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.
Asunto(s)
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.
Asunto(s)
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.
Asunto(s)
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.
Asunto(s)
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
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.
Asunto(s)
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
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.
Asunto(s)
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
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
PURPOSE: Brother of the regulator of imprinted sites (BORIS) is a novel member of the cancer-testis antigen gene family. These genes are normally expressed only in spermatocytes but abnormally activated in different malignancies, including breast cancer. The aim of this study was to investigate the expression of BORIS in the leukocytes of breast cancer patients and the correlation between BORIS levels and clinical/pathologic variables. EXPERIMENTAL DESIGN: Leukocytes were obtained from whole blood of 87 breast cancer patients and 52 donors not diagnosed with cancer. BORIS protein was detected in leukocytes by immunohistochemical staining; the immunoreactivity score (IRS) of each sample was determined. Additionally, BORIS expression was assessed by Western blot analysis and real-time reverse transcription-PCR. RESULTS: We describe significantly high levels of BORIS (IRS = 4.25 +/- 0.034) in a subpopulation of leukocytes, the neutrophil polymorphonuclear granulocytes, in 88.5% of breast cancer patients. Increased IRS for BORIS in these patients correlated with increased tumor size. In comparison, 19.2% samples from the control group were BORIS positive with only very low levels of BORIS (IRS = 0.25 +/- 0.009). CONCLUSION: We report here the novel finding of BORIS expression in polymorphonuclear granulocytes of breast cancer patients. This tumor-related occurrence is a phenomenon not observed in donors with injuries and immune and inflammatory diseases. Detection of BORIS in a high proportion of patients with various types of breast tumors indicates that BORIS can be a valuable early blood marker of breast cancer. We conclude that BORIS represents a new class of cancer biomarkers different from those currently used in medical practice.
Asunto(s)
Biomarcadores de Tumor/sangre , Neoplasias de la Mama Masculina/sangre , Neoplasias de la Mama/sangre , Proteínas de Unión al ADN/sangre , Leucocitos/química , Adulto , Anciano , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/patología , Neoplasias de la Mama Masculina/diagnóstico , Neoplasias de la Mama Masculina/patología , Femenino , Humanos , Enfermedades del Sistema Inmune/sangre , Inflamación/sangre , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Valores de ReferenciaRESUMEN
Multiple functions have been reported for the transcription factor and candidate tumour suppressor, CTCF. Among others, they include regulation of cell growth, differentiation and apoptosis, enhancer-blocking activity and control of imprinted genes. CTCF is usually localized in the nucleus and its subcellular distribution during the cell cycle is dynamic; CTCF was found associated with mitotic chromosomes and the midbody, suggesting different roles for CTCF at different stages of the cell cycle. Here we report the nucleolar localization of CTCF in several experimental model systems. Translocation of CTCF from nucleoplasm to the nucleolus was observed after differentiation of K562 myeloid cells and induction of apoptosis in MCF7 breast cancer cells. CTCF was also found in the nucleoli in terminally differentiated rat trigeminal ganglion neurons. Thus our data show that nucleolar localization of CTCF is associated with growth arrest. Interestingly, the 180 kDa poly(ADP-ribosyl)ated isoform of CTCF was predominantly found in the nucleoli fractions. By transfecting different CTCF deletion constructs into cell lines of different origin we demonstrate that the central zinc-finger domain of CTCF is the region responsible for nucleolar targeting. Analysis of subnucleolar localization of CTCF revealed that it is distributed homogeneously in both dense fibrillar and granular components of the nucleolus, but is not associated with fibrillar centres. RNA polymerase I transcription and protein synthesis were required to sustain nucleolar localization of CTCF. Notably, the labelling of active transcription sites by in situ run-on assays demonstrated that CTCF inhibits nucleolar transcription through a poly(ADP-ribosyl)ation-dependent mechanism.
Asunto(s)
Nucléolo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Poli Adenosina Difosfato Ribosa/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas/metabolismo , Proteínas Represoras/metabolismo , Transcripción Genética , Animales , Factor de Unión a CCCTC , Diferenciación Celular/fisiología , Línea Celular Tumoral , Nucléolo Celular/ultraestructura , Células Cultivadas , Proteínas de Unión al ADN/genética , Humanos , Masculino , Neuronas Aferentes/citología , Neuronas Aferentes/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Señales de Clasificación de Proteína , Transporte de Proteínas/fisiología , ARN Polimerasa I/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/genética , Ganglio del Trigémino/citología , Dedos de ZincRESUMEN
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.
Asunto(s)
Apoptosis/fisiología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteínas de Unión al ADN/biosíntesis , Proteínas Represoras/biosíntesis , Neoplasias de la Mama/genética , Factor de Unión a CCCTC , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Regulación hacia Abajo , Humanos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Proteínas Represoras/genética , Transfección , Proteína X Asociada a bcl-2RESUMEN
Chromatin insulators demarcate expression domains by blocking the cis effects of enhancers or silencers in a position-dependent manner. We show that the chromatin insulator protein CTCF carries a post-translational modification: poly(ADP-ribosyl)ation. Chromatin immunoprecipitation analysis showed that a poly(ADP-ribosyl)ation mark, which exclusively segregates with the maternal allele of the insulator domain in the H19 imprinting control region, requires the bases that are essential for interaction with CTCF. Chromatin immunoprecipitation-on-chip analysis documented that the link between CTCF and poly(ADP-ribosyl)ation extended to more than 140 mouse CTCF target sites. An insulator trap assay showed that the insulator function of most of these CTCF target sites is sensitive to 3-aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase activity. We suggest that poly(ADP-ribosyl)ation imparts chromatin insulator properties to CTCF at both imprinted and nonimprinted loci, which has implications for the regulation of expression domains and their demise in pathological lesions.