RESUMEN
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
PARylation [poly(ADP-ribosyl)ation] is involved in the maintenance of genomic methylation patterns through its control of Dnmt1 [DNA (cytosine-5)-methyltransferase 1] activity. Our previous findings indicated that Ctcf (CCCTC-binding factor) may be an important player in key events whereby PARylation controls the unmethylated status of some CpG-rich regions. Ctcf is able to activate Parp1 [poly(ADP-ribose) polymerase 1], which ADP-ribosylates itself and, in turn, inhibits DNA methylation via non-covalent interaction between its ADP-ribose polymers and Dnmt1. By such a mechanism, Ctcf may preserve the epigenetic pattern at promoters of important housekeeping genes. The results of the present study showed Dnmt1 as a new protein partner of Ctcf. Moreover, we show that Ctcf forms a complex with Dnmt1 and PARylated Parp1 at specific Ctcf target sequences and that PARylation is responsible for the maintenance of the unmethylated status of some Ctcf-bound CpGs. We suggest a mechanism by which Parp1, tethered and activated at specific DNA target sites by Ctcf, preserves their methylation-free status.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/metabolismo , Poli Adenosina Difosfato Ribosa/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Represoras/metabolismo , Factor de Unión a CCCTC , Islas de CpG/fisiología , ADN (Citosina-5-)-Metiltransferasa 1 , Metilación de ADN , Epigénesis Genética , Complejos Multiproteicos/metabolismoRESUMEN
Although the existence of histone variants has been known for quite some time, only recently are we grasping the breadth and diversity of the cellular processes in which they are involved. Of particular interest are the two variants of histone H2A, H2A.Z and H2A.X because of their roles in regulation of gene expression and in DNA double-strand break repair, respectively. We hypothesize that nucleosomes containing these variants may perform their distinct functions by interacting with different sets of proteins. Here, we present our proteome analysis aimed at identifying protein partners that interact with nucleosomes containing H2A.Z, H2A.X or their canonical H2A counterpart. Our development of a nucleosome-pull down assay and analysis of the recovered nucleosome-interacting proteins by mass spectrometry allowed us to directly compare nuclear partners of these variant-containing nucleosomes to those containing canonical H2A. To our knowledge, our data represent the first systematic analysis of the H2A.Z and H2A.X interactome in the context of nucleosome structure.
Asunto(s)
Histonas/metabolismo , Proteínas Nucleares/análisis , Proteínas Nucleares/metabolismo , Nucleosomas/química , Nucleosomas/metabolismo , Proteoma/análisis , Variación Genética/genética , Células HeLa , Histonas/química , Histonas/genética , Histonas/aislamiento & purificación , Humanos , Proteoma/metabolismoRESUMEN
BACKGROUND: Peptidylarginine deiminase 2 (PAD2) and peptidylarginine deiminase 4 (PAD4) are two members of PAD family which are over-expressed in the multiple sclerosis (MS) brain. Through its enzymatic activity PAD2 converts myelin basic protein (MBP) arginines into citrullines - an event that may favour autoimmunity - while peptidylarginine deiminase 4 (PAD4) is involved in chromatin remodelling. OBJECTIVES: Our aim was to verify whether an altered epigenetic control of PAD2, as already shown in the MS brain, can be observed in peripheral blood mononuclear cells (PBMCs) of patients with MS since some of these cells also synthesize MBP. METHODS: The expression of most suitable reference genes and of PAD2 and PAD4 was assessed by qPCR. Analysis of DNA methylation was performed by bisulfite method. RESULTS: The comparison of PAD2 expression level in PBMCs from patients with MS vs. healthy donors showed that, as well as in the white matter of MS patients, the enzyme is significantly upregulated in affected subjects. Methylation pattern analysis of a CpG island located in the PAD2 promoter showed that over-expression is associated with promoter demethylation. CONCLUSION: Defective regulation of PAD2 in the periphery, without the immunological shelter of the blood-brain barrier, may contribute to the development of the autoimmune responses in MS.
Asunto(s)
Hidrolasas/genética , Leucocitos Mononucleares/enzimología , Esclerosis Múltiple/genética , Adulto , Encéfalo/enzimología , Encéfalo/metabolismo , Islas de CpG/genética , Metilación de ADN , Femenino , Humanos , Hidrolasas/sangre , Hidrolasas/metabolismo , Leucocitos Mononucleares/metabolismo , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/enzimología , Proteína Básica de Mielina/genética , Proteína Básica de Mielina/metabolismo , Arginina Deiminasa Proteína-Tipo 2 , Desiminasas de la Arginina Proteica , Regulación hacia ArribaRESUMEN
The capability of PARP activity inhibitors to prevent DNA damage recovery suggested the use of these drugs as chemo- and radio-sensitisers for cancer therapy. Our research, carried out on cultured human M14 melanoma cells, was aimed to examine if PJ-34, a potent PARP activity inhibitor of second generation, was per se able to affect the viability of these cancer cells without any DNA damaging agents. Using time-lapse videomicroscopy, we evidenced that 10 microM PJ-34 treatment induced severe mitotic defects leading to dramatic reduction of cell proliferation and to cell death. PJ-34 cytotoxic effect was further confirmed by analysis of cell viability and clonogenic assay. Absence of canonic apoptosis markers allowed us to exclude this kind of cell death. No single and/or double stranded DNA damage was evidenced. Immunofluorescence analysis showed an aberrant mitotic scenario in several cells and subsequent multinucleation suggesting an atypical way for cells to die: the mitotic catastrophe. The detection of aberrant accumulation of polymerised actin inside the nucleolus was noteworthy. Taken together, our results demonstrate that, targeting PARP activity by PJ-34, cancer cell survival is affected independently of DNA damage repair. Two findings are remarkable: (a) cisplatin concentration can be reduced by three quarters if it is followed by treatment with 10 microM PJ-34 for 24 h to obtain the same cytotoxic effect; (b) effects dependent on PJ-34 treatment are reversible. Our data suggest that, to reduce the harm done to non-tumour cells during chemotherapy with cisplatin, the latter could be coupled with PJ-34 treatment.
Asunto(s)
Actinas/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Nucléolo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Melanoma/enzimología , Mitosis/efectos de los fármacos , Fenantrenos/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Muerte Celular , Línea Celular Tumoral , Nucléolo Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Cisplatino/farmacología , Relación Dosis-Respuesta a Droga , Técnica del Anticuerpo Fluorescente , Humanos , Melanoma/patología , Microscopía por Video , Poli(ADP-Ribosa) Polimerasas/metabolismo , Factores de TiempoRESUMEN
In the postgenome era, attention is being focused on those epigenetic modifications that modulate chromatin structure to guarantee that information present on DNA is read correctly and at the most appropriate time to meet cellular requirements. Data reviewed show that along the chain of events that induce DNA methylation-dependent chromatin condensation/decondensation, a postsynthetic modification other than histone acetylation, phosphorylation, and methylation--namely poly(ADP-ribosyl)ation (PARylation)--participates in the establishment and maintenance of a genome methylation pattern. We hypothesize that the right nuclear balance between unmodified and PARylated poly(ADP-ribose) polymerase 1 (PARP-1), which depends on the dynamics of PARPs/PARG activity, is key to maintaining genomic methylation pattern. According to our data, decreased or increased levels of PARylated PARP-1 are responsible for diffuse hypermethylation or hypomethylation of DNA, respectively. In our model, polymers present on PARP-1 interact noncovalently with DNA methyltransferase 1 (Dnmt1), preventing its enzymatic activity. In the absence of PARylated PARP-1, Dnmt1 is free to methylate DNA; if, in contrast, high levels of PARylated PARP-1 persist, Dnmt1 will be stably inhibited, preventing DNA methylation.
Asunto(s)
Epigénesis Genética , Poli Adenosina Difosfato Ribosa/metabolismo , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas/metabolismo , Metilación de ADN , Genómica , Transcripción GenéticaRESUMEN
Down syndrome (DS) is caused by the presence of part or an entire extra copy of chromosome 21, a phenomenon that can cause a wide spectrum of clinically defined phenotypes of the disease. Most of the clinical signs of DS are typical of the aging process including dysregulation of immune system. Beyond the causative genetic defect, DS persons display epigenetic alterations, particularly aberrant DNA methylation patterns that can contribute to the heterogeneity of the disease. In the present work, we investigated the levels of 5-hydroxymethylcytosine and of the Ten-eleven translocation dioxygenase enzymes, which are involved in DNA demethylation processes and are often deregulated in pathological conditions as well as in aging. Analyses were carried out on peripheral blood mononuclear cells of DS volunteers enrolled in the context of the MARK-AGE study, a large-scale cross-sectional population study with subjects representing the general population in eight European countries. We observed a decrease in 5-hydroxymethylcytosine, TET1, and other components of the DNA methylation/demethylation machinery in DS subjects, indicating that aberrant DNA methylation patterns in DS, which may have consequences on the transcriptional status of immune cells, may be due to a global disturbance of methylation control in DS.
Asunto(s)
Envejecimiento/sangre , Envejecimiento/genética , Metilación de ADN , Síndrome de Down/sangre , Síndrome de Down/genética , Leucocitos Mononucleares/metabolismo , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/sangre , Adulto , Anciano , Estudios Transversales , Epigénesis Genética , Europa (Continente) , Femenino , Humanos , Immunoblotting , Italia , Masculino , Persona de Mediana Edad , Oxigenasas de Función Mixta/sangre , Proteínas Proto-Oncogénicas/sangre , ARN Mensajero/sangreRESUMEN
Gradual changes in the DNA methylation landscape occur throughout aging virtually in all human tissues. A widespread reduction of 5-methylcytosine (5mC), associated with highly reproducible site-specific hypermethylation, characterizes the genome in aging. Therefore, an equilibrium seems to exist between general and directional deregulating events concerning DNA methylation controllers, which may underpin the age-related epigenetic changes. In this context, 5mC-hydroxylases (TET enzymes) are new potential players. In fact, TETs catalyze the stepwise oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), driving the DNA demethylation process based on thymine DNA glycosylase (TDG)-mediated DNA repair pathway. The present paper reports the expression of DNA hydroxymethylation components, the levels of 5hmC and of its derivatives in peripheral blood mononuclear cells of age-stratified donors recruited in several European countries in the context of the EU Project 'MARK-AGE'. The results provide evidence for an age-related decline of TET1, TET3 and TDG gene expression along with a decrease of 5hmC and an accumulation of 5caC. These associations were independent of confounding variables, including recruitment center, gender and leukocyte composition. The observed impairment of 5hmC-mediated DNA demethylation pathway in blood cells may lead to aberrant transcriptional programs in the elderly.
Asunto(s)
5-Metilcitosina/metabolismo , Envejecimiento/genética , Metilación de ADN , Dioxigenasas/genética , Regulación de la Expresión Génica , Oxigenasas de Función Mixta/genética , Proteínas Proto-Oncogénicas/genética , Adulto , Anciano , Envejecimiento/metabolismo , Dioxigenasas/metabolismo , Femenino , Expresión Génica , Humanos , Leucocitos Mononucleares/metabolismo , Masculino , Persona de Mediana Edad , Oxigenasas de Función Mixta/metabolismo , Proteínas Proto-Oncogénicas/metabolismoRESUMEN
Aging is associated with alterations in the content and patterns of DNA methylation virtually throughout the entire human lifespan. Reasons for these variations are not well understood. However, several lines of evidence suggest that the epigenetic instability in aging may be traced back to the alteration of the expression of DNA methyltransferases. Here, the association of the expression of DNA methyltransferases DNMT1 and DNMT3B with age has been analysed in the context of the MARK-AGE study, a large-scale cross-sectional study of the European general population. Using peripheral blood mononuclear cells, we assessed the variation of DNMT1 and DNMT3B gene expression in more than two thousand age-stratified women and men (35-75 years) recruited across eight European countries. Significant age-related changes were detected for both transcripts. The level of DNMT1 gradually dropped with aging but this was only observed up to the age of 64 years. By contrast, the expression of DNMT3B decreased linearly with increasing age and this association was particularly evident in females. We next attempted to trace the age-related changes of both transcripts to the influence of different variables that have an impact on changes of their expression in the population, including demographics, dietary and health habits, and clinical parameters. Our results indicate that age affects the expression of DNMT1 and DNMT3B as an almost independent variable in respect of all other variables evaluated.
Asunto(s)
Envejecimiento/genética , ADN (Citosina-5-)-Metiltransferasas/genética , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Leucocitos Mononucleares/enzimología , Población Blanca/genética , Adulto , Anciano , Índice de Masa Corporal , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Femenino , Ontología de Genes , Humanos , Estilo de Vida , Masculino , Persona de Mediana Edad , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Regresión , Factores de Riesgo , ADN Metiltransferasa 3BRESUMEN
To overcome cancer cells resistance to pharmacological therapy, the development of new therapeutic approaches becomes urgent. For this purpose, the use of poly(ADP-ribose) polymerase (PARP) inhibitors in combination with other cytotoxic agents could represent an efficacious strategy. Poly(ADP-ribosyl)ation (PARylation) is a post-translational modification that plays a well characterized role in the cellular decisions of life and death. Recent findings indicate that PARP-1 may control the expression of Snail, the master gene of epithelial-mesenchymal transition (EMT). Snail is highly represented in different resistant tumors, functioning as a factor regulating anti-apoptotic programmes. MDA-MB-231 is a Snail-expressing metastatic breast cancer cell line, which exhibits chemoresistance properties when treated with damaging agents. In this study, we show that the PARP inhibitor ABT-888 was capable to modulate the MDA-MB-231 cell response to doxorubicin, leading to an increase in the rate of apoptosis. Our further results indicate that PARP-1 controlled Snail expression at transcriptional level in cells exposed to doxorubicin. Given the increasing interest in the employment of PARP inhibitors as chemotherapeutic adjuvants, our in vitro results suggest that one of the mechanisms through which PARP inhibition can chemosensitize cancer cells in vivo, is targeting Snail expression thus promoting apoptosis.
Asunto(s)
Bencimidazoles/farmacología , Doxorrubicina/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Factores de Transcripción/metabolismo , Antibióticos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Western Blotting , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Células MCF-7 , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/genética , Regiones Promotoras Genéticas/genética , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción de la Familia Snail , Factores de Transcripción/genéticaRESUMEN
TET enzymes are the epigenetic factors involved in the formation of the sixth DNA base 5-hydroxymethylcytosine, whose deregulation has been associated with tumorigenesis. In particular, TET1 acts as tumor suppressor preventing cell proliferation and tumor metastasis and it has frequently been found down-regulated in cancer. Thus, considering the importance of a tight control of TET1 expression, the epigenetic mechanisms involved in the transcriptional regulation of TET1 gene are here investigated. The involvement of poly(ADP-ribosyl)ation in the control of DNA and histone methylation on TET1 gene was examined. PARP activity is able to positively regulate TET1 expression maintaining a permissive chromatin state characterized by DNA hypomethylation of TET1 CpG island as well as high levels of H3K4 trimethylation. These epigenetic modifications were affected by PAR depletion causing TET1 down-regulation and in turn reduced recruitment of TET1 protein on HOXA9 target gene. In conclusion, this work shows that PARP activity is a transcriptional regulator of TET1 gene through the control of epigenetic events and it suggests that deregulation of these mechanisms could account for TET1 repression in cancer.
Asunto(s)
Neoplasias de la Mama/genética , Proteínas de Unión al ADN/metabolismo , Epigénesis Genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Proteínas Proto-Oncogénicas/metabolismo , Adenosina Difosfato/metabolismo , Metilación de ADN/genética , Proteínas de Unión al ADN/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Histonas/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Células Jurkat , Células MCF-7 , Oxigenasas de Función Mixta , Poli(ADP-Ribosa) Polimerasas/genética , Proteínas Proto-Oncogénicas/genética , Transcripción Genética/genéticaRESUMEN
Poly(ADP-ribose) polymerase 1 (PARP-1) catalyzes a post-translational modification that plays a crucial role in coordinating the signalling cascade in response to stress stimuli. During the DNA damage response, phosphorylation by ataxia telangiectasia mutated (ATM) kinase and checkpoint kinase Chk2 induces the stabilization of Che-1 protein, which is critical for the maintenance of G2/M arrest. In this study we showed that poly(ADP-ribosyl)ation, beyond phosphorylation, is involved in the regulation of Che-1 stabilization following DNA damage. We demonstrated that Che-1 accumulation upon doxorubicin treatment is reduced after the inhibition of PARP activity in HCT116 cells and in PARP-1 knock-out or silenced cells. In accordance, impairment in Che-1 accumulation by PARP inhibition reduced Che-1 occupancy at p21 promoter and affected the expression of the corresponding gene. Epistasis experiments showed that the effect of poly(ADP-ribosyl)ation on Che-1 stabilization is independent from ATM kinase activity. Indeed we demonstrated that Che-1 protein co-immunoprecipitates with ADP-ribose polymers and that PARP-1 directly interacts with Che-1, promoting its modification in vitro and in vivo.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Daño del ADN , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Animales , Antineoplásicos/farmacología , Proteínas Reguladoras de la Apoptosis/genética , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Daño del ADN/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Activación Enzimática/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Estabilidad Proteica , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
BACKGROUND: Aberrant hypermethylation of CpG islands in housekeeping gene promoters and widespread genome hypomethylation are typical events occurring in cancer cells. The molecular mechanisms behind these cancer-related changes in DNA methylation patterns are not well understood. Two questions are particularly important: (i) how are CpG islands protected from methylation in normal cells, and how is this protection compromised in cancer cells, and (ii) how does the genome-wide demethylation in cancer cells occur. The latter question is especially intriguing since so far no DNA demethylase enzyme has been found. METHODOLOGY/PRINCIPAL FINDINGS: Our data show that the absence of ADP-ribose polymers (PARs), caused by ectopic over-expression of poly(ADP-ribose) glycohydrolase (PARG) in L929 mouse fibroblast cells leads to aberrant methylation of the CpG island in the promoter of the Dnmt1 gene, which in turn shuts down its transcription. The transcriptional silencing of Dnmt1 may be responsible for the widespread passive hypomethylation of genomic DNA which we detect on the example of pericentromeric repeat sequences. Chromatin immunoprecipitation results show that in normal cells the Dnmt1 promoter is occupied by poly(ADP-ribosyl)ated Parp1, suggesting that PARylated Parp1 plays a role in protecting the promoter from methylation. CONCLUSIONS/SIGNIFICANCE: In conclusion, the genome methylation pattern following PARG over-expression mirrors the pattern characteristic of cancer cells, supporting our idea that the right balance between Parp/Parg activities maintains the DNA methylation patterns in normal cells. The finding that in normal cells Parp1 and ADP-ribose polymers localize on the Dnmt1 promoter raises the possibility that PARylated Parp1 marks those sequences in the genome that must remain unmethylated and protects them from methylation, thus playing a role in the epigenetic regulation of gene expression.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/genética , Metilación de ADN , Epigénesis Genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Regiones Promotoras Genéticas , Animales , Línea Celular , ADN (Citosina-5-)-Metiltransferasa 1 , Fibroblastos , Genoma , Glicósido Hidrolasas/metabolismo , Ratones , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/genéticaRESUMEN
Our previous data have shown that in L929 mouse fibroblasts the control of methylation pattern depends in part on poly(ADP-ribosyl)ation and that ADP-ribose polymers (PARs), both present on poly(ADP-ribosyl)ated PARP-1 and/or protein-free, have an inhibitory effect on Dnmt1 activity. Here we show that transient ectopic overexpression of CCCTC-binding factor (CTCF) induces PAR accumulation, PARP-1, and CTCF poly(ADP-ribosyl)ation in the same mouse fibroblasts. The persistence in time of a high PAR level affects the DNA methylation machinery; the DNA methyltransferase activity is inhibited with consequences for the methylation state of genome, which becomes diffusely hypomethylated affecting centromeric minor satellite and B1 DNA repeats. In vitro data show that CTCF is able to activate PARP-1 automodification even in the absence of nicked DNA. Our new finding that CTCF is able per se to activate PARP-1 automodification in vitro is of great interest as so far a burst of poly(ADP-ribosyl)ated PARP-1 has generally been found following introduction of DNA strand breaks. CTCF is unable to inhibit DNMT1 activity, whereas poly(ADP-ribosyl)ated PARP-1 plays this inhibitory role. These data suggest that CTCF is involved in the cross-talk between poly(ADP-ribosyl)ation and DNA methylation and underscore the importance of a rapid reversal of PARP activity, as DNA methylation pattern is responsible for an important epigenetic code.