caroline Bartman, Aaron J. Stonestrom, Christopher R. Edwards, Thomas Gilgenast, Daniel Emerson, Peng Huang, et al. "The BET Protein BRD2 Cooperates with CTCF to Enforce a Transcriptional Boundary in Erythroid Cells." Blood 128, no. 22 (December 2, 2016): 1034. http://dx.doi.org/10.1182/blood.v128.22.1034.1034.
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Abstract Pharmacologic inhibitors of the bromodomain and extraterminal motif (BET) family of proteins have shown promise in the treatment of hematologic and other malignancies and are being developed for clinical use. However, BET inhibitors do not discriminate between the family members BRD2, BRD3, and BRD4, and thus the mechanistic basis for their therapeutic efficacy is not well understood. In addition, BRD2 and BRD4 are individually required for the activation of genes driven by the erythroid transcription factor GATA1 (Stonestrom et al., Blood 2015), yet how BRD2 in particular contributes to this process has not been studied. We examined BRD2 occupancy genome-wide in erythroid cells and find that BRD2 colocalizes extensively with the architectural/insulator protein CCCTC-binding factor (CTCF). We define a functional hierarchy whereby CTCF is required for BRD2 to occupy co-bound sites, while CTCF binding is BRD2-independent. Using CRISPR/Cas9-based genome editing we identify a boundary element occupied by CTCF and BRD2 that is adjacent to a GATA1-driven enhancer and ensures appropriate transcriptional regulation at the locus. Employing single-molecule RNA FISH we show that either site-specific CTCF disruption or BRD2 depletion leads to increased correlation in mature mRNA levels between the genes flanking this boundary, suggesting that they become inappropriately coregulated. Taken together these findings indicate that BRD2 collaborates with CTCF to constrain the activity of an erythroid enhancer and reveal a potential new role for BET proteins in chromatin domain boundary function. Disclosures No relevant conflicts of interest to declare.
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Filippova, G. N., S. Fagerlie, E. M. Klenova, C. Myers, Y. Dehner, G. Goodwin, P. E. Neiman, S. J. Collins, and V. V. Lobanenkov. "An exceptionally conserved transcriptional repressor, CTCF, employs different combinations of zinc fingers to bind diverged promoter sequences of avian and mammalian c-myc oncogenes." Molecular and Cellular Biology 16, no. 6 (June 1996): 2802–13. http://dx.doi.org/10.1128/mcb.16.6.2802.
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We have isolated and analyzed human CTCF cDNA clones and show here that the ubiquitously expressed 11-zinc-finger factor CTCF is an exceptionally highly conserved protein displaying 93% identity between avian and human amino acid sequences. It binds specifically to regulatory sequences in the promoter-proximal regions of chicken, mouse, and human c-myc oncogenes. CTCF contains two transcription repressor domains transferable to a heterologous DNA binding domain. One CTCF binding site, conserved in mouse and human c-myc genes, is found immediately downstream of the major P2 promoter at a sequence which maps precisely within the region of RNA polymerase II pausing and release. Gel shift assays of nuclear extracts from mouse and human cells show that CTCF is the predominant factor binding to this sequence. Mutational analysis of the P2-proximal CTCF binding site and transient-cotransfection experiments demonstrate that CTCF is a transcriptional repressor of the human c-myc gene. Although there is 100% sequence identity in the DNA binding domains of the avian and human CTCF proteins, the regulatory sequences recognized by CTCF in chicken and human c-myc promoters are clearly diverged. Mutating the contact nucleotides confirms that CTCF binding to the human c-myc P2 promoter requires a number of unique contact DNA bases that are absent in the chicken c-myc CTCF binding site. Moreover, proteolytic-protection assays indicate that several more CTCF Zn fingers are involved in contacting the human CTCF binding site than the chicken site. Gel shift assays utilizing successively deleted Zn finger domains indicate that CTCF Zn fingers 2 to 7 are involved in binding to the chicken c-myc promoter, while fingers 3 to 11 mediate CTCF binding to the human promoter. This flexibility in Zn finger usage reveals CTCF to be a unique "multivalent" transcriptional factor and provides the first feasible explanation of how certain homologous genes (i.e., c-myc) of different vertebrate species are regulated by the same factor and maintain similar expression patterns despite significant promoter sequence divergence.
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Fresan, U., S. Cuartero, M. B. O'Connor, and M. L. Espinas. "The insulator protein CTCF regulates Drosophila steroidogenesis." Biology Open 4, no. 7 (May 15, 2015): 852–57. http://dx.doi.org/10.1242/bio.012344.
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Ortabozkoyun, Havva, Pin-Yao Huang, Hyunwoo Cho, Varun Narendra, Gary LeRoy, Edgar Gonzalez-Buendia, Jane A. Skok, Aristotelis Tsirigos, Esteban O. Mazzoni, and Danny Reinberg. "CRISPR and biochemical screens identify MAZ as a cofactor in CTCF-mediated insulation at Hox clusters." Nature Genetics 54, no. 2 (February 2022): 202–12. http://dx.doi.org/10.1038/s41588-021-01008-5.
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AbstractCCCTC-binding factor (CTCF) is critical to three-dimensional genome organization. Upon differentiation, CTCF insulates active and repressed genes within Hox gene clusters. We conducted a genome-wide CRISPR knockout (KO) screen to identify genes required for CTCF-boundary activity at the HoxA cluster, complemented by biochemical approaches. Among the candidates, we identified Myc-associated zinc-finger protein (MAZ) as a cofactor in CTCF insulation. MAZ colocalizes with CTCF at chromatin borders and, similar to CTCF, interacts with the cohesin subunit RAD21. MAZ KO disrupts gene expression and local contacts within topologically associating domains. Similar to CTCF motif deletions, MAZ motif deletions lead to derepression of posterior Hox genes immediately after CTCF boundaries upon differentiation, giving rise to homeotic transformations in mouse. Thus, MAZ is a factor contributing to appropriate insulation, gene expression and genomic architecture during development.
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Ribeiro de Almeida, Claudia, Ralph Stadhouders, Supat Thongjuea, Eric Soler, and Rudi W. Hendriks. "DNA-binding factor CTCF and long-range gene interactions in V(D)J recombination and oncogene activation." Blood 119, no. 26 (June 28, 2012): 6209–18. http://dx.doi.org/10.1182/blood-2012-03-402586.
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Abstract Regulation of V(D)J recombination events at immunoglobulin (Ig) and T-cell receptor loci in lymphoid cells is complex and achieved via changes in substrate accessibility. Various studies over the last year have identified the DNA-binding zinc-finger protein CCCTC-binding factor (CTCF) as a crucial regulator of long-range chromatin interactions. CTCF often controls specific interactions by preventing inappropriate communication between neighboring regulatory elements or independent chromatin domains. Although recent gene targeting experiments demonstrated that the presence of the CTCF protein is not required for the process of V(D)J recombination per se, CTCF turned out to be essential to control order, lineage specificity and to balance the Ig V gene repertoire. Moreover, CTCF was shown to restrict activity of κ enhancer elements to the Ig κ locus. In this review, we discuss CTCF function in the regulation of V(D)J recombination on the basis of established knowledge on CTCF-mediated chromatin loop domains in various other loci, including the imprinted H19-Igf2 locus as well as the complex β-globin, MHC class II and IFN-γ loci. Moreover, we discuss that loss of CTCF-mediated restriction of enhancer activity may well contribute to oncogenic activation, when in chromosomal translocations Ig enhancer elements and oncogenes appear in a novel genomic context.
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Kang, Mi Ae, and Jong-Soo Lee. "A Newly Assigned Role of CTCF in Cellular Response to Broken DNAs." Biomolecules 11, no. 3 (February 27, 2021): 363. http://dx.doi.org/10.3390/biom11030363.
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Best known as a transcriptional factor, CCCTC-binding factor (CTCF) is a highly conserved multifunctional DNA-binding protein with 11 zinc fingers. It functions in diverse genomic processes, including transcriptional activation/repression, insulation, genome imprinting and three-dimensional genome organization. A big surprise has recently emerged with the identification of CTCF engaging in the repair of DNA double-strand breaks (DSBs) and in the maintenance of genome fidelity. This discovery now adds a new dimension to the multifaceted attributes of this protein. CTCF facilitates the most accurate DSB repair via homologous recombination (HR) that occurs through an elaborate pathway, which entails a chain of timely assembly/disassembly of various HR-repair complexes and chromatin modifications and coordinates multistep HR processes to faithfully restore the original DNA sequences of broken DNA sites. Understanding the functional crosstalks between CTCF and other HR factors will illuminate the molecular basis of various human diseases that range from developmental disorders to cancer and arise from impaired repair. Such knowledge will also help understand the molecular mechanisms underlying the diverse functions of CTCF in genome biology. In this review, we discuss the recent advances regarding this newly assigned versatile role of CTCF and the mechanism whereby CTCF functions in DSB repair.
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Sun, Xiaoyue, Jing Zhang, and Chunwei Cao. "CTCF and Its Partners: Shaper of 3D Genome during Development." Genes 13, no. 8 (August 2, 2022): 1383. http://dx.doi.org/10.3390/genes13081383.
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The 3D genome organization and its dynamic modulate genome function, playing a pivotal role in cell differentiation and development. CTCF and cohesin, acting as the core architectural components involved in chromatin looping and genome folding, can also recruit other protein or RNA partners to fine-tune genome structure during development. Moreover, systematic screening for partners of CTCF has been performed through high-throughput approaches. In particular, several novel protein and RNA partners, such as BHLHE40, WIZ, MAZ, Aire, MyoD, YY1, ZNF143, and Jpx, have been identified, and these partners are mostly implicated in transcriptional regulation and chromatin remodeling, offering a unique opportunity for dissecting their roles in higher-order chromatin organization by collaborating with CTCF and cohesin. Here, we review the latest advancements with an emphasis on features of CTCF partners and also discuss the specific functions of CTCF-associated complexes in chromatin structure modulation, which may extend our understanding of the functions of higher-order chromatin architecture in developmental processes.
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Holwerda, Sjoerd Johannes Bastiaan, and Wouter de Laat. "CTCF: the protein, the binding partners, the binding sites and their chromatin loops." Philosophical Transactions of the Royal Society B: Biological Sciences 368, no. 1620 (June 19, 2013): 20120369. http://dx.doi.org/10.1098/rstb.2012.0369.
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CTCF has it all. The transcription factor binds to tens of thousands of genomic sites, some tissue-specific, others ultra-conserved. It can act as a transcriptional activator, repressor and insulator, and it can pause transcription. CTCF binds at chromatin domain boundaries, at enhancers and gene promoters, and inside gene bodies. It can attract many other transcription factors to chromatin, including tissue-specific transcriptional activators, repressors, cohesin and RNA polymerase II, and it forms chromatin loops. Yet, or perhaps therefore, CTCF's exact function at a given genomic site is unpredictable. It appears to be determined by the associated transcription factors, by the location of the binding site relative to the transcriptional start site of a gene, and by the site's engagement in chromatin loops with other CTCF-binding sites, enhancers or gene promoters. Here, we will discuss genome-wide features of CTCF binding events, as well as locus-specific functions of this remarkable transcription factor.
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Ferguson, Jack, Karen Campos-León, Ieisha Pentland, Joanne D. Stockton, Thomas Günther, Andrew D. Beggs, Adam Grundhoff, Sally Roberts, Boris Noyvert, and Joanna L. Parish. "The chromatin insulator CTCF regulates HPV18 transcript splicing and differentiation-dependent late gene expression." PLOS Pathogens 17, no. 11 (November 4, 2021): e1010032. http://dx.doi.org/10.1371/journal.ppat.1010032.
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The ubiquitous host protein, CCCTC-binding factor (CTCF), is an essential regulator of cellular transcription and functions to maintain epigenetic boundaries, stabilise chromatin loops and regulate splicing of alternative exons. We have previously demonstrated that CTCF binds to the E2 open reading frame (ORF) of human papillomavirus (HPV) 18 and functions to repress viral oncogene expression in undifferentiated keratinocytes by co-ordinating an epigenetically repressed chromatin loop within HPV episomes. Keratinocyte differentiation disrupts CTCF-dependent chromatin looping of HPV18 episomes promoting induction of enhanced viral oncogene expression. To further characterise CTCF function in HPV transcription control we utilised direct, long-read Nanopore RNA-sequencing which provides information on the structure and abundance of full-length transcripts. Nanopore analysis of primary human keratinocytes containing HPV18 episomes before and after synchronous differentiation allowed quantification of viral transcript species, including the identification of low abundance novel transcripts. Comparison of transcripts produced in wild type HPV18 genome-containing cells to those identified in CTCF-binding deficient genome-containing cells identifies CTCF as a key regulator of differentiation-dependent late promoter activation, required for efficient E1^E4 and L1 protein expression. Furthermore, our data show that CTCF binding at the E2 ORF promotes usage of the downstream weak splice donor (SD) sites SD3165 and SD3284, to the dominant E4 splice acceptor site at nucleotide 3434. These findings demonstrate that in the HPV life cycle both early and late virus transcription programmes are facilitated by recruitment of CTCF to the E2 ORF.
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Bailey, Charles, Cynthia Metierre, Yue Feng, Kinsha Baidya, Galina Filippova, Dmitri Loukinov, Victor Lobanenkov, Crystal Semaan, and John Rasko. "CTCF Expression is Essential for Somatic Cell Viability and Protection Against Cancer." International Journal of Molecular Sciences 19, no. 12 (November 30, 2018): 3832. http://dx.doi.org/10.3390/ijms19123832.
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CCCTC-binding factor (CTCF) is a conserved transcription factor that performs diverse roles in transcriptional regulation and chromatin architecture. Cancer genome sequencing reveals diverse acquired mutations in CTCF, which we have shown functions as a tumour suppressor gene. While CTCF is essential for embryonic development, little is known of its absolute requirement in somatic cells and the consequences of CTCF haploinsufficiency. We examined the consequences of CTCF depletion in immortalised human and mouse cells using shRNA knockdown and CRISPR/Cas9 genome editing as well as examined the growth and development of heterozygous Ctcf (Ctcf+/−) mice. We also analysed the impact of CTCF haploinsufficiency by examining gene expression changes in CTCF-altered endometrial carcinoma. Knockdown and CRISPR/Cas9-mediated editing of CTCF reduced the cellular growth and colony-forming ability of K562 cells. CTCF knockdown also induced cell cycle arrest and a pro-survival response to apoptotic insult. However, in p53 shRNA-immortalised Ctcf+/− MEFs we observed the opposite: increased cellular proliferation, colony formation, cell cycle progression, and decreased survival after apoptotic insult compared to wild-type MEFs. CRISPR/Cas9-mediated targeting in Ctcf+/− MEFs revealed a predominance of in-frame microdeletions in Ctcf in surviving clones, however protein expression could not be ablated. Examination of CTCF mutations in endometrial cancers showed locus-specific alterations in gene expression due to CTCF haploinsufficiency, in concert with downregulation of tumour suppressor genes and upregulation of estrogen-responsive genes. Depletion of CTCF expression imparts a dramatic negative effect on normal cell function. However, CTCF haploinsufficiency can have growth-promoting effects consistent with known cancer hallmarks in the presence of additional genetic hits. Our results confirm the absolute requirement for CTCF expression in somatic cells and provide definitive evidence of CTCF’s role as a haploinsufficient tumour suppressor gene. CTCF genetic alterations in endometrial cancer indicate that gene dysregulation is a likely consequence of CTCF loss, contributing to, but not solely driving cancer growth.
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Zhang, Xizhe, Sergio Branciamore, Grigoriy Gogoshin, Andrei S. Rodin, and Arthur D. Riggs. "Analysis of high-resolution 3D intrachromosomal interactions aided by Bayesian network modeling." Proceedings of the National Academy of Sciences 114, no. 48 (November 13, 2017): E10359—E10368. http://dx.doi.org/10.1073/pnas.1620425114.
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Long-range intrachromosomal interactions play an important role in 3D chromosome structure and function, but our understanding of how various factors contribute to the strength of these interactions remains poor. In this study we used a recently developed analysis framework for Bayesian network (BN) modeling to analyze publicly available datasets for intrachromosomal interactions. We investigated how 106 variables affect the pairwise interactions of over 10 million 5-kb DNA segments in the B-lymphocyte cell line GB12878. Strictly data-driven BN modeling indicates that the strength of intrachromosomal interactions (hic_strength) is directly influenced by only four types of factors: distance between segments, Rad21 or SMC3 (cohesin components),transcription at transcription start sites (TSS), and the number of CCCTC-binding factor (CTCF)–cohesin complexes between the interacting DNA segments. Subsequent studies confirmed that most high-intensity interactions have a CTCF–cohesin complex in at least one of the interacting segments. However, 46% have CTCF on only one side, and 32% are without CTCF. As expected, high-intensity interactions are strongly dependent on the orientation of the ctcf motif, and, moreover, we find that the interaction between enhancers and promoters is similarly dependent on ctcf motif orientation. Dependency relationships between transcription factors were also revealed, including known lineage-determining B-cell transcription factors (e.g., Ebf1) as well as potential novel relationships. Thus, BN analysis of large intrachromosomal interaction datasets is a useful tool for gaining insight into DNA–DNA, protein–DNA, and protein–protein interactions.
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Kotova, E. S., I. V. Sorokina, S. B. Akopov, L. G. Nikolaev, and E. D. Sverdlov. "Expression of chicken CTCF gene in COS-1 cells and partial purification of CTCF protein." Biochemistry (Moscow) 78, no. 8 (August 2013): 879–83. http://dx.doi.org/10.1134/s0006297913080038.
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Zampieri, Michele, Tiziana Guastafierro, Roberta Calabrese, Fabio Ciccarone, Maria G. Bacalini, Anna Reale, Mariagrazia Perilli, Claudio Passananti, and Paola Caiafa. "ADP-ribose polymers localized on Ctcf–Parp1–Dnmt1 complex prevent methylation of Ctcf target sites." Biochemical Journal 441, no. 2 (December 21, 2011): 645–52. http://dx.doi.org/10.1042/bj20111417.
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PARylation [poly(ADP-ribosyl)ation] is involved in the maintenance of genomic methylation patterns through its control of Dnmt1 [DNA (cytosine-5)-methyltransferase 1] activity. Our previous findings indicated that Ctcf (CCCTC-binding factor) may be an important player in key events whereby PARylation controls the unmethylated status of some CpG-rich regions. Ctcf is able to activate Parp1 [poly(ADP-ribose) polymerase 1], which ADP-ribosylates itself and, in turn, inhibits DNA methylation via non-covalent interaction between its ADP-ribose polymers and Dnmt1. By such a mechanism, Ctcf may preserve the epigenetic pattern at promoters of important housekeeping genes. The results of the present study showed Dnmt1 as a new protein partner of Ctcf. Moreover, we show that Ctcf forms a complex with Dnmt1 and PARylated Parp1 at specific Ctcf target sequences and that PARylation is responsible for the maintenance of the unmethylated status of some Ctcf-bound CpGs. We suggest a mechanism by which Parp1, tethered and activated at specific DNA target sites by Ctcf, preserves their methylation-free status.
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Li, Tao, Ji-Fan Hu, Xinwen Qiu, Jianqun Ling, Huiling Chen, Shukui Wang, Aiju Hou, Thanh H. Vu, and Andrew R. Hoffman. "CTCF Regulates Allelic Expression of Igf2 by Orchestrating a Promoter-Polycomb Repressive Complex 2 Intrachromosomal Loop." Molecular and Cellular Biology 28, no. 20 (July 28, 2008): 6473–82. http://dx.doi.org/10.1128/mcb.00204-08.
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ABSTRACT CTCF is a zinc finger DNA-binding protein that regulates the epigenetic states of numerous target genes. Using allelic regulation of mouse insulin-like growth factor II (Igf2) as a model, we demonstrate that CTCF binds to the unmethylated maternal allele of the imprinting control region (ICR) in the Igf2/H19 imprinting domain and forms a long-range intrachromosomal loop to interact with the three clustered Igf2 promoters. Polycomb repressive complex 2 is recruited through the interaction of CTCF with Suz12, leading to allele-specific methylation at lysine 27 of histone H3 (H3-K27) and to suppression of the maternal Igf2 promoters. Targeted mutation or deletion of the maternal ICR abolishes this chromatin loop, decreases allelic H3-K27 methylation, and causes loss of Igf2 imprinting. RNA interference knockdown of Suz12 also leads to reactivation of the maternal Igf2 allele and biallelic Igf2 expression. CTCF and Suz12 are coprecipitated from nuclear extracts with antibodies specific for either protein, and they interact with each other in a two-hybrid system. These findings offer insight into general epigenetic mechanisms by which CTCF governs gene expression by orchestrating chromatin loop structures and by serving as a DNA-binding protein scaffold to recruit and bind polycomb repressive complexes.
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Day, Latasha, Charles M. Chau, Michael Nebozhyn, Andrew J. Rennekamp, Michael Showe, and Paul M. Lieberman. "Chromatin Profiling of Epstein-Barr Virus Latency Control Region." Journal of Virology 81, no. 12 (April 4, 2007): 6389–401. http://dx.doi.org/10.1128/jvi.02172-06.
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ABSTRACT Epstein-Barr virus (EBV) escapes host immunity by the reversible and epigenetic silencing of immunogenic viral genes. We previously presented evidence that a dynamic chromatin domain, which we have referred to as the latency control region (LCR), contributes to the reversible repression of EBNA2 and LMP1 gene transcription. We now explore the protein-DNA interaction profiles for a few known regulatory factors and histone modifications that regulate LCR structure and activity. A chromatin immunoprecipitation assay combined with real-time PCR analysis was used to analyze protein-DNA interactions at ∼500-bp intervals across the first 60,000 bp of the EBV genome. We compared the binding patterns of EBNA1 with those of the origin recognition complex protein ORC2, the chromatin boundary factor CTCF, the linker histone H1, and several histone modifications. We analyzed three EBV-positive cell lines (MutuI, Raji, and LCL3459) with distinct transcription patterns reflecting different latency types. Our findings suggest that histone modification patterns within the LCR are complex but reflect differences in each latency type. The most striking finding was the identification of CTCF sites immediately upstream of the Qp, Cp, and EBER transcription initiation regions in all three cell types. In transient assays, CTCF facilitated EBNA1-dependent transcription activation of Cp, suggesting that CTCF coordinates interactions between different chromatin domains. We also found that histone H3 methyl K4 clustered with CTCF and EBNA1 at sites of active transcription or DNA replication initiation. Our findings support a model where CTCF delineates multiple domains within the LCR and regulates interactions between these domains that correlate with changes in gene expression.
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Hyle, Judith, Yang Zhang, Shaela Wright, Beisi Xu, Ying Shao, John Easton, Liqing Tian, Ruopeng Feng, Peng Xu, and Chunliang Li. "Acute depletion of CTCF directly affects MYC regulation through loss of enhancer–promoter looping." Nucleic Acids Research 47, no. 13 (May 25, 2019): 6699–713. http://dx.doi.org/10.1093/nar/gkz462.
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Abstract Numerous pieces of evidence support the complex, 3D spatial organization of the genome dictates gene expression. CTCF is essential to define topologically associated domain boundaries and to facilitate the formation of insulated chromatin loop structures. To understand CTCF’s direct role in global transcriptional regulation, we integrated the miniAID-mClover3 cassette to the endogenous CTCF locus in a human pediatric B-ALL cell line, SEM, and an immortal erythroid precursor cell line, HUDEP-2, to allow for acute depletion of CTCF protein by the auxin-inducible degron system. In SEM cells, CTCF loss notably disrupted intra-TAD loops and TAD integrity in concurrence with a reduction in CTCF-binding affinity, while showing no perturbation to nuclear compartment integrity. Strikingly, the overall effect of CTCF’s loss on transcription was minimal. Whole transcriptome analysis showed hundreds of genes differentially expressed in CTCF-depleted cells, among which MYC and a number of MYC target genes were specifically downregulated. Mechanically, acute depletion of CTCF disrupted the direct interaction between the MYC promoter and its distal enhancer cluster residing ∼1.8 Mb downstream. Notably, MYC expression was not profoundly affected upon CTCF loss in HUDEP-2 cells suggesting that CTCF could play a B-ALL cell line specific role in maintaining MYC expression.
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Zlatanova, J., and P. Caiafa. "CTCF and its protein partners: divide and rule?" Journal of Cell Science 122, no. 9 (April 22, 2009): 1275–84. http://dx.doi.org/10.1242/jcs.039990.
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Yang, Bobae, Tae-Gyun Kim, Sueun Kim, and Hyoung-Pyo Kim. "CCCTC-binding factor regulates the development and function of dendritic cells." Journal of Immunology 202, no. 1_Supplement (May 1, 2019): 118.5. http://dx.doi.org/10.4049/jimmunol.202.supp.118.5.
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Abstract Dendritic cells (DCs) are professional antigen presenting cells, which present antigen to cognate T cells. DC activation through diverse toll-like receptors is prerequisite for triggering efficient immune responses to foreign antigens. CCCTC-binding factor (CTCF) is a DNA binding protein that regulates 3D genome structure which is believed to be important to control of gene expression. Here we described that CTCF is required for development and function in DCs. CTCF is critically required for the FLT3L-dependent CD11c+ DCs (FL-DCs) development, unlike for those in the GM-CSF-dependent CD11c+ DC (GM-DCs) development. However, the resultant CTCF-deficient DC number was decreased by 50% in both FLT3L- and GM-CSF-supplemented culture conditions. Although CTCF was required for bone marrow (BM) progenitor proliferation in both FLT3L-and GM-CSF-supplemented cultures, CTCF-deficient GM-DCs showed normal cellular apoptosis rate and higher CD11c+ DC differentiation. Upon TLR4 ligation, CTCF-deficient DCs showed higher expression levels of surface MHCII and co-stimulatory molecules compared to those WT DCs. Thus, our data indicate that CTCF plays a critical role in the overall differentiation, survival, and maturation of DCs.
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Amiri Roudbar, M., H. Dehghani, M. Tahmoorespur, A. Zahmatkesh, H. Adeldust, S. Ansari Majd, and M. Daliri Joupari. "Quantitative analysis of RNA abondance for CTCF during reprogramming of bovine embryo from oocyte to blastocyst." Archives Animal Breeding 58, no. 1 (April 28, 2015): 171–75. http://dx.doi.org/10.5194/aab-58-171-2015.
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Abstract. CTCF is a highly conserved protein among eukaryotes and it is involved in many of regulatory functions including, transcriptional repression and activation, chromatin insulation, imprinting, X chromosome inactivation, higher-order chromatin organization, and alternative splicing. Studies performed on mouse embryos indicate that CTCF can be a maternal-effect gene, and is essential for normal development of embryos. CTCF can be used as a molecular effector for the proper epigenetic establishment of embryonic development. The aim of this study was to determine changes in transcript levels of the CTCF gene in bovine preimplantation embryos. RNA was extracted from immature and mature oocytes and embryos at various developmental stages (two-cell, four-cell, eight-cell, and blastocysts). Results showed that the amounts of CTCF transcripts decreased in mature oocyte in comparison with immature oocytes, but this change was not significant. In addition, the amount of CTCF transcript in embryos at two-cell, four-cell, eight-cell, and blastocyst stages significantly increased in comparison with immature oocytes. These data show that CTCF expression in bovine embryo begins at minor embryonic genome activation.
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Mamberti, Stefania, Maruthi K. Pabba, Alexander Rapp, M. Cristina Cardoso, and Michael Scholz. "The Chromatin Architectural Protein CTCF Is Critical for Cell Survival upon Irradiation-Induced DNA Damage." International Journal of Molecular Sciences 23, no. 7 (March 31, 2022): 3896. http://dx.doi.org/10.3390/ijms23073896.
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CTCF is a nuclear protein initially discovered for its role in enhancer-promoter insulation. It has been shown to play a role in genome architecture and in fact, its DNA binding sites are enriched at the borders of chromatin domains. Recently, we showed that depletion of CTCF impairs the DNA damage response to ionizing radiation. To investigate the relationship between chromatin domains and DNA damage repair, we present here clonogenic survival assays in different cell lines upon CTCF knockdown and ionizing irradiation. The application of a wide range of ionizing irradiation doses (0–10 Gy) allowed us to investigate the survival response through a biophysical model that accounts for the double-strand breaks’ probability distribution onto chromatin domains. We demonstrate that the radiosensitivity of different cell lines is increased upon lowering the amount of the architectural protein. Our model shows that the deficiency in the DNA repair ability is related to the changes in the size of chromatin domains that occur when different amounts of CTCF are present in the nucleus.
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Mao, Albert, Carrie Chen, Stephanie Portillo-Ledesma, and Tamar Schlick. "Effect of Single-Residue Mutations on CTCF Binding to DNA: Insights from Molecular Dynamics Simulations." International Journal of Molecular Sciences 24, no. 7 (March 29, 2023): 6395. http://dx.doi.org/10.3390/ijms24076395.
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In humans and other eukaryotes, DNA is condensed into chromatin fibers that are further wound into chromosomes. This organization allows regulatory elements in the genome, often distant from each other in the linear DNA, to interact and facilitate gene expression through regions known as topologically associating domains (TADs). CCCTC–binding factor (CTCF) is one of the major components of TAD formation and is responsible for recruiting a partner protein, cohesin, to perform loop extrusion and facilitate proper gene expression within TADs. Because single-residue CTCF mutations have been linked to the development of a variety of cancers in humans, we aim to better understand how these mutations affect the CTCF structure and its interaction with DNA. To this end, we compare all-atom molecular dynamics simulations of a wildtype CTCF–DNA complex to those of eight different cancer-linked CTCF mutant sequences. We find that most mutants have lower binding energies compared to the wildtype protein, leading to the formation of less stable complexes. Depending on the type and position of the mutation, this loss of stability can be attributed to major changes in the electrostatic potential, loss of hydrogen bonds between the CTCF and DNA, and/or destabilization of specific zinc fingers. Interestingly, certain mutations in specific fingers can affect the interaction with the DNA of other fingers, explaining why mere single mutations can impair CTCF function. Overall, these results shed mechanistic insights into experimental observations and further underscore CTCF’s importance in the regulation of chromatin architecture and gene expression.
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Klenova, Elena M., Igor V. Chernukhin, Ayman El-Kady, Robin E. Lee, Elena M. Pugacheva, Dmitri I. Loukinov, Graham H. Goodwin, et al. "Functional Phosphorylation Sites in the C-Terminal Region of the Multivalent Multifunctional Transcriptional Factor CTCF." Molecular and Cellular Biology 21, no. 6 (March 15, 2001): 2221–34. http://dx.doi.org/10.1128/mcb.21.6.2221-2234.2001.
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ABSTRACT CTCF is a widely expressed and highly conserved multi-Zn-finger (ZF) nuclear factor. Binding to various CTCF target sites (CTSs) is mediated by combinatorial contributions of different ZFs. Different CTSs mediate distinct CTCF functions in transcriptional regulation, including promoter repression or activation and hormone-responsive gene silencing. In addition, the necessary and sufficient core sequences of diverse enhancer-blocking (insulator) elements, including CpG methylation-sensitive ones, have recently been pinpointed to CTSs. To determine whether a posttranslational modification may modulate CTCF functions, we studied CTCF phosphorylation. We demonstrated that most of the modifications that occur at the carboxy terminus in vivo can be reproduced in vitro with casein kinase II (CKII). Major modification sites map to four serines within the S604KKEDS609S610DS612E motif that is highly conserved in vertebrates. Specific mutations of these serines abrogate phosphorylation of CTCF in vivo and CKII-induced phosphorylation in vitro. In addition, we showed that completely preventing phosphorylation by substituting all serines within this site resulted in markedly enhanced repression of the CTS-bearing vertebrate c-myc promoters, but did not alter CTCF nuclear localization or in vitro DNA-binding characteristics assayed with c-myc CTSs. Moreover, these substitutions manifested a profound effect on negative cell growth regulation by wild-type CTCF. CKII may thus be responsible for attenuation of CTCF activity, either acting on its own or by providing the signal for phosphorylation by other kinases and for CTCF-interacting protein partners.
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Millau, Jean-François, and Luc Gaudreau. "CTCF, cohesin, and histone variants: connecting the genome." Biochemistry and Cell Biology 89, no. 5 (October 2011): 505–13. http://dx.doi.org/10.1139/o11-052.
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During the last decades our view of the genome organization has changed. We moved from a linear view to a looped view of the genome. It is now well established that inter- and intra-connections occur between chromosomes and play a major role in gene regulations. These interconnections are mainly orchestrated by the CTCF protein, which is also known as the “master weaver” of the genome. Recent advances in sequencing and genome-wide studies revealed that CTCF binds to DNA at thousands of sites within the human genome, providing the possibility to form thousands of genomic connection hubs. Strikingly, two histone variants, namely H2A.Z and H3.3, strongly co-localize at CTCF binding sites. In this article, we will review the recent advances in CTCF biology and discuss the role of histone variants H2A.Z and H3.3 at CTCF binding sites.
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Salamon, Daniel, Ferenc Banati, Anita Koroknai, Mate Ravasz, Kalman Szenthe, Zoltan Bathori, Agnes Bakos, Hans Helmut Niller, Hans Wolf, and Janos Minarovits. "Binding of CCCTC-binding factor in vivo to the region located between Rep* and the C promoter of Epstein–Barr virus is unaffected by CpG methylation and does not correlate with Cp activity." Journal of General Virology 90, no. 5 (May 1, 2009): 1183–89. http://dx.doi.org/10.1099/vir.0.007344-0.
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In this study, the binding of the insulator protein CCCTC-binding factor (CTCF) to the region located between Rep* and the C promoter (Cp) of Epstein–Barr virus (EBV) was analysed using chromatin immunoprecipitation and in vivo footprinting. CTCF binding was found to be independent of Cp usage in cell lines corresponding to the major EBV latency types. Bisulfite sequencing and an electrophoretic mobility-shift assay (using methylated and unmethylated probes) revealed that CTCF binding was insufficient to induce local CpG demethylation in certain cell lines and was unaffected by CpG methylation in the region between Rep* and Cp. In addition, CTCF binding to the latency promoter, Qp, did not correlate with Qp activity.
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Williams, Adam, and Richard A. Flavell. "The role of CTCF in regulating nuclear organization." Journal of Experimental Medicine 205, no. 4 (March 17, 2008): 747–50. http://dx.doi.org/10.1084/jem.20080066.
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The spatial organization of the genome is thought to play an important part in the coordination of gene regulation. New techniques have been used to identify specific long-range interactions between distal DNA sequences, revealing an ever-increasing complexity to nuclear organization. CCCTC-binding factor (CTCF) is a versatile zinc finger protein with diverse regulatory functions. New data now help define how CTCF mediates both long-range intrachromosomal and interchromosomal interactions, and highlight CTCF as an important factor in determining the three-dimensional structure of the genome.
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Cha, Hye Ji, Özgün Uyan, Job Dekker, and Stuart H. Orkin. "Inner Nuclear Protein Matrin-3 Coordinates Hematopoietic Cell Transcription and Differentiation By Stabilizing Chromatin Architecture." Blood 138, Supplement 1 (November 5, 2021): 285. http://dx.doi.org/10.1182/blood-2021-151070.
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Abstract The nucleus is spatially organized by chromosome and interchromatin functional components. Global reorganization of chromatin interactions and compartmentalization occurring during differentiation requires proper chromosome positioning, but the involvement of nuclear components in this process remains largely underexplored. In particular, blood cell development exemplifies a coordinated process accompanied by dramatic chromatin reorganization, thereby providing a model in which to interrogate chromatin dynamics during differentiation. Here, we show that an abundant inner nuclear protein Matrin-3 (Matr3) plays a critical role in the maintenance of chromatin structure and has a broad effect on erythroid cell differentiation by coordinating gene expression. First, we deleted the entire gene body by CRISPR/Cas9 in mouse erythroleukemia (MEL) cells. The Matr3 knockout (KO) cells proliferate normally and exhibit morphological changes on differentiation suggestive of accelerated maturation. Consistently, erythroid-specific genes were expressed at a higher level in MEL Matr3 KO cells than in parental cells. The consequences of Matr3 deletion were also determined in G1ER cells, in which differentiation is conditional on activation of GATA-1. To assess the global impact of Matr3 loss on erythroid cell maturation, we measured global RNA expression changes. Erythroid-specific genes were expressed at a much higher level upon differentiation of Matr3 KO cells. Differentiation is typically accompanied by specific changes in nuclear architecture. Using super-resolution microscopy, we observed that heterochromatin protein 1α (HP1α) was more dispersed and irregular in appearance in Matr3 KO cells, suggesting that Matr3 loss alters morphological boundaries of heterochromatin. Analysis of the interactions between different regions of chromatin identifies topologically associating domains and classifies the genome into two compartments (A and B). The A and B compartments correspond to the structures and characteristics of known euchromatin and heterochromatin, respectively. We next explored global chromatin structure using a high-throughput chromosome conformation capture (Hi-C) assay. In Matr3 KO cells, insulation at the domain boundaries was reduced, and the compartment strengths between the B compartments became stronger, while those between A-type domains were reduced. Remarkably, we found that these changes in cells lacking Matr3 were similar to changes in chromatin contact during differentiation. To access the genomic features at a higher resolution, we performed the assay for transposase-accessible chromatin with high throughput sequencing (ATAC-seq). Notably, the newly opened regions in Matr3 KO, as compared to parental, cells were enriched for GATA motifs, which are generally more accessible in differentiated erythroid cells. Architectural proteins function cooperatively to organize chromatin. Using affinity purification followed by mass spectrometry and immunoblotting, we found that Matr3 interacts with proteins involved in chromatin remodeling, such as CTCF and cohesin. To identify whether Matr3 loss alters chromatin occupancy of its interacting partners, we performed ChIP-seq for CTCF and the core cohesin component Rad21. In the absence of Matr3, occupancy of CTCF and Rad21 was perturbed in a subset of genomic regions. Moreover, destabilization of CTCF and cohesin binding correlated with altered transcription and accelerated erythroid differentiation. Most sites with disrupted CTCF and Rad21 binding during differentiation were also sensitive to the absence of the scaffold protein Matr3. Our data demonstrate that the nucleoplasmic protein Matr3 stabilizes the binding of the architectural proteins (CTCF and cohesin) to chromatin and serves to maintain chromatin structure. We speculate that Matr3 negatively regulates cell fate transitions by maintaining cellular state through fine-tuning the binding of CTCF/cohesin to chromatin and associated 3D interactions. Our work reveals a previously unrecognized role of Matr3 in chromatin organization and responses to developmental cues. Disclosures No relevant conflicts of interest to declare.