RESUMEN
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.
Asunto(s)
Factor de Unión a CCCTC/genética , Factor de Unión a CCCTC/metabolismo , Supervivencia Celular/fisiología , Neoplasias Endometriales/genética , Edición Génica , Animales , Sistemas CRISPR-Cas , Proliferación Celular/genética , Proliferación Celular/fisiología , Supervivencia Celular/genética , Femenino , Haploinsuficiencia/genética , Haploinsuficiencia/fisiología , Humanos , Células K562 , Ratones , ARN Interferente Pequeño/genéticaRESUMEN
High-grade serous carcinoma (HGSC) is the most aggressive and predominant form of epithelial ovarian cancer and the leading cause of gynecologic cancer-related death. We have previously shown that CTCFL (also known as BORIS, Brother of the Regulator of Imprinted Sites) is expressed in most ovarian cancers, and is associated with global and promoter-specific DNA hypomethylation, advanced tumor stage, and poor prognosis. To explore its role in HGSC, we expressed BORIS in human fallopian tube secretory epithelial cells (FTSEC), the presumptive cells of origin for HGSC. BORIS-expressing cells exhibited increased motility and invasion, and BORIS expression was associated with alterations in several cancer-associated gene expression networks, including fatty acid metabolism, TNF signaling, cell migration, and ECM-receptor interactions. Importantly, GALNT14, a glycosyltransferase gene implicated in cancer cell migration and invasion, was highly induced by BORIS, and GALNT14 knockdown significantly abrogated BORIS-induced cell motility and invasion. In addition, in silico analyses provided evidence for BORIS and GALNT14 coexpression in several cancers. Finally, ChIP-seq demonstrated that expression of BORIS was associated with de novo and enhanced binding of CTCF at hundreds of loci, many of which correlated with activation of transcription at target genes, including GALNT14. Taken together, our data indicate that BORIS may promote cell motility and invasion in HGSC via upregulation of GALNT14, and suggests BORIS as a potential therapeutic target in this malignancy. IMPLICATIONS: These studies provide evidence that aberrant expression of BORIS may play a role in the progression to HGSC by enhancing the migratory and invasive properties of FTSEC.
Asunto(s)
Factor de Unión a CCCTC/genética , Proteínas de Unión al ADN/genética , N-Acetilgalactosaminiltransferasas/genética , Neoplasias Ováricas/genética , Factor de Unión a CCCTC/metabolismo , Carcinoma Epitelial de Ovario/genética , Carcinoma Epitelial de Ovario/metabolismo , Carcinoma Epitelial de Ovario/patología , Línea Celular Tumoral , Proliferación Celular/fisiología , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/patología , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/metabolismo , Trompas Uterinas/metabolismo , Trompas Uterinas/patología , Femenino , Humanos , N-Acetilgalactosaminiltransferasas/metabolismo , Invasividad Neoplásica , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Regiones Promotoras Genéticas , TransfecciónRESUMEN
Imprinted methylation of the paternal Rasgrf1 allele in mice occurs at a differentially methylated domain (DMD) 30 kbp 5' of the promoter. A repeated sequence 3' of the DMD regulates imprinted methylation, which is required for imprinted expression. Here we identify the mechanism by which methylation controls imprinting. The DMD is an enhancer blocker that binds CTCF in a methylation-sensitive manner. CTCF bound to the unmethylated maternal allele silences expression. CTCF binding to the paternal allele is prevented by repeat-mediated methylation, allowing expression. Optimal in vitro enhancer-blocking activity requires CTCF binding sites. The enhancer blocker can be bypassed in vivo and imprinting abolished by placing an extra enhancer proximal to the promoter. Together, the repeats and the DMD constitute a binary switch that regulates Rasgrf1 imprinting.
Asunto(s)
Proteínas Portadoras/genética , Metilación de ADN , Proteínas de Unión al ADN/metabolismo , Elementos de Facilitación Genéticos/genética , Impresión Genómica , Proteínas Represoras/metabolismo , Animales , Factor de Unión a CCCTC , Proteínas Activadoras de GTPasa , Ratones , Modelos GenéticosRESUMEN
Brother of the Regulator of Imprinted Sites (BORIS) is a mammalian CTCF paralog with the same central 11Zn fingers (11ZF) that mediate specific interactions with varying approximately 50-bp target sites. Regulated in vivo occupancy of such sites may yield structurally and functionally distinct CTCF/DNA complexes involved in various aspects of gene regulation, including epigenetic control of gene imprinting and X chromosome inactivation. The latter functions are mediated by meCpG-sensitive 11ZF binding. Because CTCF is normally present in all somatic cells, whereas BORIS is active only in CTCF- and 5-methylcytosine-deficient adult male germ cells, switching DNA occupancy from CTCF to BORIS was suggested to regulate site specificity and timing of epigenetic reprogramming. In addition to 11ZF-binding paternal imprinting control regions, cancer-testis gene promoters also undergo remethylation during CTCF/BORIS switching in germ cells. Only promoters of cancer testis genes are normally silenced in all somatic cells but activated during spermatogenesis when demethylated in BORIS-positive germ cells and are found aberrantly derepressed in various tumors. We show here that BORIS is also expressed in multiple cancers and is thus itself a cancer-testis gene and that conditional expression of BORIS in normal fibroblasts activates cancer-testis genes selectively. We tested if replacement of CTCF by BORIS on regulatory DNA occurs in vivo on activation of a prototype cancer-testis gene, MAGE-A1. Transition from a hypermethylated/silenced to a hypomethylated/activated status induced in normal cells by 5-aza-2'-deoxycytidine (5-azadC) was mimicked by conditional input of BORIS and is associated with complete switching from CTCF to BORIS occupancy at a single 11ZF target. This site manifested a novel type of CTCF/BORIS 11ZF binding insensitive to CpG methylation. Whereas 5-azadC induction of BORIS takes only few hours, derepression of MAGE-A1 occurred 1 to 2 days later, suggesting that BORIS mediates cancer-testis gene activation by 5-azadC. Indeed, infection of normal fibroblasts with anti-BORIS short hairpin RNA retroviruses before treatment with 5-azadC blocked reactivation of MAGE-A1. We suggest that BORIS is likely tethering epigenetic machinery to a novel class of CTCF/BORIS 11ZF target sequences that mediate induction of cancer-testis genes.
Asunto(s)
Metilación de ADN , Proteínas de Unión al ADN/biosíntesis , Regulación Neoplásica de la Expresión Génica/genética , Proteínas de Neoplasias/genética , Animales , Antígenos de Neoplasias , Azacitidina/análogos & derivados , Azacitidina/farmacología , Secuencia de Bases , Línea Celular Tumoral , Metilación de ADN/efectos de los fármacos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Decitabina , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/fisiología , Vectores Genéticos/genética , Humanos , Inmunohistoquímica , Antígenos Específicos del Melanoma , Datos de Secuencia Molecular , Proteínas de Neoplasias/antagonistas & inhibidores , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas , Unión Proteica , ARN Interferente Pequeño/genética , Retroviridae/genética , Activación Transcripcional , TransfecciónRESUMEN
Regulatory sequences recognized by the unique pair of paralogous factors, CTCF and BORIS, have been implicated in epigenetic regulation of imprinting and X chromosome inactivation. Lung cancers exhibit genome-wide demethylation associated with derepression of a specific class of genes encoding cancer-testis (CT) antigens such as NY-ESO-1. CT genes are normally expressed in BORIS-positive male germ cells deficient in CTCF and meCpG contents, but are strictly silenced in somatic cells. The present study was undertaken to ascertain if aberrant activation of BORIS contributes to derepression of NY-ESO-1 during pulmonary carcinogenesis. Preliminary experiments indicated that NY-ESO-1 expression coincided with derepression of BORIS in cultured lung cancer cells. Quantitative reverse transcription-PCR analysis revealed robust, coincident induction of BORIS and NY-ESO-1 expression in lung cancer cells, but not normal human bronchial epithelial cells following 5-aza-2'-deoxycytidine (5-azadC), Depsipeptide FK228 (DP), or sequential 5-azadC/DP exposure under clinically relevant conditions. Bisulfite sequencing, methylation-specific PCR, and chromatin immunoprecipitation (ChIP) experiments showed that induction of BORIS coincided with direct modulation of chromatin structure within a CpG island in the 5'-flanking noncoding region of this gene. Cotransfection experiments using promoter-reporter constructs confirmed that BORIS modulates NY-ESO-1 expression in lung cancer cells. Gel shift and ChIP experiments revealed a novel CTCF/BORIS-binding site in the NY-ESO-1 promoter, which unlike such sites in the H19-imprinting control region and X chromosome, is insensitive to CpG methylation in vitro. In vivo occupancy of this site by CTCF was associated with silencing of the NY-ESO-1 promoter, whereas switching from CTCF to BORIS occupancy coincided with derepression of NY-ESO-1. Collectively, these data indicate that reciprocal binding of CTCF and BORIS to the NY-ESO-1 promoter mediates epigenetic regulation of this CT gene in lung cancer cells, and suggest that induction of BORIS may be a novel strategy to augment immunogenicity of pulmonary carcinomas.
Asunto(s)
Antígenos de Neoplasias/genética , Proteínas de Unión al ADN/metabolismo , Neoplasias Pulmonares/genética , Proteínas de la Membrana/genética , Proteínas Represoras/metabolismo , Antígenos de Neoplasias/biosíntesis , Antígenos de Neoplasias/metabolismo , Secuencia de Bases , Factor de Unión a CCCTC , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Metilación de ADN , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Histonas/metabolismo , Humanos , Inmunohistoquímica , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sulfitos/farmacologíaRESUMEN
CTCF is a widely expressed 11-zinc finger (ZF) transcription factor that is involved in different aspects of gene regulation including promoter activation or repression, hormone-responsive gene silencing, methylation-dependent chromatin insulation, and genomic imprinting. Because CTCF targets include oncogenes and tumor suppressor genes, we screened over 100 human tumor samples for mutations that might disrupt CTCF activity. We did not observe any CTCF mutations leading to truncations/premature stops. Rather, in breast, prostate, and Wilms' tumors, we observed four different CTCF somatic missense mutations involving amino acids within the ZF domain. Each ZF mutation abrogated CTCF binding to a subset of target sites within the promoters/insulators of certain genes involved in regulating cell proliferation but did not alter binding to the regulatory sequences of other genes. These observations suggest that CTCF may represent a novel tumor suppressor gene that displays tumor-specific "change of function" rather than complete "loss of function."
Asunto(s)
ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Mutación Missense , Proteínas Represoras , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Dedos de Zinc/genética , Secuencia de Aminoácidos , Secuencia de Bases , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Factor de Unión a CCCTC , Proteínas de Ciclo Celular/genética , Femenino , Genes Supresores de Tumor , Globinas/genética , Humanos , Masculino , Datos de Secuencia Molecular , Muramidasa/genética , Regiones Promotoras Genéticas , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Conformación Proteica , Especificidad por Sustrato , Tumor de Wilms/genética , Tumor de Wilms/metabolismoRESUMEN
Insulator elements affect gene expression by preventing the spread of heterochromatin and restricting transcriptional enhancers from activation of unrelated promoters. In vertebrates, insulator's function requires association with the CCCTC-binding factor (CTCF), a protein that recognizes long and diverse nucleotide sequences. While insulators are critical in gene regulation, only a few have been reported. Here, we describe 13,804 CTCF-binding sites in potential insulators of the human genome, discovered experimentally in primary human fibroblasts. Most of these sequences are located far from the transcriptional start sites, with their distribution strongly correlated with genes. The majority of them fit to a consensus motif highly conserved and suitable for predicting possible insulators driven by CTCF in other vertebrate genomes. In addition, CTCF localization is largely invariant across different cell types. Our results provide a resource for investigating insulator function and possible other general and evolutionarily conserved activities of CTCF sites.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Genoma Humano , Elementos Aisladores/genética , Proteínas Represoras/metabolismo , Animales , Factor de Unión a CCCTC , Línea Celular , Inmunoprecipitación de Cromatina , Secuencia Conservada , Evolución Molecular , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Células U937 , Vertebrados/genéticaRESUMEN
The choice mechanisms that determine the future inactive X chromosome in somatic cells of female mammals involve the regulated expression of the XIST gene. A familial C(-43)G mutation in the XIST promoter results in skewing of X chromosome inactivation (XCI) towards the inactive X chromosome of heterozygous females, whereas a C(-43)A mutation found primarily in the active X chromosome results in the opposite skewing pattern. Both mutations point to the existence of a factor that might be responsible for the skewed patterns. Here we identify this factor as CTCF, a conserved protein with a 11 Zn-finger (ZF) domain that can mediate multiple sequence-specificity and interactions between DNA-bound CTCF molecules. We show that mouse and human Xist/XIST promoters contain one homologous CTCF-binding sequence with the matching dG-contacts, which in the human XIST include the -43 position within the DNase I footprint of CTCF. While the C(-43)A mutation abrogates CTCF binding, the C(-43)G mutation results in a dramatic increase in CTCF-binding efficiency by altering ZF-usage mode required for recognition of the altered dG-contacts of the mutant site. Thus, the skewing effect of the two -43C mutations correlates with their effects on CTCF binding. Finally, CTCF interacts with the XIST/Xist promoter only in female human and mouse cells. The interpretation that this reflected a preferential interaction with the promoter of the active Xist allele was confirmed in mouse fetal placenta. These observations are in keeping with the possibility that the choice of X chromosome inactivation reflects stabilization of a higher order chromatin conformation impinging on the CTCF-XIST promoter complex.
Asunto(s)
Cromosomas Humanos X , Proteínas de Unión al ADN/metabolismo , Compensación de Dosificación (Genética) , Mutación , Mutación Puntual , Regiones Promotoras Genéticas , ARN no Traducido/genética , Proteínas Represoras/metabolismo , Alelos , Animales , Secuencia de Bases , Factor de Unión a CCCTC , Núcleo Celular/metabolismo , Cromatina/metabolismo , Inmunoprecipitación de Cromatina , Metilación de ADN , Proteínas de Unión al ADN/genética , Desoxirribonucleasa I/metabolismo , Salud de la Familia , Femenino , Heterocigoto , Humanos , Inmunoprecipitación , Masculino , Ratones , Modelos Genéticos , Datos de Secuencia Molecular , Plásmidos/metabolismo , Unión Proteica , Biosíntesis de Proteínas , Conformación Proteica , Estructura Terciaria de Proteína , ARN Largo no Codificante , Proteínas Represoras/genética , Homología de Secuencia de Ácido Nucleico , Factores Sexuales , Transcripción Genética , Dedos de ZincRESUMEN
CTCF, a conserved, ubiquitous, and highly versatile 11-zinc-finger factor involved in various aspects of gene regulation, forms methylation-sensitive insulators that regulate X chromosome inactivation and expression of imprinted genes. We document here the existence of a paralogous gene with the same exons encoding the 11-zinc-finger domain as mammalian CTCF genes and thus the same DNA-binding potential, but with distinct amino and carboxy termini. We named this gene BORIS for Brother of the Regulator of Imprinted Sites. BORIS is present only in the testis, and expressed in a mutually exclusive manner with CTCF during male germ cell development. We show here that erasure of methylation marks during male germ-line development is associated with dramatic up-regulation of BORIS and down-regulation of CTCF expression. Because BORIS bears the same DNA-binding domain that CTCF employs for recognition of methylation marks in soma, BORIS is a candidate protein for the elusive epigenetic reprogramming factor acting in the male germ line.