RESUMEN
DNA methylation and histone modifications are key epigenetic regulators of gene expression, and tight connections are known between the two. DNA methyltransferases are upregulated in several tumors and aberrant DNA methylation profiles are a cancer hallmark. On the other hand, histone demethylases are upregulated in cancer cells. Previous work on ES cells has shown that the lysine demethylase KDM1A binds to DNMT1, thereby affecting DNA methylation. In cancer cells, the occurrence of this interaction has not been explored. Here we demonstrate in several tumor cell lines an interaction between KDM1A and both DNMT1 and DNMT3B. Intriguingly and in contrast to what is observed in ES cells, KDM1A depletion in cancer cells was found not to trigger any reduction in the DNMT1 or DNMT3B protein level or any change in DNA methylation. In the S-phase, furthermore, KDM1A and DNMT1 were found, to co-localize within the heterochromatin. Using P-LISA, we revealed substantially increased binding of KDM1A to DNMT1 during the S-phase. Together, our findings propose a mechanistic link between KDM1A and DNA methyltransferases in cancer cells and suggest that the KDM1A/DNMT1 interaction may play a role during replication. Our work also strengthens the idea that DNMTs can exert functions unrelated to act on DNA methylation.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Histona Demetilasas/metabolismo , Neoplasias/metabolismo , Puntos de Control de la Fase S del Ciclo Celular , Animales , Carcinogénesis , Metilación de ADN , Células HeLa , Histona Demetilasas/genética , Histonas/metabolismo , Humanos , Lisina , Ratones , Células 3T3 NIH , Unión Proteica , ADN Metiltransferasa 3BRESUMEN
In plants, as in mammals, mutations in SNF2-like DNA helicases/ATPases were shown to affect not only chromatin structure but also global methylation patterns, suggesting a potential functional link between chromatin structure and epigenetic marks. The SNF2-like ATPase containing nucleosome remodeling and deacetylase corepressor complex (NuRD) is involved in gene transcriptional repression and chromatin remodeling. We have previously shown that the leukemogenic protein PML-RARa represses target genes through recruitment of DNA methytransferases and Polycomb complex. Here, we demonstrate a direct role of the NuRD complex in aberrant gene repression and transmission of epigenetic repressive marks in acute promyelocytic leukemia (APL). We show that PML-RARa binds and recruits NuRD to target genes, including to the tumor-suppressor gene RARbeta2. In turn, the NuRD complex facilitates Polycomb binding and histone methylation at lysine 27. Retinoic acid treatment, which is often used for patients at the early phase of the disease, reduced the promoter occupancy of the NuRD complex. Knockdown of the NuRD complex in leukemic cells not only prevented histone deacetylation and chromatin compaction but also impaired DNA and histone methylation, as well as stable silencing, thus favoring cellular differentiation. These results unveil an important role for NuRD in the establishment of altered epigenetic marks in APL, demonstrating an essential link between chromatin structure and epigenetics in leukemogenesis that could be exploited for therapeutic intervention.
Asunto(s)
Ensamble y Desensamble de Cromatina , Proteínas de Unión al ADN/metabolismo , Epigénesis Genética , Histona Desacetilasas/metabolismo , Leucemia Promielocítica Aguda/genética , Línea Celular Tumoral , Humanos , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2 , Proteínas de Fusión Oncogénica/metabolismoRESUMEN
It is well established that two marks of silent chromatin, DNA methylation and histone H3 methylation at lysine 9, engage in an epigenetic conversation. In a recent issue of Genes & Development, Smallwood et al. (2007) report that the mammalian HP1 adaptor "translates" methylation information from histone to DNA, helping to cement epigenetic expression states.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Silenciador del Gen , Histonas/metabolismo , Lisina/metabolismo , Metilación , Transcripción Genética/genética , Acetilación , Cromatina , Homólogo de la Proteína Chromobox 5 , HumanosRESUMEN
The establishment and maintenance of epigenetic gene silencing is fundamental to cell determination and function. The essential epigenetic systems involved in heritable repression of gene activity are the Polycomb group (PcG) proteins and the DNA methylation systems. Here we show that the corresponding silencing pathways are mechanistically linked. We find that the PcG protein EZH2 (Enhancer of Zeste homolog 2) interacts-within the context of the Polycomb repressive complexes 2 and 3 (PRC2/3)-with DNA methyltransferases (DNMTs) and associates with DNMT activity in vivo. Chromatin immunoprecipitations indicate that binding of DNMTs to several EZH2-repressed genes depends on the presence of EZH2. Furthermore, we show by bisulphite genomic sequencing that EZH2 is required for DNA methylation of EZH2-target promoters. Our results suggest that EZH2 serves as a recruitment platform for DNA methyltransferases, thus highlighting a previously unrecognized direct connection between two key epigenetic repression systems.
Asunto(s)
Metilación de ADN , Proteínas de Unión al ADN/clasificación , Proteínas de Unión al ADN/metabolismo , Silenciador del Gen , Proteínas Represoras/clasificación , Proteínas Represoras/metabolismo , Factores de Transcripción/clasificación , Factores de Transcripción/metabolismo , Inmunoprecipitación de Cromatina , Islas de CpG/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Proteína Potenciadora del Homólogo Zeste 2 , Epigénesis Genética , Células HeLa , Humanos , Complejo Represivo Polycomb 2 , Proteínas del Grupo Polycomb , Regiones Promotoras Genéticas/genética , Unión Proteica , Especificidad por SustratoRESUMEN
The Myc transcription factor is an essential mediator of cell growth and proliferation through its ability to both positively and negatively regulate transcription. The mechanisms by which Myc silences gene expression are not well understood. The current model is that Myc represses transcription through functional interference with transcriptional activators. Here we show that Myc binds the corepressor Dnmt3a and associates with DNA methyltransferase activity in vivo. In cells with reduced Dnmt3a levels, we observe specific reactivation of the Myc-repressed p21Cip1 gene, whereas the expression of Myc-activated E-boxes genes is unchanged. In addition, we find that Myc can target Dnmt3a selectively to the promoter of p21Cip1. Myc is known to be recruited to the p21Cip1 promoter by the DNA-binding factor Miz-1. Consistent with this, we observe that Myc and Dnmt3a form a ternary complex with Miz-1 and that this complex can corepress the p21Cip1 promoter. Finally, we show that DNA methylation is required for Myc-mediated repression of p21Cip1. Our data identify a new mechanism by which Myc can silence gene expression not only by passive functional interference but also by active recruitment of corepressor proteins. Furthermore, these findings suggest that targeting of DNA methyltransferases by transcription factors is a wide and general mechanism for the generation of specific DNA methylation patterns within a cell.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/fisiología , Proteínas Proto-Oncogénicas c-myc/fisiología , Transcripción Genética/fisiología , Animales , Secuencia de Bases , Western Blotting , Proteínas de Ciclo Celular/genética , Línea Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , ADN (Citosina-5-)-Metiltransferasas/antagonistas & inhibidores , ADN Metiltransferasa 3A , Cartilla de ADN , Silenciador del Gen/fisiología , Inmunoprecipitación , Regiones Promotoras Genéticas , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Erm, a member of the PEA3 group within the Ets family of transcription factors, is expressed in murine and human lymphocytes. Here, we show that in the human Molt4 lymphoblastic cell line, the erm gene expression is regulated by the conventional PKC (cPKC) pathway. To better characterize the molecular mechanism by which cPKC regulates Erm transcription in Molt4 cells, we tested proximal promoter deletions of the human gene, and identified a specific cPKC-regulated region between positions -420 and -115 upstream of the first exon.
Asunto(s)
Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica/fisiología , Regiones Promotoras Genéticas/genética , Proteína Quinasa C/fisiología , Transducción de Señal/fisiología , Linfocitos T/metabolismo , Acetato de Tetradecanoilforbol/análogos & derivados , Factores de Transcripción/genética , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Dactinomicina/farmacología , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras , Proteína Quinasa C/antagonistas & inhibidores , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Eliminación de Secuencia/genética , Acetato de Tetradecanoilforbol/farmacología , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacos , Transcripción Genética/fisiología , Regulación hacia ArribaRESUMEN
Although most Ets transcription factors have been characterized as transcriptional activators, some of them display repressor activity. Here we characterize an Ets-family member, the very specifically expressed human Fifth Ewing Variant (FEV), as a transcriptional repressor. We show that among a broad range of human cell lines, only Dami megakaryocytic cells express FEV. This nuclear protein binds to Ets-binding sites, such as that of the human ICAM-1 promoter. We used this promoter to demonstrate that FEV can repress both basal transcription and, even more strongly, ectopically Ets-activated transcription. We identified two domains responsible for FEV-mediated repression: the ETS domain, responsible for passive repression, and the carboxy-terminal alanine-rich domain, involved in active repression. In the Ets-independent LEXA system also, FEV acts as a transcriptional repressor via its alanine-rich carboxy-terminal domain. The mechanism by which FEV actively represses transcription is currently unknown, since FEV-triggered repression is not reversed by the histone deacetylase inhibitor trichostatin A. We also showed that long-term overexpression of FEV proteins containing the alanine-rich domain prevents cell clones from growing, whereas clones expressing a truncated FEV protein lacking this domain develop like control cells. This confirms the importance of this domain in FEV-triggered repression.
Asunto(s)
Alanina/análisis , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/fisiología , Silenciador del Gen , Proteínas Nucleares/química , Proteínas Nucleares/fisiología , Proteínas Represoras/química , Proteínas Represoras/fisiología , Animales , Células COS , Línea Celular , Núcleo Celular/química , Proteínas de Unión al ADN/genética , Humanos , Proteínas Nucleares/genética , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-ets , Conejos , Elementos de Respuesta , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Transcripción Genética , Transfección , Células Tumorales CultivadasRESUMEN
The Dnmt3L protein belongs to the Dnmt3 family of DNA methyltransferases by virtue of its sequence homology in the plant homeodomain (PHD)-like motif. Dnmt3L is essential for the establishment of maternal genomic imprints and, given its lack of key methyltransferase motifs, is more likely to act as a regulator of methylation rather than as an enzyme that methylates DNA. Here, we show that Dnmt3L, like Dnmt3a and Dnmt3b, interacts both in vitro and in vivo with the histone deacetylase HDAC1. Consistent with the binding to a deacetylase, Dnmt3L purifies histone deacetylase activity from nuclear extracts. We find that Dnmt3L can repress transcription and that this repression is dependent on HDAC1 and is relieved by treatment with the HDAC inhibitor trichostatin A. Binding of Dnmt3L to HDAC1 as well as its repressive function require the PHD-like motif. Our results indicate that Dnmt3L plays a role in transcriptional regulation and that recruitment of the HDAC repressive machinery is a shared and conserved feature of the Dnmt3 family. The fact that, despite the absence of a methyltransferase domain, Dnmt3L retains the capacity to contact deacetylase further substantiates the notion that the Dnmts can repress transcription independently of their methylating activities.