The Experts below are selected from a list of 13254 Experts worldwide ranked by ideXlab platform
Victor V Lobanenkov - One of the best experts on this subject based on the ideXlab platform.
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CTCF mediates chromatin looping via n terminal domain dependent cohesin retention
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Elena M Pugacheva, Naoki Kubo, Dmitri Loukinov, Sungyun Kang, Alexander L Kovalchuk, Alexander V Strunnikov, Gabriel E Zentner, Bing Ren, Victor V LobanenkovAbstract: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.
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CTCF Expression is Essential for Somatic Cell Viability and Protection Against Cancer.
International Journal of Molecular Sciences, 2018Co-Authors: Charles G. Bailey, Dmitri Loukinov, Victor V Lobanenkov, Cynthia Metierre, Kinsha Baidya, Galina N. Filippova, Crystal Semaan, Yue Feng, John E.j. RaskoAbstract: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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CTCF Expression Is Essential for Somatic Cell Viability and Protection against Cancer
2018Co-Authors: Charles G. Bailey, Dmitri Loukinov, Victor V Lobanenkov, Cynthia Metierre, Julie Feng, Kinsha Baidya, Galina N. Filippova, Crystal Semaan, John E.j. RaskoAbstract: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 and 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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A Novel Mechanism for CTCF in the Epigenetic Regulation of Bax in Breast Cancer Cells
Neoplasia, 2013Co-Authors: Claudia Fabiola Méndez-catalá, Elena M Pugacheva, Victor V Lobanenkov, Dawn Farrar, Alexander A. Vostrov, Yoko Ito, G-x Kita, A Murrell, Svetlana Gretton, Elena KlenovaAbstract:We previously reported the association of elevated levels of the multifunctional transcription factor, CCCTC binding factor (CTCF), in breast cancer cells with the specific anti-apoptotic function of CTCF. To understand the molecular mechanisms of this phenomenon, we investigated regulation of the human Bax gene by CTCF in breast and non-breast cells. Two CTCF binding sites (CTSs) within the Bax promoter were identified. In all cells, breast and non-breast, active histone modifications were present at these CTSs, DNA harboring this region was unmethylated, and levels of Bax mRNA and protein were similar. Nevertheless, up-regulation of Bax mRNA and protein and apoptotic cell death were observed only in breast cancer cells depleted of CTCF. We proposed that increased CTCF binding to the Bax promoter in breast cancer cells, by comparison with non-breast cells, may be mechanistically linked to the specific apoptotic phenotype in CTCF-depleted breast cancer cells. In this study, we show that CTCF binding was enriched at the Bax CTSs in breast cancer cells and tumors; in contrast, binding of other transcription factors (SP1, WT1, EGR1, and c-Myc) was generally increased in non-breast cells and normal breast tissues. Our findings suggest a novel mechanism for CTCF in the epigenetic regulation of Bax in breast cancer cells, whereby elevated levels of CTCF support preferential binding of CTCF to the Bax CTSs. In this context, CTCF functions as a transcriptional repressor counteracting influences of positive regulatory factors; depletion of breast cancer cells from CTCF therefore results in the activation of Bax and apoptosis.
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A Novel Mechanism for CTCF in the Epigenetic Regulation of
2013Co-Authors: Claudia Fabiola Méndez-catalá, Elena M Pugacheva, Victor V Lobanenkov, Dawn Farrar, Alexander A. Vostrov, Yoko Ito, G-x Kita, A Murrell, Svetlana Gretton, Elena KlenovaAbstract:Wepreviouslyreportedtheassociation ofelevatedlevelsofthemultifunctionaltranscription factor,CCCTCbindingfactor (CTCF), in breast cancercells with thespecific anti-apoptotic functionof CTCF.To understandthemolecular mechanisms of this phenomenon, we investigated regulation of the human Bax gene by CTCF in breast and non–breast cells. Two CTCF binding sites (CTSs) within theBaxpromoter were identified. In all cells, breast and non-breast, active histone modifications were present at these CTSs, DNA harboring this region was unmethylated, and levels of Bax mRNA and protein were similar. Nevertheless, up-regulation of Bax mRNA and protein and apoptotic cell death were observed only in breast cancer cells depleted of CTCF. We proposed that increased CTCF binding to the Bax promoter in breast cancer cells, by comparison with non–breast cells, may be mechanistically linked to the specific apoptotic phenotype in CTCF-depleted breast cancer cells. In this study, we show that CTCF binding was enriched at the Bax CTSs in breast cancer cells and tumors; in contrast, binding of other transcription factors (SP1, WT1, EGR1, and c-Myc) was generally increased in non– breast cells and normal breast tissues. Our findings suggest a novel mechanism for CTCF in the epigenetic regulation of Bax in breast cancer cells, whereby elevated levels of CTCF support preferential binding of CTCF to the Bax CTSs. In this context, CTCF functions as a transcriptional repressor counteracting influences of positive regulatory factors; depletion of breast cancer cells from CTCF therefore results in the activation of Bax and apoptosis.
John E.j. Rasko - One of the best experts on this subject based on the ideXlab platform.
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CTCF Haploinsufficiency Mediates Intron Retention in A Tissue-specific Manner
2019Co-Authors: Adel B Alharbi, Amy D. Marshall, Charles G. Bailey, M Vellozzi, Ulf Schmitz, Darya Vanichkina, Rajini Nagarajah, Justin J.-l. Wong, John E.j. RaskoAbstract:CTCF is a master regulator of gene transcription and chromatin organization with occupancy at thousands of DNA target sites. CTCF is essential for embryonic development and somatic cell viability and has been characterized as a haploinsufficient tumor suppressor. Increasing evidence demonstrates CTCF as a key player in several alternative splicing (AS) regulatory mechanisms, including transcription elongation, regulation of splicing factors, and epigenetic regulation. However, the genome-wide impact of CTCF dosage on AS has not been investigated. We examined the effect of CTCF haploinsufficiency on gene expression and AS in multiple tissues from CTCF hemizygous (CTCF+/-) mice. Distinct tissue-specific differences in gene expression and AS were observed in CTCF+/- mice compared to wildtype mice. We observed a surprisingly large number of increased intron retention events in CTCF+/- liver and kidney, specifically in genes associated with cytoskeletal organization, splicing and metabolism. This study provides further evidence for CTCF dose-dependent and tissue-specific regulation of gene expression and AS. Our data provide a strong foundation for elucidating the mechanistic role of CTCF in AS regulation and its biological consequences.
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CTCF Expression is Essential for Somatic Cell Viability and Protection Against Cancer.
International Journal of Molecular Sciences, 2018Co-Authors: Charles G. Bailey, Dmitri Loukinov, Victor V Lobanenkov, Cynthia Metierre, Kinsha Baidya, Galina N. Filippova, Crystal Semaan, Yue Feng, John E.j. RaskoAbstract: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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CTCF Expression Is Essential for Somatic Cell Viability and Protection against Cancer
2018Co-Authors: Charles G. Bailey, Dmitri Loukinov, Victor V Lobanenkov, Cynthia Metierre, Julie Feng, Kinsha Baidya, Galina N. Filippova, Crystal Semaan, John E.j. RaskoAbstract: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 and 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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CTCF and BORIS in genome regulation and cancer.
Current Opinion in Genetics & Development, 2014Co-Authors: Amy D. Marshall, Charles G. Bailey, John E.j. RaskoAbstract:CTCF plays a vital role in chromatin structure and function. CTCF is ubiquitously expressed and plays diverse roles in gene regulation, imprinting, insulation, intra/interchromosomal interactions, nuclear compartmentalisation, and alternative splicing. CTCF has a single paralogue, the testes-specific CTCF-like gene (CTCFL)/BORIS. CTCF and BORIS can be deregulated in cancer. The tumour suppressor gene CTCF can be mutated or deleted in cancer, or CTCF DNA binding can be altered by epigenetic changes. BORIS is aberrantly expressed frequently in cancer, leading some to propose a pro-tumourigenic role for BORIS. However, BORIS can inhibit cell proliferation, and is mutated in cancer similarly to CTCF suggesting BORIS activation in cancer may be due to global genetic or epigenetic changes typical of malignant transformation.
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Dynamic association of the mammalian insulator protein CTCF with centrosomes and the midbody.
Experimental Cell Research, 2004Co-Authors: Ru Zhang, John E.j. Rasko, Victor V Lobanenkov, Les J. Burke, Rainer RenkawitzAbstract:CTCF is a highly conserved, ubiquitously expressed DNA-binding protein that has widespread capabilities in gene regulation. CTCF plays important roles in cell growth regulatory processes and epigenetic functions. Ectopic expression of CTCF results in severe cell growth inhibition at multiple points within the cell cycle, indicating that CTCF levels must be stringently monitored. We have investigated the subcellular localization of CTCF in detail. Interestingly, we observe that CTCF shows a dynamic cell cycle-dependent distribution. Immunofluorescent staining reveals that in interphase CTCF is a nuclear protein, which is mainly excluded from the nucleolus. Strikingly, CTCF is associated with the centrosome during mitosis, especially from metaphase to anaphase. At telophase, CTCF dissociates from the centrosome and localizes to the midbody and the reformed nuclei. The association of CTCF with centrosomes and the midbody is further confirmed by biochemical fractionation. Moreover, subcellular fractions of CTCF show cell cycle and organelle-specific posttranslational modifications, suggesting different roles for CTCF at different stages of the cell cycle.
Dawn Farrar - One of the best experts on this subject based on the ideXlab platform.
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ADP-ribose polymer depletion leads to nuclear CTCF re-localization and chromatin rearrangement(1).
Biochemical Journal, 2013Co-Authors: Tiziana Guastafierro, Roberta Calabrese, Anna Reale, Michele Zampieri, Maria Giulia Bacalini, Angela Catizone, Oliviano Martella, Maria Di Girolamo, Margherita Miccheli, Dawn FarrarAbstract:CTCF (CCCTC-binding factor) directly induces Parp [poly(ADP-ribose) polymerase] 1 activity and its PARylation [poly(ADPribosyl)ation] in the absence of DNA damage. CTCF, in turn, is a substrate for this post-synthetic modification and as such it is covalently and non-covalently modified by PARs (ADP-ribose polymers). Moreover, PARylation is able to protect certain DNA regions bound by CTCF from DNA methylation. We recently reported that de novo methylation of CTCF target sequences due to overexpression of Parg [poly(ADP-ribose)glycohydrolase] induces loss of CTCF binding. Considering this, we investigate to what extent PARP activity is able to affect nuclear distribution of CTCF in the present study. Notably, CTCF lost its diffuse nuclear localization following PAR (ADP-ribose polymer) depletion and accumulated at the periphery of the nucleus where it was linked with nuclear pore complex proteins remaining external to the perinuclear Lamin B1 ring. We demonstrated that PAR depletion-dependent perinuclear localization of CTCF was due to its blockage from entering the nucleus. Besides CTCF nuclear delocalization, the outcome of PAR depletion led to changes in chromatin architecture. Immunofluorescence analyses indicated DNA redistribution, a generalized genomic hypermethylation and an increase of inactive compared with active chromatin marks in Parg-overexpressing or CTCF-silenced cells. Together these results underline the importance of the cross-talk between Parp1 and CTCF in the maintenance of nuclear organization.
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A Novel Mechanism for CTCF in the Epigenetic Regulation of Bax in Breast Cancer Cells
Neoplasia, 2013Co-Authors: Claudia Fabiola Méndez-catalá, Elena M Pugacheva, Victor V Lobanenkov, Dawn Farrar, Alexander A. Vostrov, Yoko Ito, G-x Kita, A Murrell, Svetlana Gretton, Elena KlenovaAbstract:We previously reported the association of elevated levels of the multifunctional transcription factor, CCCTC binding factor (CTCF), in breast cancer cells with the specific anti-apoptotic function of CTCF. To understand the molecular mechanisms of this phenomenon, we investigated regulation of the human Bax gene by CTCF in breast and non-breast cells. Two CTCF binding sites (CTSs) within the Bax promoter were identified. In all cells, breast and non-breast, active histone modifications were present at these CTSs, DNA harboring this region was unmethylated, and levels of Bax mRNA and protein were similar. Nevertheless, up-regulation of Bax mRNA and protein and apoptotic cell death were observed only in breast cancer cells depleted of CTCF. We proposed that increased CTCF binding to the Bax promoter in breast cancer cells, by comparison with non-breast cells, may be mechanistically linked to the specific apoptotic phenotype in CTCF-depleted breast cancer cells. In this study, we show that CTCF binding was enriched at the Bax CTSs in breast cancer cells and tumors; in contrast, binding of other transcription factors (SP1, WT1, EGR1, and c-Myc) was generally increased in non-breast cells and normal breast tissues. Our findings suggest a novel mechanism for CTCF in the epigenetic regulation of Bax in breast cancer cells, whereby elevated levels of CTCF support preferential binding of CTCF to the Bax CTSs. In this context, CTCF functions as a transcriptional repressor counteracting influences of positive regulatory factors; depletion of breast cancer cells from CTCF therefore results in the activation of Bax and apoptosis.
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A Novel Mechanism for CTCF in the Epigenetic Regulation of
2013Co-Authors: Claudia Fabiola Méndez-catalá, Elena M Pugacheva, Victor V Lobanenkov, Dawn Farrar, Alexander A. Vostrov, Yoko Ito, G-x Kita, A Murrell, Svetlana Gretton, Elena KlenovaAbstract:Wepreviouslyreportedtheassociation ofelevatedlevelsofthemultifunctionaltranscription factor,CCCTCbindingfactor (CTCF), in breast cancercells with thespecific anti-apoptotic functionof CTCF.To understandthemolecular mechanisms of this phenomenon, we investigated regulation of the human Bax gene by CTCF in breast and non–breast cells. Two CTCF binding sites (CTSs) within theBaxpromoter were identified. In all cells, breast and non-breast, active histone modifications were present at these CTSs, DNA harboring this region was unmethylated, and levels of Bax mRNA and protein were similar. Nevertheless, up-regulation of Bax mRNA and protein and apoptotic cell death were observed only in breast cancer cells depleted of CTCF. We proposed that increased CTCF binding to the Bax promoter in breast cancer cells, by comparison with non–breast cells, may be mechanistically linked to the specific apoptotic phenotype in CTCF-depleted breast cancer cells. In this study, we show that CTCF binding was enriched at the Bax CTSs in breast cancer cells and tumors; in contrast, binding of other transcription factors (SP1, WT1, EGR1, and c-Myc) was generally increased in non– breast cells and normal breast tissues. Our findings suggest a novel mechanism for CTCF in the epigenetic regulation of Bax in breast cancer cells, whereby elevated levels of CTCF support preferential binding of CTCF to the Bax CTSs. In this context, CTCF functions as a transcriptional repressor counteracting influences of positive regulatory factors; depletion of breast cancer cells from CTCF therefore results in the activation of Bax and apoptosis.
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Regulation of the apoptotic genes in breast cancer cells by the transcription factor CTCF
Breast Cancer Research, 2010Co-Authors: Claudia Fabiola Méndez-catalá, Elena M Pugacheva, Igor Chernukhin, Dawn Farrar, Alexander A. Vostrov, Yoko Ito, F Docquier, G-x Kita, A Murrell, V LobanankovAbstract: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.
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CTCF interacts with and recruits the largest subunit of RNA polymerase II to CTCF target sites genome-wide.
Molecular and Cellular Biology, 2007Co-Authors: Igor Chernukhin, Sungyun Kang, Joanne Whitehead, Dawn Farrar, Rituparna Mukhopadhyay, Shaharum Shamsuddin, Rosita Bergström, Yoo Wook Kwon, Ian E. G. MorrisonAbstract: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 beta-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.
Dmitri Loukinov - One of the best experts on this subject based on the ideXlab platform.
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CTCF mediates chromatin looping via n terminal domain dependent cohesin retention
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Elena M Pugacheva, Naoki Kubo, Dmitri Loukinov, Sungyun Kang, Alexander L Kovalchuk, Alexander V Strunnikov, Gabriel E Zentner, Bing Ren, Victor V LobanenkovAbstract: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.
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CTCF Expression is Essential for Somatic Cell Viability and Protection Against Cancer.
International Journal of Molecular Sciences, 2018Co-Authors: Charles G. Bailey, Dmitri Loukinov, Victor V Lobanenkov, Cynthia Metierre, Kinsha Baidya, Galina N. Filippova, Crystal Semaan, Yue Feng, John E.j. RaskoAbstract: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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CTCF Expression Is Essential for Somatic Cell Viability and Protection against Cancer
2018Co-Authors: Charles G. Bailey, Dmitri Loukinov, Victor V Lobanenkov, Cynthia Metierre, Julie Feng, Kinsha Baidya, Galina N. Filippova, Crystal Semaan, John E.j. RaskoAbstract: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 and 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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Loss of maternal CTCF is associated with peri-implantation lethality of CTCF null embryos.
PLoS ONE, 2012Co-Authors: James M. Moore, Dmitri Loukinov, Natalia A. Rabaia, Leslie E. Smith, Sara R. Fagerlie, Kay E. Gurley, Christine M. Disteche, Steven J. Collins, Christopher J. Kemp, Victor V LobanenkovAbstract:CTCF is a highly conserved, multifunctional zinc finger protein involved in critical aspects of gene regulation including transcription regulation, chromatin insulation, genomic imprinting, X-chromosome inactivation, and higher order chromatin organization. Such multifunctional properties of CTCF suggest an essential role in development. Indeed, a previous report on maternal depletion of CTCF suggested that CTCF is essential for pre-implantation development. To distinguish between the effects of maternal and zygotic expression of CTCF, we studied pre-implantation development in mice harboring a complete loss of function CTCF knockout allele. Although we demonstrated that homozygous deletion of CTCF is early embryonically lethal, in contrast to previous observations, we showed that the CTCF nullizygous embryos developed up to the blastocyst stage (E3.5) followed by peri-implantation lethality (E4.5–E5.5). Moreover, one-cell stage CTCF nullizygous embryos cultured ex vivo developed to the 16–32 cell stage with no obvious abnormalities. Using a single embryo assay that allowed both genotype and mRNA expression analyses of the same embryo, we demonstrated that pre-implantation development of the CTCF nullizygous embryos was associated with the retention of the maternal wild type CTCF mRNA. Loss of this stable maternal transcript was temporally associated with loss of CTCF protein expression, apoptosis of the developing embryo, and failure to further develop an inner cell mass and trophoectoderm ex vivo. This indicates that CTCF expression is critical to early embryogenesis and loss of its expression rapidly leads to apoptosis at a very early developmental stage. This is the first study documenting the presence of the stable maternal CTCF transcript in the blastocyst stage embryos. Furthermore, in the presence of maternal CTCF, zygotic CTCF expression does not seem to be required for pre-implantation development.
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Heightened expression of CTCF in breast cancer cells is associated with resistance to apoptosis.
Cancer research, 2005Co-Authors: Dawn Farrar, Dmitri Loukinov, Igor Chernukhin, Sergei Vatolin, Abigail F. Robinson, Vivien D'arcy, Svetlana Pack, Alan Mackay, Robert A. Harris, Heather DorricottAbstract:CTCF is a candidate tumor suppressor gene encoding a multifunctional transcription factor. Surprisingly for a tumor suppressor, the levels of CTCF in breast cancer cell lines and tumors were found elevated compared with breast cell lines with finite life span and normal breast tissues. In this study, we aimed to investigate the possible cause for this increase in CTCF content and in particular to test the hypothesis that up-regulation of CTCF may be linked to resistance of breast cancer cells to apoptosis. For this purpose, apoptotic cell death was monitored following alterations of CTCF levels induced by transient transfection and conditional knockdown of CTCF in various cell lines. We observed apoptotic cell death in all breast cancer cell lines examined following CTCF down-regulation. In addition, overexpression of CTCF partially protected cells from apoptosis induced by overexpression of Bax or treatment with sodium butyrate. To elucidate possible mechanisms of this phenomenon, we used a proteomics approach and observed that levels of the proapoptotic protein, Bax, were increased following CTCF down-regulation in MCF7 cells. Taken together, these results suggest that in some cellular contexts CTCF shows antiapoptotic characteristics, most likely exerting its functions through regulation of apoptotic genes. We hypothesize that CTCF overexpression may have evolved as a compensatory mechanism to protect breast cancer cells from apoptosis, thus providing selective survival advantages to these cells. The observations reported in this study may lead to development of therapies based on selective reduction of CTCF in breast cancer cells.
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CTCF sites display cell cycle dependent dynamics in factor binding and nucleosome positioning
Genome Research, 2019Co-Authors: Marlies E Oomen, Anders S Hansen, Yu Liu, Xavier Darzacq, Job DekkerAbstract:CCCTC-binding factor (CTCF) plays a key role in the formation of topologically associating domains (TADs) and loops in interphase. During mitosis TADs are absent, but how TAD formation is dynamically controlled during the cell cycle is not known. Several contradicting observations have been made regarding CTCF binding to mitotic chromatin using both genomics- and microscopy-based techniques. Here, we have used four different assays to address this debate. First, using 5C, we confirmed that TADs and CTCF loops are readily detected in interphase, but absent during prometaphase. Second, ATAC-seq analysis showed that CTCF sites display greatly reduced accessibility and lose the CTCF footprint in prometaphase, suggesting loss of CTCF binding and rearrangement of the nucleosomal array around the binding motif. In contrast, transcription start sites remain accessible in prometaphase, although adjacent nucleosomes can also become repositioned and occupy at least a subset of start sites during mitosis. Third, loss of site-specific CTCF binding was directly demonstrated using CUT&RUN. Histone modifications and histone variants are maintained in mitosis, suggesting a role in bookmarking of active CTCF sites. Finally, live-cell imaging, fluorescence recovery after photobleaching, and single molecule tracking showed that almost all CTCF chromatin binding is lost in prometaphase. Combined, our results demonstrate loss of CTCF binding to CTCF sites during prometaphase and rearrangement of the chromatin landscape around CTCF motifs. This, combined with loss of cohesin, would contribute to the observed loss of TADs and CTCF loops during mitosis and reveals that CTCF sites, key architectural cis-elements, display cell cycle stage-dependent dynamics in factor binding and nucleosome positioning.
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CTCF sites display cell cycle dependent dynamics in factor binding and nucleosome positioning
bioRxiv, 2018Co-Authors: Marlies E Oomen, Anders S Hansen, Yu Liu, Xavier Darzacq, Job DekkerAbstract:CTCF plays a key role in formation of topologically associating domains (TADs) and loops in interphase. During mitosis TADs are absent, but how TAD formation is dynamically controlled during the cell cycle is not known. Several contradicting observations have been made regarding CTCF binding to mitotic chromatin using both genomics and microscopy-based techniques. Here we have used 4 different assays to address this debate. First, using 5C we confirmed that TADs and CTCF loops are readily detected in interphase, but absent during prometaphase. Second, ATAC-seq analysis showed that CTCF sites display greatly reduced accessibility and lose the CTCF footprint in prometaphase, suggesting loss of CTCF binding and rearrangement of the nucleosomal array around the binding motif. In contrast, transcription start sites remain accessible in prometaphase, although adjacent nucleosomes can also become repositioned and occupy at least a subset of start sites during mitosis. Third, loss of site-specific CTCF binding was directly demonstrated using CUT&RUN. Histone modifications and histone variants are maintained in mitosis, suggesting a role in bookmarking of active CTCF sites. Finally, live-cell imaging, fluorescence recovery after photobleaching and single molecule tracking showed that almost all CTCF chromatin binding is lost in prometaphase. Combined, our results demonstrate loss of CTCF binding to CTCF sites during prometaphase and rearrangement of the chromatin landscape around CTCF motifs. This contributes to loss of TADs and CTCF loops during mitosis, and reveals that CTCF sites, a key architectural cis-element of the genome, display cell cycle stage-dependent dynamics in factor binding and nucleosome positioning.