RESUMEN
Deciphering the spatiotemporal coordination between nuclear functions is important to understand its role in the maintenance of human genome. In this context, super-resolution microscopy has gained considerable interest because it can be used to probe the spatial organization of functional sites in intact single-cell nuclei in the 20-250 nm range. Among the methods that quantify colocalization from multicolor images, image cross-correlation spectroscopy (ICCS) offers several advantages, namely it does not require a presegmentation of the image into objects and can be used to detect dynamic interactions. However, the combination of ICCS with super-resolution microscopy has not been explored yet. Here, we combine dual-color stimulated emission depletion (STED) nanoscopy with ICCS (STED-ICCS) to quantify the nanoscale distribution of functional nuclear sites. We show that super-resolved ICCS provides not only a value of the colocalized fraction but also the characteristic distances associated to correlated nuclear sites. As a validation, we quantify the nanoscale spatial distribution of three different pairs of functional nuclear sites in MCF10A cells. As expected, transcription foci and a transcriptionally repressive histone marker (H3K9me3) are not correlated. Conversely, nascent DNA replication foci and the proliferating cell nuclear antigen(PCNA) protein have a high level of proximity and are correlated at a nanometer distance scale that is close to the limit of our experimental approach. Finally, transcription foci are found at a distance of 130 nm from replication foci, indicating a spatial segregation at the nanoscale. Overall, our data demonstrate that STED-ICCS can be a powerful tool for the analysis of the nanoscale distribution of functional sites in the nucleus.
Asunto(s)
Núcleo Celular/metabolismo , Microscopía/métodos , Nanotecnología/métodos , Análisis Espectral , Color , Humanos , Células MCF-7RESUMEN
UNLABELLED: The identification of genes maintaining cancer growth is critical to our understanding of tumorigenesis. We report the first in vivo genetic screen of patient-derived tumors, using metastatic melanomas and targeting 236 chromatin genes by expression of specific shRNA libraries. Our screens revealed unprecedented numerosity of genes indispensable for tumor growth (â¼50% of tested genes) and unexpected functional heterogeneity among patients (<15% in common). Notably, these genes were not activated by somatic mutations in the same patients and are therefore distinguished from mutated cancer driver genes. We analyzed underlying molecular mechanisms of one of the identified genes, the Histone-lysine N-methyltransferase KMT2D, and showed that it promotes tumorigenesis by dysregulating a subset of transcriptional enhancers and target genes involved in cell migration. The assembly of enhancer genomic patterns by activated KMT2D was highly patient-specific, regardless of the identity of transcriptional targets, suggesting that KMT2D might be activated by distinct upstream signaling pathways. SIGNIFICANCE: Drug targeting of biologically relevant cancer-associated mutations is considered a critical strategy to control cancer growth. Our functional in vivo genetic screens of patient-derived tumors showed unprecedented numerosity and interpatient heterogeneity of genes that are essential for tumor growth, but not mutated, suggesting that multiple, patient-specific signaling pathways are activated in tumors. Cancer Discov; 6(6); 650-63. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 561.
Asunto(s)
Transformación Celular Neoplásica/genética , Estudios de Asociación Genética , Pruebas Genéticas , Neoplasias/diagnóstico , Neoplasias/genética , Fenotipo , Animales , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Biología Computacional/métodos , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Elementos de Facilitación Genéticos , Epigénesis Genética , Epigenómica/métodos , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad , Xenoinjertos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Metástasis de la Neoplasia , Proteínas de Neoplasias/metabolismo , Unión Proteica , ARN Interferente Pequeño/genética , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: ChIP-seq experiments are widely used to detect and study DNA-protein interactions, such as transcription factor binding and chromatin modifications. However, downstream analysis of ChIP-seq data is currently restricted to the evaluation of signal intensity and the detection of enriched regions (peaks) in the genome. Other features of peak shape are almost always neglected, despite the remarkable differences shown by ChIP-seq for different proteins, as well as by distinct regions in a single experiment. RESULTS: We hypothesize that statistically significant differences in peak shape might have a functional role and a biological meaning. Thus, we design five indices able to summarize peak shapes and we employ multivariate clustering techniques to divide peaks into groups according to both their complexity and the intensity of their coverage function. In addition, our novel analysis pipeline employs a range of statistical and bioinformatics techniques to relate the obtained peak shapes to several independent genomic datasets, including other genome-wide protein-DNA maps and gene expression experiments. To clarify the meaning of peak shape, we apply our methodology to the study of the erythroid transcription factor GATA-1 in K562 cell line and in megakaryocytes. CONCLUSIONS: Our study demonstrates that ChIP-seq profiles include information regarding the binding of other proteins beside the one used for precipitation. In particular, peak shape provides new insights into cooperative transcriptional regulation and is correlated to gene expression.
Asunto(s)
Biología Computacional , Inmunoprecipitación de Cromatina , Análisis por Conglomerados , ADN/química , ADN/metabolismo , Factor de Transcripción GATA1/antagonistas & inhibidores , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Células K562 , Megacariocitos/citología , Megacariocitos/metabolismo , Unión Proteica , Análisis de Secuencia de ADNRESUMEN
Polycomb group proteins are transcriptional repressors that control many developmental genes. The Polycomb group protein Enhancer of Zeste has been shown in vitro to methylate specifically lysine 27 and lysine 9 of histone H3 but the role of this modification in Polycomb silencing is unknown. We show that H3 trimethylated at lysine 27 is found on the entire Ubx gene silenced by Polycomb. However, Enhancer of Zeste and other Polycomb group proteins stay primarily localized at their response elements, which appear to be the least methylated parts of the silenced gene. Our results suggest that, contrary to the prevailing view, the Polycomb group proteins and methyltransferase complexes are recruited to the Polycomb response elements independently of histone methylation and then loop over to scan the entire region, methylating all accessible nucleosomes. We propose that the Polycomb chromodomain is required for the looping mechanism that spreads methylation over a broad domain, which in turn is required for the stability of the Polycomb group protein complex. Both the spread of methylation from the Polycomb response elements, and the silencing effect can be blocked by the gypsy insulator.
Asunto(s)
Metilación de ADN , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiología , Proteínas de Homeodominio/genética , Factores de Transcripción/genética , Animales , Sitios de Unión , Cromatina/química , Cromatina/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiología , Drosophila melanogaster/metabolismo , Silenciador del Gen , Histonas/química , Histonas/metabolismo , Proteínas de Homeodominio/metabolismo , Modelos Genéticos , Complejo Represivo Polycomb 1 , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Polycomb (PcG) complexes maintain the silent state of target genes. The mechanism of silencing is not known but has been inferred to involve chromatin packaging to block the access of transcription factors. We have studied the effect of PcG silencing on the hsp26 heat shock promoter. While silencing does decrease the accessibility of some restriction enzyme sites to some extent, it does not prevent the binding of TBP, RNA polymerase, or the heat shock factor to the hsp26 promoter, as shown by chromatin immunoprecipitation. However, we find that in the repressed state, the RNA polymerase cannot initiate transcription. We conclude that, rather than altering chromatin structure to block accessibility, PcG silencing in this construct targets directly the activity of the transcriptional machinery at the promoter.
Asunto(s)
Proteínas de Drosophila/metabolismo , Silenciador del Gen , Genes de Insecto , Transcripción Genética , Animales , Biomarcadores , Cromatina/metabolismo , Huella de ADN , Elementos Transponibles de ADN , ARN Polimerasas Dirigidas por ADN/metabolismo , Drosophila , Proteínas de Drosophila/genética , Proteínas de Choque Térmico/genética , Complejo Represivo Polycomb 1 , Pruebas de Precipitina , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , TransgenesRESUMEN
The Polycomb Response Element (PRE) is the nucleation site for the Polycomb silencing complexes. The sequences responsible for the recruitment of the components of the Polycomb complex are not well understood. A comparison of the bxd PRE sequences from several different Drosophila species shows that some changes have occurred during phylogeny but large blocks of sequence are conserved after a divergence of some 60 million years. We compare the PRE sequences, the sites of some known PRE binding proteins, the conservation of DNasel hypersensitive sites and relate them to the sequence of the Ultrabithorax promoter which these PREs regulate.