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BACKGROUND: Nucleosome repositioning in cancer is believed to cause many changes in genome organisation and gene expression. Understanding these changes is important to elucidate fundamental aspects of cancer. It is also important for medical diagnostics based on cell-free DNA (cfDNA), which originates from genomic DNA regions protected from digestion by nucleosomes. RESULTS: We have generated high-resolution nucleosome maps in paired tumour and normal tissues from the same breast cancer patients using MNase-assisted histone H3 ChIP-seq and compared them with the corresponding cfDNA from blood plasma. This analysis has detected single-nucleosome repositioning at key regulatory regions in a patient-specific manner and common cancer-specific patterns across patients. The nucleosomes gained in tumour versus normal tissue were particularly informative of cancer pathways, with ~ 20-fold enrichment at CpG islands, a large fraction of which marked promoters of genes encoding DNA-binding proteins. The tumour tissues were characterised by a 5-10 bp decrease in the average distance between nucleosomes (nucleosome repeat length, NRL), which is qualitatively similar to the differences between pluripotent and differentiated cells. This effect was correlated with gene activity, differential DNA methylation and changes in local occupancy of linker histone variants H1.4 and H1X. CONCLUSIONS: Our study offers a novel resource of high-resolution nucleosome maps in breast cancer patients and reports for the first time the effect of systematic decrease of NRL in paired tumour versus normal breast tissues from the same patient. Our findings provide a new mechanistic understanding of nucleosome repositioning in tumour tissues that can be valuable for patient diagnostics, stratification and monitoring.
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Neoplasias de la Mama , Ácidos Nucleicos Libres de Células , Humanos , Femenino , Nucleosomas/genética , Neoplasias de la Mama/genética , Metilación de ADN , Histonas/genética , Histonas/metabolismo , ADN/metabolismo , Ácidos Nucleicos Libres de Células/metabolismo , CromatinaRESUMEN
Malignant tumors often escape anticancer immune surveillance by suppressing the cytotoxic functions of T lymphocytes. While many of these immune evasion networks include checkpoint proteins, small molecular weight compounds, such as the amino acid L-kynurenine (LKU), could also substantially contribute to the suppression of anti-cancer immunity. However, the biochemical mechanisms underlying the suppressive effects of LKU on T-cells remain unclear. Here, we report for the first time that LKU suppresses T cell function as an aryl hydrocarbon receptor (AhR) ligand. The presence of LKU in T cells is associated with AhR activation, which results in competition between AhR and hypoxia-inducible factor 1 alpha (HIF-1α) for the AhR nuclear translocator, ARNT, leading to T cell exhaustion. The expression of indoleamine 2,3-dioxygenase 1 (IDO1, the enzyme that leads to LKU generation) is induced by the TGF-ß-Smad-3 pathway. We also show that IDO-negative cancers utilize an alternative route for LKU production via the endogenous inflammatory mediator, the high mobility group box 1 (HMGB-1)-interferon-gamma (IFN-γ) axis. In addition, other IDO-negative tumors (like T-cell lymphomas) trigger IDO1 activation in eosinophils present in the tumor microenvironment (TME). These mechanisms suppress cytotoxic T cell function, and thus support the tumor immune evasion machinery.
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Quinurenina , Neoplasias , Humanos , Quinurenina/metabolismo , Quinurenina/farmacología , Evasión Inmune , Transducción de Señal , Linfocitos T , Microambiente TumoralRESUMEN
High mobility group box 1 (HMGB1) is a non-histone protein which is predominantly localised in the cell nucleus. However, stressed, dying, injured or dead cells can release this protein into the extracellular matrix passively. In addition, HMGB1 release was observed in cancer and immune cells where this process can be triggered by various endogenous as well as exogenous stimuli. Importantly, released HMGB1 acts as a so-called "danger signal" and could impact on the ability of cancer cells to escape host immune surveillance. However, the molecular mechanisms underlying the functional role of HMGB1 in determining the capability of human cancer cells to evade immune attack remain unclear. Here we report that the involvement of HMGB1 in anti-cancer immune evasion is determined by Toll-like receptor (TLR) 4, which recognises HMGB1 as a ligand. We found that HGMB1 induces TLR4-mediated production of transforming growth factor beta type 1 (TGF-ß), displaying autocrine/paracrine activities. TGF-ß induces production of the immunosuppressive protein galectin-9 in cancer cells. In TLR4-positive cancer cells, HMGB1 triggers the formation of an autocrine loop which induces galectin-9 expression. In malignant cells lacking TLR4, the same effect could be triggered by HMGB1 indirectly through TLR4-expressing myeloid cells present in the tumour microenvironment (e. g. tumour-associated macrophages).
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Galectinas/biosíntesis , Proteína HMGB1/fisiología , Neoplasias/inmunología , Receptor Toll-Like 4/fisiología , Humanos , Tolerancia Inmunológica , Células THP-1 , Factor de Crecimiento Transformador beta1/fisiologíaRESUMEN
Galectin-9 is one of the key proteins employed by a variety of human malignancies to suppress anti-cancer activities of cytotoxic lymphoid cells and thus escape immune surveillance. Human cancer cells in most cases express higher levels of galectin-9 compared to non-transformed cells. However, the biochemical mechanisms underlying this phenomenon remain unclear. Here we report for the first time that in human cancer as well as embryonic cells, the transcription factors hypoxia-inducible factor 1 (HIF-1) and activator protein 1 (AP-1) are involved in upregulation of transforming growth factor beta 1 (TGF-ß1) expression, leading to activation of the transcription factor Smad3 through autocrine action. This process triggers upregulation of galectin-9 expression in both malignant (mainly in breast and colorectal cancer as well as acute myeloid leukaemia (AML)) and embryonic cells. The effect, however, was not observed in mature non-transformed human cells. TGF-ß1-activated Smad3 therefore displays differential behaviour in human cancer and embryonic vs non-malignant cells. This study uncovered a self-supporting biochemical mechanism underlying high levels of galectin-9 expression operated by the human cancer and embryonic cells employed in our investigations. Our results suggest the possibility of using the TGF-ß1 signalling pathway as a potential highly efficient target for cancer immunotherapy.
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Galectinas/metabolismo , Células Madre Embrionarias Humanas/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neoplasias/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Comunicación Autocrina , Galectinas/genética , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Células HaCaT , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Células MCF-7 , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/patología , Transducción de Señal , Proteína smad3/genética , Proteína smad3/metabolismo , Células THP-1 , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismo , Factor de Crecimiento Transformador beta1/genética , Escape del Tumor , Hipoxia Tumoral , Microambiente TumoralRESUMEN
Human cancer cells operate a variety of effective molecular and signaling mechanisms which allow them to escape host immune surveillance and thus progress the disease. We have recently reported that the immune receptor Tim-3 and its natural ligand galectin-9 are involved in the immune escape of human acute myeloid leukemia (AML) cells. These cells use the neuronal receptor latrophilin 1 (LPHN1) and its ligand fibronectin leucine rich transmembrane protein 3 (FLRT3, and possibly other ligands) to trigger the pathway. We hypothesized that the Tim-3-galectin-9 pathway may be involved in the immune escape of cancer cells of different origins. We found that studied breast tumors expressed significantly higher levels of both galectin-9 and Tim-3 compared to healthy breast tissues of the same patients and that these proteins were co-localized. Increased levels of LPHN2 and expressions of LPHN3 as well as FLRT3 were also detected in breast tumor cells. Activation of this pathway facilitated the translocation of galectin-9 onto the tumor cell surface, however no secretion of galectin-9 by tumor cells was observed. Surface-based galectin-9 was able to protect breast carcinoma cells against cytotoxic T cell-induced death. Furthermore, we found that cell lines from brain, colorectal, kidney, blood/mast cell, liver, prostate, lung, and skin cancers expressed detectable amounts of both Tim-3 and galectin-9 proteins. The majority of cell lines expressed one of the LPHN isoforms and FLRT3. We conclude that the Tim-3-galectin-9 pathway is operated by a wide range of human cancer cells and is possibly involved in prevention of anti-tumor immunity.
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Neoplasias de la Mama/metabolismo , Galectinas/metabolismo , Receptor 2 Celular del Virus de la Hepatitis A/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Leucemia Mieloide Aguda/metabolismo , Células MCF-7 , Glicoproteínas de Membrana/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/fisiología , Linfocitos T Citotóxicos/metabolismoRESUMEN
CTCF is an evolutionarily conserved and ubiquitously expressed architectural protein regulating a plethora of cellular functions via different molecular mechanisms. CTCF can undergo a number of post-translational modifications which change its properties and functions. One such modifications linked to cancer is poly(ADP-ribosyl)ation (PARylation). The highly PARylated CTCF form has an apparent molecular mass of 180â¯kDa (referred to as CTCF180), which can be distinguished from hypo- and non-PARylated CTCF with the apparent molecular mass of 130â¯kDa (referred to as CTCF130). The existing data accumulated so far have been mainly related to CTCF130. However, the properties of CTCF180 are not well understood despite its abundance in a number of primary tissues. In this study we performed ChIP-seq and RNA-seq analyses in human breast cells 226LDM, which display predominantly CTCF130 when proliferating, but CTCF180 upon cell cycle arrest. We observed that in the arrested cells the majority of sites lost CTCF, whereas fewer sites gained CTCF or remain bound (i.e. common sites). The classical CTCF binding motif was found in the lost and common, but not in the gained sites. The changes in CTCF occupancies in the lost and common sites were associated with increased chromatin densities and altered expression from the neighboring genes. Based on these results we propose a model integrating the CTCF130/180 transition with CTCF-DNA binding and gene expression changes. This study also issues an important cautionary note concerning the design and interpretation of any experiments using cells and tissues where CTCF180 may be present.
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Mama/metabolismo , Factor de Unión a CCCTC/metabolismo , Cromatina/metabolismo , Expresión Génica , Poli ADP Ribosilación , Mama/efectos de los fármacos , Línea Celular , ADN/química , Femenino , Humanos , Hidroxiurea/farmacología , Nocodazol/farmacología , Nucleosomas/metabolismo , Motivos de NucleótidosRESUMEN
INTRODUCTION: CTCF is a candidate tumor suppressor gene encoding a multifunctional transcription factor. CTCF function is controlled by posttranslational modification and interaction with other proteins. Research findings suggested that CTCF function can be regulated by poly(ADP-ribosyl)ation (PARlation) and has specific anti-apoptotic function in breast cancer cells. The aim of this study is to assess the effect of CTCF-wild type (WT) and CTCF complete mutant, which is deficient of PARlation in regulating apoptosis in breast cancer cells. MATERIALS AND METHODS: The effect of CTCF-WT and CTCF complete mutant was expressed in breast cancer cell-lines by DNA-mediated transfection technique monitored by enhanced green fluorescent protein fluorescence. Evaluation of apoptotic cell death was carried out with immunohistochemical staining using 4'-6'-diamino-2 phenylindole and scoring by fluorescent microscopy. RESULTS: CTCF-WT supports survival of breast cancer cells and was observed that CTCF complete mutant interferes with the functions of the CTCF-WT and there was a considerable apoptotic cell death in the breast cancer cell lines such as MDA-MB-435, CAMA-1 and MCF-7. CONCLUSION: The study enlighten CTCF as a Biological Marker for breast cancer and the role of CTCF PARlation may be involved in breast carcinogenesis.
RESUMEN
BACKGROUND: BORIS, a paralogue of the transcription factor CTCF, is a member of the cancer-testis antigen (CT) family. BORIS is normally present at high levels in the testis; however it is aberrantly expressed in various tumors and cancer cell lines. The main objectives of this study were to investigate BORIS expression together with sub-cellular localization in both prostate cell lines and tumor tissues, and assess correlations between BORIS and clinical/pathological characteristics. METHODS: We examined BORIS mRNA expression, protein levels and cellular localization in a panel of human prostate tissues, cancer and benign, together with a panel prostate cell lines. We also compared BORIS levels and localization with clinical/pathological characteristics in prostate tumors. RESULTS: BORIS was detected in all inspected prostate cancer cell lines and tumors, but was absent in benign prostatic hyperplasia. Increased levels of BORIS protein positively correlated with Gleason score, T-stage and androgen receptor (AR) protein levels in prostate tumors. The relationship between BORIS and AR was further highlighted in prostate cell lines by the ability of ectopically expressed BORIS to activate the endogenous AR mRNA and protein. BORIS localization in the nucleus plus cytoplasm was also associated with higher BORIS levels and Gleason score. CONCLUSIONS: Detection of BORIS in prostate tumors suggests potential applications of BORIS as a biomarker for prostate cancer diagnosis, as an immunotherapy target and, potentially, a prognostic marker of more aggressive prostate cancer. The ability of BORIS to activate the AR gene indicates BORIS involvement in the growth and development of prostate tumors.
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Adenocarcinoma/metabolismo , Biomarcadores de Tumor/metabolismo , Proteínas de Unión al ADN/metabolismo , Neoplasias de la Próstata/metabolismo , Adenocarcinoma/patología , Anciano , Línea Celular Tumoral , Humanos , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Próstata/metabolismo , Próstata/patología , Hiperplasia Prostática/metabolismo , Hiperplasia Prostática/patología , Neoplasias de la Próstata/patología , ARN Mensajero/metabolismo , Receptores Androgénicos/metabolismoRESUMEN
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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Neoplasias de la Mama/genética , Epigénesis Genética , Proteínas Represoras/genética , Proteína X Asociada a bcl-2/genética , Apoptosis/genética , Secuencia de Bases , Sitios de Unión/genética , Unión Competitiva , Western Blotting , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Factor de Unión a CCCTC , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Células K562 , Células MCF-7 , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Unión Proteica , Interferencia de ARN , Proteínas Represoras/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína X Asociada a bcl-2/metabolismoRESUMEN
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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Adenosina Difosfato Ribosa/metabolismo , Proteínas Represoras/metabolismo , Transporte Activo de Núcleo Celular , Sustitución de Aminoácidos , Animales , Factor de Unión a CCCTC , Línea Celular , Núcleo Celular/metabolismo , Ensamble y Desensamble de Cromatina , Metilación de ADN , Técnicas de Silenciamiento del Gen , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Laminas/metabolismo , Ratones , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Poli(ADP-Ribosa) Polimerasa-1 , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/genéticaRESUMEN
Generation of the mutated versions of proteins deficient for poly(ADP-ribosyl)ation (PARylation) is a major challenge because there is no clearly defined consensus site for PARylation. In this chapter, we describe possible strategies to produce such mutants, demonstrated by the example of CTCF, a transcription factor. To achieve this in our study, the protein domain modified by PARylation was mapped and the amino acids, which can be potentially PARylated, selected. Mutations of such individual amino acids as either single or combinatorial mutations were introduced by site-directed mutagenesis, using mutagenic primers and the wild-type sequences as a template. Mutants were validated by DNA sequencing and assessed for the presence of the PARylation mark. The latter was achieved by ectopic expression of mutated proteins in cells, followed by immunoprecipitation with the polyclonal anti-PAR antibody and Western analysis with a protein-specific antibody. The PARylation-deficient CTCF mutant was identified and compared with the wild-type protein. Based on several general characteristics (nuclear distribution/localisation, stability and levels of expression in the cell), the PARylation-deficient mutant was comparable with the wild-type CTCF.
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Poli Adenosina Difosfato Ribosa/metabolismo , Proteínas Represoras/metabolismo , Animales , Western Blotting , Factor de Unión a CCCTC , Línea Celular , Inmunoprecipitación de Cromatina , Humanos , Ratones , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa , Unión Proteica , Ratas , Proteínas Represoras/química , Proteínas Represoras/genéticaRESUMEN
Chromatin immunoprecipitation (ChIP) is an important technique in the study of DNA/protein interactions. The ChIP procedure, however, has limitations in that it is lengthy, can be inconsistent, and is prone to nonspecific binding of DNA and proteins to the bead-based solid-phase matrices that are often used for the immunoprecipitation step. In this investigation, we examined the utility of a new matrix for ChIP assays, BioVyon Protein A, a solid support based on porous polyethylene. In ChIP experiments carried out using two antibodies and seven DNA loci, the performance of BioVyon Protein A was significantly better, with a greater percentage of DNA pull-down in all of the assays tested compared with bead-based matrices, Protein A Sepharose, and Dynabeads Protein A. Furthermore, the rigid porous disc format within a column made the BioVyon matrix much easier to use with fewer steps and less equipment requirements, resulting in a significant reduction in the time taken to process the ChIP samples. In summary, BioVyon Protein A provides a column-based assay method for ChIP and other immunoprecipitation-based procedures; the rigid porous structure of BioVyon enables a fast and robust protocol with higher ChIP enrichment ratios.
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Inmunoprecipitación de Cromatina/métodos , Cromatografía de Afinidad , Polietilenos/metabolismo , Proteína Estafilocócica A/metabolismo , Animales , Línea Celular Tumoral , ADN/metabolismo , Humanos , Ratones , Células 3T3 NIH , Reacción en Cadena de la PolimerasaRESUMEN
The multifunctional zinc-finger protein CCCTC-binding factor (CTCF) is a very strong candidate for the role of coordinating the expression level of coding sequences with their three-dimensional position in the nucleus, apparently responding to a "code" in the DNA itself. Dynamic interactions between chromatin fibers in the context of nuclear architecture have been implicated in various aspects of genome functions. However, the molecular basis of these interactions still remains elusive and is a subject of intense debate. Here we discuss the nature of CTCF-DNA interactions, the CTCF-binding specificity to its binding sites and the relationship between CTCF and chromatin, and we examine data linking CTCF with gene regulation in the three-dimensional nuclear space. We discuss why these features render CTCF a very strong candidate for the role and propose a unifying model, the "CTCF code," explaining the mechanistic basis of how the information encrypted in DNA may be interpreted by CTCF into diverse nuclear functions.
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Núcleo Celular/genética , Núcleo Celular/metabolismo , Expresión Génica , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Animales , Secuencia de Bases , Sitios de Unión/genética , Factor de Unión a CCCTC , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Epigénesis Genética , Humanos , Ratones , Modelos Biológicos , Modelos Genéticos , Nucleosomas/genética , Nucleosomas/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteínas Represoras/química , CohesinasRESUMEN
Poly(ADP-ribosyl)ation of the conserved multifunctional transcription factor CTCF was previously identified as important to maintain CTCF insulator and chromatin barrier functions. However, the molecular mechanism of this regulation and also the necessity of this modification for other CTCF functions remain unknown. In this study, we identified potential sites of poly(ADP-ribosyl)ation within the N-terminal domain of CTCF and generated a mutant deficient in poly(ADP-ribosyl)ation. Using this CTCF mutant, we demonstrated the requirement of poly(ADP-ribosyl)ation for optimal CTCF function in transcriptional activation of the p19ARF promoter and inhibition of cell proliferation. By using a newly generated isogenic insulator reporter cell line, the CTCF insulator function at the mouse Igf2-H19 imprinting control region (ICR) was found to be compromised by the CTCF mutation. The association and simultaneous presence of PARP-1 and CTCF at the ICR, confirmed by single and serial chromatin immunoprecipitation assays, were found to be independent of CTCF poly(ADP-ribosyl)ation. These results suggest a model of CTCF regulation by poly(ADP-ribosyl)ation whereby CTCF and PARP-1 form functional complexes at sites along the DNA, producing a dynamic reversible modification of CTCF. By using bioinformatics tools, numerous sites of CTCF and PARP-1 colocalization were demonstrated, suggesting that such regulation of CTCF may take place at the genome level.
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Poli Adenosina Difosfato Ribosa/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión/genética , Factor de Unión a CCCTC , Línea Celular , Proliferación Celular , Cartilla de ADN/genética , Impresión Genómica , Células HeLa , Humanos , Células Híbridas , Factor II del Crecimiento Similar a la Insulina/genética , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/metabolismo , ARN Largo no Codificante , ARN no Traducido/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Represoras/química , TransfecciónRESUMEN
Two distinct variable number tandem repeats (VNTRs) within the human serotonin transporter gene (SLC6A4) have been implicated as predisposing factors for CNS disorders. The linked polymorphic region in the 5'-promoter exists as short (s) and long (l) alleles of a 22 or 23 bp elements. The second within intron 2 (Stin2) exists as three variants containing 9, 10 or 12 copies of a 16 or 17 bp element. These VNTRs, individually or in combination, supported differential reporter gene expression in rat neonate prefrontal cortical cultures. The level of reporter gene activity from the dual VNTR constructs indicated combinatorial action between the two domains. Chromatin immunoprecipitation demonstrated that both these VNTR domains can bind the CCCTC-binding factor and this correlated with the ability of exogenously supplied CCCTC-binding factor to modulate the expression supported by these reporter gene constructs. We suggest that the potential for interaction between multiple polymorphic domains should be incorporated into genetic association studies.
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Variación Genética/fisiología , Repeticiones de Minisatélite/fisiología , Proteínas Represoras/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Animales , Secuencia de Bases , Factor de Unión a CCCTC , Línea Celular Tumoral , Células Cultivadas , Técnicas Químicas Combinatorias , Humanos , Masculino , Datos de Secuencia Molecular , Unión Proteica/fisiología , Ratas , Ratas Wistar , Proteínas Represoras/fisiología , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genéticaRESUMEN
PURPOSE: There is compelling evidence of a relationship between poly(ADP-ribosyl)ation and tumorigenesis; however, much less is known about the role of specific targets of poly(ADP-ribosyl)ation in tumor development. Two forms of the multifunctional transcription factor, CTCF, were previously identified: a 130-kDa protein (CTCF-130), characteristic for cell lines, and a 180-kDa protein (CTCF-180), modified by poly(ADP-ribosyl)ation. This study was aimed to investigate differential poly(ADP-ribosyl)ation of CTCF in normal and tumor breast tissues. EXPERIMENTAL DESIGN: Western blot analysis, mass spectrometry, and immunohistochemical and immunofluorescent stainings were used to characterize CTCF-130 and CTCF-180 in breast cell lines, primary cultures, and normal and tumor breast tissues. The immunoreactivity score was used for CTCF-130 quantification in tissues. RESULTS: We discovered that only CTCF-180 is detected in the normal breast tissues, whereas both CTCF-130 and CTCF-180 are present in breast tumors. Using an antibody specific for CTCF-130, we observed that 87.7% of breast tumors were positive for CTCF-130. A negative correlation existed between the levels of CTCF-130, tumor stage, and tumor size. Significantly, a transition from CTCF-180 to CTCF-130 was discovered in primary cultures generated from normal breast tissues, indicating a link between CTCF-130 and proliferation. Conversely, the appearance of CTCF-180 was observed following growth arrest in breast cell lines. CONCLUSIONS: Collectively, our data suggest that the loss of CTCF poly(ADP-ribosyl)ation is associated with cell proliferation and breast tumor development. We propose the use of CTCF-130 as a marker for tumor breast cells and lower levels of CTCF-130 as an indicator of unfavorable prognosis.
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Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Poli Adenosina Difosfato Ribosa/metabolismo , Proteínas Represoras/metabolismo , Neoplasias de la Mama/enzimología , Factor de Unión a CCCTC , Proliferación Celular , Humanos , Fenotipo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Células Tumorales CultivadasRESUMEN
The imprinted insulin-like growth factor 2 (IGF2) gene is expressed predominantly from the paternal allele. Loss of imprinting (LOI) associated with hypomethylation at the promoter proximal sequence (DMR0) of the IGF2 gene was proposed as a predisposing constitutive risk biomarker for colorectal cancer. We used pyrosequencing to assess whether IGF2 DMR0 methylation is either present constitutively prior to cancer or whether it is acquired tissue-specifically after the onset of cancer. DNA samples from tumour tissues and matched non-tumour tissues from 22 breast and 42 colorectal cancer patients as well as peripheral blood samples obtained from colorectal cancer patients [SEARCH (n=case 192, controls 96)], breast cancer patients [ABC (n=case 364, controls 96)] and the European Prospective Investigation of Cancer [EPIC-Norfolk (n=breast 228, colorectal 225, controls 895)] were analysed. The EPIC samples were collected 2-5 years prior to diagnosis of breast or colorectal cancer. IGF2 DMR0 methylation levels in tumours were lower than matched non-tumour tissue. Hypomethylation of DMR0 was detected in breast (33%) and colorectal (80%) tumour tissues with a higher frequency than LOI indicating that methylation levels are a better indicator of cancer than LOI. In the EPIC population, the prevalence of IGF2 DMR0 hypomethylation was 9.5% and this correlated with increased age not cancer risk. Thus, IGF2 DMR0 hypomethylation occurs as an acquired tissue-specific somatic event rather than a constitutive innate epimutation. These results indicate that IGF2 DMR0 hypomethylation has diagnostic potential for colon cancer rather than value as a surrogate biomarker for constitutive LOI.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias Colorrectales/genética , Metilación de ADN , Regulación Neoplásica de la Expresión Génica , Factor II del Crecimiento Similar a la Insulina/genética , Estudios de Casos y Controles , Femenino , Impresión Genómica , Humanos , Factor II del Crecimiento Similar a la Insulina/metabolismoRESUMEN
The serotoninergic pathways are possible targets for the action of lithium, a therapeutic agent for treatment of bipolar affective disorders. This study aimed to investigate the molecular mechanisms regulating human serotonin transporter gene (SLC6A4) expression by lithium and, specifically, the role of the variable number tandem repeat (VNTR) polymorphic region in intron 2, which is potentially a predisposing genetic factor for bipolar affective disorders. We demonstrated that addition of lithium to human JAr cells led to changes in the levels of SLC6A4 mRNA and protein. Additional investigations revealed that the intron 2 VNTR domain was a potential target for mediation of a transcriptional response to lithium. Properties of two transcription factors, CCCTC binding protein (CTCF) and Y-box binding protein 1 (YB-1), previously shown to be involved in the regulation of SLC6A4 VNTR, were found to be modulated by LiCl. Thus, levels of CTCF and YB-1 mRNA and protein were altered in vivo in response to LiCl. Furthermore, CTCF and YB-1 showed differential binding to the polymorphic alleles of the VNTR on exposure to LiCl. Our data suggest a model in which differential binding of CTCF and YB-1 to the allelic variants of the intron 2 VNTR can be regulated by lithium and in part result in differential and even aberrant expression of SLC6A4. Our study of the regulation of the SLC6A4 VNTR by lithium may improve the understanding of psychiatric disorders and enable the development of novel therapies for conditions such as bipolar affective disorder to target only the at-risk allele.
Asunto(s)
Proteínas de Unión al ADN/genética , Litio/farmacología , Repeticiones de Minisatélite/genética , Polimorfismo Genético/genética , Proteínas Represoras/genética , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Proteína 1 de Unión a la Caja Y/genética , Factor de Unión a CCCTC , Línea Celular , Proteínas de Unión al ADN/biosíntesis , Humanos , Intrones/efectos de los fármacos , Intrones/genética , Repeticiones de Minisatélite/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Proteínas Represoras/biosíntesis , Proteínas de Transporte de Serotonina en la Membrana Plasmática/biosíntesis , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Proteína 1 de Unión a la Caja Y/biosíntesisRESUMEN
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.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Genoma Humano , ARN Polimerasa II/metabolismo , Proteínas Represoras/metabolismo , Animales , Sitios de Unión , Neoplasias de la Mama/patología , Factor de Unión a CCCTC , Línea Celular Tumoral , Núcleo Celular/metabolismo , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/química , Genes Reporteros , Células HeLa , Humanos , Inmunohistoquímica , Células K562 , Ratones , Células 3T3 NIH , Análisis de Secuencia por Matrices de Oligonucleótidos , Estructura Terciaria de Proteína , ARN Polimerasa II/química , ARN Polimerasa II/genética , Proteínas Represoras/química , TransfecciónRESUMEN
We have shown previously that AWT1 and WT1-AS are functionally imprinted in human kidney. In the adult kidney, expression of both transcripts is restricted to the paternal allele, with the silent maternal allele retaining methylation at the WT1 antisense regulatory region (WT1 ARR). Here, we report characterization of the WT1 ARR differentially methylated region and show that it contains a transcriptional silencer element acting on both the AWT1 and WT1-AS promoters. DNA methylation of the silencer results in increased transcriptional repression, and the silencer is also shown to be an in vitro and in vivo target site for the imprinting regulator protein CTCF. Binding of CTCF is methylation-sensitive and limited to the unmethylated silencer. Potentiation of the silencer activity is demonstrated after CTCF protein is knocked down, suggesting a novel silencer-blocking activity for CTCF. We also report assessment of WT1 ARR methylation in developmental and tumour tissues, including the first analysis of Wilms' tumour precursor lesions, nephrogenic rests. Nephrogenic rests show increases in methylation levels relative to foetal kidney and reductions relative to the adult kidney, together with biallelic expression of AWT1 and WT1-AS. Notably, the methylation status of CpG residues within the CTCF target site appears to distinguish monoallelic and biallelic expression states. Our data suggest that failure of methylation spreading at the WT1 ARR early in renal development, followed by imprint erasure, occurs during Wilms' tumourigenesis. We propose a model wherein imprinting defects at chromosome 11p13 may contribute to Wilms' tumourigenesis.