RESUMEN
In chronic lymphocytic leukemia (CLL), epigenetic alterations are considered to centrally shape the transcriptional signatures that drive disease evolution and underlie its biological and clinical subsets. Characterizations of epigenetic regulators, particularly histone-modifying enzymes, are very rudimentary in CLL. In efforts to establish effectors of the CLL-associated oncogene T-cell leukemia 1A (TCL1A), we identified here the lysine-specific histone demethylase KDM1A to interact with the TCL1A protein in B cells in conjunction with an increased catalytic activity of KDM1A. We demonstrate that KDM1A is upregulated in malignant B cells. Elevated KDM1A and associated gene expression signatures correlated with aggressive disease features and adverse clinical outcomes in a large prospective CLL trial cohort. Genetic Kdm1a knockdown in Eµ-TCL1A mice reduced leukemic burden and prolonged animal survival, accompanied by upregulated p53 and proapoptotic pathways. Genetic KDM1A depletion also affected milieu components (T, stromal, and monocytic cells), resulting in significant reductions in their capacity to support CLL-cell survival and proliferation. Integrated analyses of differential global transcriptomes (RNA sequencing) and H3K4me3 marks (chromatin immunoprecipitation sequencing) in Eµ-TCL1A vs iKdm1aKD;Eµ-TCL1A mice (confirmed in human CLL) implicate KDM1A as an oncogenic transcriptional repressor in CLL which alters histone methylation patterns with pronounced effects on defined cell death and motility pathways. Finally, pharmacologic KDM1A inhibition altered H3K4/9 target methylation and revealed marked anti-B-cell leukemic synergisms. Overall, we established the pathogenic role and effector networks of KDM1A in CLL via tumor-cell intrinsic mechanisms and its impacts in cells of the microenvironment. Our data also provide rationales to further investigate therapeutic KDM1A targeting in CLL.
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Leucemia Linfocítica Crónica de Células B , Humanos , Ratones , Animales , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Histonas/metabolismo , Lisina , Estudios Prospectivos , Histona Demetilasas/genética , Histona Demetilasas/metabolismo , Microambiente TumoralRESUMEN
BACKGROUND: The tight junction proteins claudin-2 and claudin-10a form paracellular cation and anion channels, respectively, and are expressed in the proximal tubule. However, the physiologic role of claudin-10a in the kidney has been unclear. METHODS: To investigate the physiologic role of claudin-10a, we generated claudin-10a-deficient mice, confirmed successful knockout by Southern blot, Western blot, and immunofluorescence staining, and analyzed urine and serum of knockout and wild-type animals. We also used electrophysiologic studies to investigate the functionality of isolated proximal tubules, and studied compensatory regulation by pharmacologic intervention, RNA sequencing analysis, Western blot, immunofluorescence staining, and respirometry. RESULTS: Mice deficient in claudin-10a were fertile and without overt phenotypes. On knockout, claudin-10a was replaced by claudin-2 in all proximal tubule segments. Electrophysiology showed conversion from paracellular anion preference to cation preference and a loss of paracellular Cl- over HCO3- preference. As a result, there was tubular retention of calcium and magnesium, higher urine pH, and mild hypermagnesemia. A comparison with other urine and serum parameters under control conditions and sequential pharmacologic transport inhibition, and unchanged fractional lithium excretion, suggested compensative measures in proximal and distal tubular segments. Changes in proximal tubular oxygen handling and differential expression of genes regulating fatty acid metabolism indicated proximal tubular adaptation. Western blot and immunofluorescence revealed alterations in distal tubular transport. CONCLUSIONS: Claudin-10a is the major paracellular anion channel in the proximal tubule and its deletion causes calcium and magnesium hyper-reabsorption by claudin-2 redistribution. Transcellular transport in proximal and distal segments and proximal tubular metabolic adaptation compensate for loss of paracellular anion permeability.
Asunto(s)
Claudina-2 , Claudinas/metabolismo , Animales , Cationes/metabolismo , Túbulos Renales Proximales/metabolismo , Ratones , Permeabilidad , Uniones Estrechas/fisiologíaRESUMEN
Lysine-specific demethylase 1 (LSD1) is highly expressed in many cancer types and strongly associated with cancer progression and metastasis. Circular RNAs (circRNAs) are produced by back-splicing and influence the interactive RNA network by microRNA and protein sponging. In the present study, we aimedto identify circRNAs that derive from the LSD1-encoding KDM1A gene, and to investigate their potential to be released and uptaken by lung cancer versus non-cancer epithelial cells. We identified four circLSD1-RNAs by RT-PCR with divergent primers, followed by sequencing. The expression level of circLSD1-RNAs was then studied by quantitative PCR on cellular and extracellular fractions of lung cancer (PC9) and non-cancer primary small airway epithelial (PSAE) cells. Moreover, we established the transgenic overexpression of circLSD1-RNAs. We show that circLSD1-RNAs are primarily located in the cytoplasm, but are packaged and released from lung cancer and non-cancer cells by extracellular vesicles (EVs) and ribonucleoprotein (RNP) complexes, respectively. Proteomics demonstrated a different protein pattern of EV fractions released from PC9 versus PSAE cells. Importantly, released circLSD1-RNAs were differently taken up by PSAE and PC9 cells. In conclusion, our findings provide primary evidence that circLSD1-RNAs participate in the intercellular communication of lung cancer cells with the tumor environment.
RESUMEN
The evolutionary conserved Polo-like kinase 4 (PLK4) is essential for centriole duplication, spindle assembly, and de novo centriole formation. In man, homozygous mutations in PLK4 lead to primary microcephaly, altered PLK4 expression is associated with aneuploidy in human embryos. Here, we report on a consanguineous four-generation family with 8 affected individuals compound heterozygous for a novel missense variant, c.881 T > G, and a deletion of the PLK4 gene. The clinical phenotype of the adult patients is mild compared to individuals with previously described PLK4 mutations. One individual was homozygous for the variant c.881G and phenotypically unaffected. The deletion was inherited by 14 of 16 offspring and thus exhibits transmission ratio distortion (TRD). Moreover, based on the already published families with PLK4 mutations, it could be shown that due to the preferential transmission of the mutant alleles, the number of affected offspring is significantly increased. It is assumed that reduced expression of PLK4 decreases the intrinsically high error rate of the first cell divisions after fertilization, increases the number of viable embryos and thus leads to preferential transmission of the deleted/mutated alleles.
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Proteínas de Ciclo Celular , Centriolos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , División Celular , Centriolos/genética , Centriolos/metabolismo , Humanos , Mutación , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
Neuroblastoma is a malignant paediatric tumour of the sympathetic nervous system. Roughly half of these tumours regress spontaneously or are cured by limited therapy. By contrast, high-risk neuroblastomas have an unfavourable clinical course despite intensive multimodal treatment, and their molecular basis has remained largely elusive. Here we have performed whole-genome sequencing of 56 neuroblastomas (high-risk, n = 39; low-risk, n = 17) and discovered recurrent genomic rearrangements affecting a chromosomal region at 5p15.33 proximal of the telomerase reverse transcriptase gene (TERT). These rearrangements occurred only in high-risk neuroblastomas (12/39, 31%) in a mutually exclusive fashion with MYCN amplifications and ATRX mutations, which are known genetic events in this tumour type. In an extended case series (n = 217), TERT rearrangements defined a subgroup of high-risk tumours with particularly poor outcome. Despite a large structural diversity of these rearrangements, they all induced massive transcriptional upregulation of TERT. In the remaining high-risk tumours, TERT expression was also elevated in MYCN-amplified tumours, whereas alternative lengthening of telomeres was present in neuroblastomas without TERT or MYCN alterations, suggesting that telomere lengthening represents a central mechanism defining this subtype. The 5p15.33 rearrangements juxtapose the TERT coding sequence to strong enhancer elements, resulting in massive chromatin remodelling and DNA methylation of the affected region. Supporting a functional role of TERT, neuroblastoma cell lines bearing rearrangements or amplified MYCN exhibited both upregulated TERT expression and enzymatic telomerase activity. In summary, our findings show that remodelling of the genomic context abrogates transcriptional silencing of TERT in high-risk neuroblastoma and places telomerase activation in the centre of transformation in a large fraction of these tumours.
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Regulación Neoplásica de la Expresión Génica/genética , Genoma Humano/genética , Neuroblastoma/genética , Neuroblastoma/patología , Recombinación Genética/genética , Telomerasa/genética , Telomerasa/metabolismo , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Cromatina/genética , Cromatina/metabolismo , Cromosomas Humanos Par 5/genética , ADN Helicasas/genética , Metilación de ADN , Elementos de Facilitación Genéticos/genética , Activación Enzimática/genética , Amplificación de Genes/genética , Silenciador del Gen , Humanos , Lactante , Proteína Proto-Oncogénica N-Myc , Neuroblastoma/clasificación , Neuroblastoma/enzimología , Proteínas Nucleares/genética , Proteínas Oncogénicas/genética , Pronóstico , ARN Mensajero/análisis , ARN Mensajero/genética , Riesgo , Translocación Genética/genética , Regulación hacia Arriba/genética , Proteína Nuclear Ligada al Cromosoma XRESUMEN
Mutations in splicing factor genes have a severe impact on the survival of cancer patients. Splicing factor 3b subunit 1 (SF3B1) is one of the most frequently mutated genes in chronic lymphocytic leukemia (CLL); patients carrying these mutations have a poor prognosis. Since the splicing machinery and the epigenome are closely interconnected, we investigated whether these alterations may affect the epigenomes of CLL patients. While an overall hypomethylation during CLL carcinogenesis has been observed, the interplay between the epigenetic stage of the originating B cells and SF3B1 mutations, and the subsequent effect of the mutations on methylation alterations in CLL, have not been investigated. We profiled the genome-wide DNA methylation patterns of 27 CLL patients with and without SF3B1 mutations and identified local decreases in methylation levels in SF3B1mut CLL patients at 67 genomic regions, mostly in proximity to telomeric regions. These differentially methylated regions (DMRs) were enriched in gene bodies of cancer-related signaling genes, e.g., NOTCH1, HTRA3, and BCL9L. In our study, SF3B1 mutations exclusively emerged in two out of three epigenetic stages of the originating B cells. However, not all the DMRs could be associated with the methylation programming of B cells during development, suggesting that mutations in SF3B1 cause additional epigenetic aberrations during carcinogenesis.
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Biomarcadores de Tumor/genética , Metilación de ADN , Regulación Leucémica de la Expresión Génica , Leucemia Linfocítica Crónica de Células B/patología , Mutación , Fosfoproteínas/genética , Factores de Empalme de ARN/genética , Epigénesis Genética , Humanos , Leucemia Linfocítica Crónica de Células B/genética , PronósticoRESUMEN
5-Methylcytosine (5mC), the central epigenetic mark of mammalian DNA, plays fundamental roles in chromatin regulation. 5mC is written onto genomes by DNA methyltransferases (DNMT), and perturbation of this process is an early event in carcinogenesis. However, studying 5mC functions is limited by the inability to control individual DNMTs with spatiotemporal resolution in vivo. We report light-control of DNMT catalysis by genetically encoding a photocaged cysteine as a catalytic residue. This enables translation of inactive DNMTs, their rapid activation by light-decaging, and subsequent monitoring of de novo DNA methylation. We provide insights into how cancer-related DNMT mutations alter de novo methylation in vivo, and demonstrate local and tuneable cytosine methylation by light-controlled DNMTs fused to a programmable transcription activator-like effector domain targeting pericentromeric satellite-3 DNA. We further study early events of transcriptome alterations upon DNMT-catalyzed cytosine methylation. Our study sets a basis to dissect the order and kinetics of diverse chromatin-associated events triggered by normal and aberrant DNA methylation.
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ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN/efectos de la radiación , Luz , 5-Metilcitosina/metabolismo , Biocatálisis , Línea Celular Tumoral , ADN (Citosina-5-)-Metiltransferasas/química , ADN (Citosina-5-)-Metiltransferasas/genética , Células HEK293 , Humanos , Mutación , Transcriptoma/efectos de la radiaciónRESUMEN
Aberrant DNA methylation in stem cells is a hallmark of aging and tumor development. Recently, we have suggested that promoter DNA hyper-methylation originates in DNA repair and that even successful DNA repair might confer this kind of epigenetic long-term change. Here, we ask for interrelations between promoter DNA methylation and histone modification changes observed in the intestine weeks after irradiation and/or following Msh2 loss. We focus on H3K4me3 recruitment to the promoter of H3K27me3 target genes. By RNA- and histone ChIP-sequencing, we demonstrate that this recruitment occurs without changes of the average gene transcription and does not involve H3K9me3. Applying a mathematical model of epigenetic regulation of transcription, we show that the recruitment can be explained by stronger DNA binding of H3K4me3 and H3K27me3 histone methyl-transferases as a consequence of lower DNA methylation. This scenario implicates stable transcription despite of H3K4me3 recruitment, in agreement with our RNA-seq data. Following several kinds of stress, including moderate irradiation, stress-sensitive intestinal stem cell (ISCs) are known to become replaced by more resistant populations. Our simulation results suggest that the stress-resistant ISCs are largely protected against promoter hyper-methylation of H3K27me3 target genes.
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Metilación de ADN/genética , ADN/genética , Histonas/genética , Intestinos/fisiología , Regiones Promotoras Genéticas/genética , Células Madre/fisiología , Animales , Epigénesis Genética/genética , Código de Histonas/genética , RatonesRESUMEN
The epithelial sodium channel (ENaC) can increase the colonic absorptive capacity for salt and water. Campylobacter concisus is a common pathogenic epsilonproteobacterium, causing enteritis and diarrhea. It can induce barrier dysfunction in the intestine, but its influence on intestinal transport function is still unknown. Therefore, our study aimed to characterize C. concisus effects on ENaC using the HT-29/B6-GR/MR (epithelial cell line HT-29/B6 transfected with glucocorticoid and mineralocorticoid receptors) cell model and mouse colon. In Ussing chambers, C. concisus infection inhibited ENaC-dependent Na+ transport as indicated by a reduction in amiloride-sensitive short circuit current (-55%, n = 15, p < 0.001). This occurred via down-regulation of ß- and γ-ENaC mRNA expression and ENaC ubiquitination due to extracellular signal-regulated kinase (ERK)1/2 activation, predicted by Ingenuity Pathway Analysis (IPA). In parallel, C. concisus reduced the expression of the sealing tight junction (TJ) protein claudin-8 and induced claudin-8 redistribution off the TJ domain of the enterocytes, which facilitates the back leakage of Na+ ions into the intestinal lumen. In conclusion, C. concisus caused ENaC dysfunction via interleukin-32-regulated ERK1/2, as well as claudin-8-dependent barrier dysfunction-both of which contribute to Na+ malabsorption and diarrhea.
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Infecciones por Campylobacter/metabolismo , Campylobacter/fisiología , Claudinas/metabolismo , Canales Epiteliales de Sodio/metabolismo , Sodio/metabolismo , Animales , Infecciones por Campylobacter/microbiología , Colon/metabolismo , Colon/microbiología , Diarrea/metabolismo , Diarrea/microbiología , Células HT29 , Interacciones Huésped-Patógeno , Humanos , Absorción Intestinal , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Ratones , Ratones Endogámicos C57BLRESUMEN
We report programmable receptors for the imaging-based analysis of 5-methylcytosine (5mC) in user-defined DNA sequences of single cells. Using fluorescent transcription-activator-like effectors (TALEs) that can recognize sequences of canonical and epigenetic nucleobases through selective repeats, we imaged cellular SATIII DNA, the origin of nuclear stress bodies (nSB). We achieve high nucleobase selectivity of natural repeats in imaging and demonstrate universal nucleobase binding by an engineered repeat. We use TALE pairs differing in only one such repeat in co-stains to detect 5mC in SATIII sequences with nucleotide resolution independently of differences in target accessibility. Further, we directly correlate the presence of heat shock factor 1 with 5mC at its recognition sequence, revealing a potential function of 5mC in its recruitment as initial step of nSB formation. This opens a new avenue for studying 5mC functions in chromatin regulation inâ situ with nucleotide, locus, and cell resolution.
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5-Metilcitosina/metabolismo , Genómica , Imagen Molecular , Nucleótidos/metabolismo , Células HeLa , Humanos , Análisis de la Célula IndividualRESUMEN
The cellular response to heat stress is an ancient and evolutionarily highly conserved defence mechanism characterised by the transcriptional up-regulation of cyto-protective genes and a partial inhibition of splicing. These features closely resemble the proteotoxic stress response during tumor development. The bromodomain protein BRD4 has been identified as an integral member of the oxidative stress as well as of the inflammatory response, mainly due to its role in the transcriptional regulation process. In addition, there are also several lines of evidence implicating BRD4 in the splicing process. Using RNA-sequencing we found a significant increase in splicing inhibition, in particular intron retentions (IR), following heat treatment in BRD4-depleted cells. This leads to a decrease of mRNA abundancy of the affected transcripts, most likely due to premature termination codons. Subsequent experiments revealed that BRD4 interacts with the heat shock factor 1 (HSF1) such that under heat stress BRD4 is recruited to nuclear stress bodies and non-coding SatIII RNA transcripts are up-regulated. These findings implicate BRD4 as an important regulator of splicing during heat stress. Our data which links BRD4 to the stress induced splicing process may provide novel mechanisms of BRD4 inhibitors in regard to anti-cancer therapies.
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Proteínas de Unión al ADN/genética , Respuesta al Choque Térmico/genética , Proteínas Nucleares/genética , Empalme del ARN , ARN Mensajero/genética , ARN no Traducido/genética , Factores de Transcripción/genética , Proteínas de Unión al ADN/metabolismo , Exones , Células HeLa , Factores de Transcripción del Choque Térmico , Histona Acetiltransferasas , Chaperonas de Histonas , Calor , Humanos , Intrones , Proteínas Nucleares/metabolismo , Dominios Proteicos , ARN Mensajero/metabolismo , ARN no Traducido/metabolismo , Análisis de Secuencia de ARN , Factores de Transcripción/metabolismoRESUMEN
Genome-wide enrichment of methylated DNA followed by sequencing (MeDIP-seq) offers a reasonable compromise between experimental costs and genomic coverage. However, the computational analysis of these experiments is complex, and quantification of the enrichment signals in terms of absolute levels of methylation requires specific transformation. In this work, we present QSEA, Quantitative Sequence Enrichment Analysis, a comprehensive workflow for the modelling and subsequent quantification of MeDIP-seq data. As the central part of the workflow we have developed a Bayesian statistical model that transforms the enrichment read counts to absolute levels of methylation and, thus, enhances interpretability and facilitates comparison with other methylation assays. We suggest several calibration strategies for the critical parameters of the model, either using additional data or fairly general assumptions. By comparing the results with bisulfite sequencing (BS) validation data, we show the improvement of QSEA over existing methods. Additionally, we generated a clinically relevant benchmark data set consisting of methylation enrichment experiments (MeDIP-seq), BS-based validation experiments (Methyl-seq) as well as gene expression experiments (RNA-seq) derived from non-small cell lung cancer patients, and show that the workflow retrieves well-known lung tumour methylation markers that are causative for gene expression changes, demonstrating the applicability of QSEA for clinical studies. QSEA is implemented in R and available from the Bioconductor repository 3.4 (www.bioconductor.org/packages/qsea).
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Metilación de ADN , Genómica/métodos , Análisis de Secuencia de ADN/métodos , Animales , Teorema de Bayes , Regulación de la Expresión Génica , Humanos , Neoplasias Pulmonares/genética , Ratones , Regiones Promotoras Genéticas , Sulfitos , Flujo de TrabajoRESUMEN
This case report presents a male patient with epithelioid mesothelioma that was initially misdiagnosed as cancer of unknown primary (CUP) and correctly identified using molecular panel sequencing. The patient had a prior history of colon and breast cancer. To assess the enlarged mediastinal lymph nodes, retrosternal lymphadenectomy was performed in 2016. The lymph nodes were histologically deemed unrelated to the known breast cancer by the reference pathologist, thus leading to the diagnosis of a CUP syndrome. When the patient presented to our center, targeted deep sequencing of both breast cancer and presumed CUP was performed to address the clonal relationship between both malignancies. A missense mutation in BAP1 was revealed in both samples, with coverage data indicating a germline event. The patient was subsequently counseled by a human geneticist and underwent genetic testing, which confirmed the germline nature of this mutation. Collectively, these data led to the diagnosis of BAP1 (BRCA1-associated protein-1) tumor predisposition syndrome (TPDS). With the knowledge of an underlying BAP1 mutation and its known frequent association with epithelioid mesothelioma, the histology was reassessed and the diagnosis was revised to epithelioid mesothelioma. At this point, peritoneal involvement of mesothelioma could be diagnosed and histologically confirmed. This case illustrates the potential of integrated histopathologic and molecular diagnostics in helping to decipher CUP syndromes and establish the correct diagnosis. Additionally, this case highlights typical features of BAP1 TPDS with its general susceptibility to cancers, with pleural and peritoneal mesotheliomas as most prevalent clinical entities and the typically more benign course of these epithelioid mesotheliomas compared with BAP1-unrelated cases of mesotheliomas.
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Biomarcadores de Tumor/genética , Neoplasias Pulmonares/genética , Mesotelioma/genética , Neoplasias Primarias Desconocidas/genética , Síndromes Neoplásicos Hereditarios/genética , Proteínas Supresoras de Tumor/genética , Ubiquitina Tiolesterasa/genética , Anciano , Predisposición Genética a la Enfermedad , Humanos , Neoplasias Pulmonares/secundario , Masculino , Mesotelioma/secundario , Mesotelioma Maligno , Mutación , Mutación Missense , Neoplasias Primarias Desconocidas/patología , Síndromes Neoplásicos Hereditarios/patologíaRESUMEN
Colorectal cancer (CRC) arising in Lynch syndrome (LS) comprises tumours with constitutional mutations in DNA mismatch repair genes. There is still a lack of whole-genome and transcriptome studies of LS-CRC to address questions about similarities and differences in mutation and gene expression characteristics between LS-CRC and sporadic CRC, about the molecular heterogeneity of LS-CRC, and about specific mechanisms of LS-CRC genesis linked to dysfunctional mismatch repair in LS colonic mucosa and the possible role of immune editing. Here, we provide a first molecular characterization of LS tumours and of matched tumour-distant reference colonic mucosa based on whole-genome DNA-sequencing and RNA-sequencing analyses. Our data support two subgroups of LS-CRCs, G1 and G2, whereby G1 tumours show a higher number of somatic mutations, a higher amount of microsatellite slippage, and a different mutation spectrum. The gene expression phenotypes support this difference. Reference mucosa of G1 shows a strong immune response associated with the expression of HLA and immune checkpoint genes and the invasion of CD4+ T cells. Such an immune response is not observed in LS tumours, G2 reference and normal (non-Lynch) mucosa, and sporadic CRC. We hypothesize that G1 tumours are edited for escape from a highly immunogenic microenvironment via loss of HLA presentation and T-cell exhaustion. In contrast, G2 tumours seem to develop in a less immunogenic microenvironment where tumour-promoting inflammation parallels tumourigenesis. Larger studies on non-neoplastic mucosa tissue of mutation carriers are required to better understand the early phases of emerging tumours. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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Neoplasias Colorrectales/genética , Mutación/genética , Antígenos de Neoplasias/genética , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Neoplasias Colorrectales Hereditarias sin Poliposis/inmunología , Expresión Génica/genética , Genes Relacionados con las Neoplasias/genética , Genoma Humano/genética , Humanos , Inmunidad Celular , Fenotipo , Recurrencia , Transcriptoma/genética , Escape del Tumor/genética , Escape del Tumor/inmunologíaRESUMEN
BACKGROUND: In 30-50% of patients with colorectal adenomatous polyposis, no germline mutation in the known genes APC, causing familial adenomatous polyposis, MUTYH, causing MUTYH-associated polyposis, or POLE or POLD1, causing polymerase-proofreading-associated polyposis can be identified, although a hereditary aetiology is likely. This study aimed to explore the impact of APC mutational mosaicism in unexplained polyposis. METHODS: To comprehensively screen for somatic low-level APC mosaicism, high-coverage next-generation sequencing of the APC gene was performed using DNA from leucocytes and a total of 53 colorectal tumours from 20 unrelated patients with unexplained sporadic adenomatous polyposis. APC mosaicism was assumed if the same loss-of-function APC mutation was present in ≥ 2 anatomically separated colorectal adenomas/carcinomas per patient. All mutations were validated using diverse methods. RESULTS: In 25% (5/20) of patients, somatic mosaicism of a pathogenic APC mutation was identified as underlying cause of the disease. In 2/5 cases, the mosaic level in leucocyte DNA was slightly below the sensitivity threshold of Sanger sequencing; while in 3/5 cases, the allelic fraction was either very low (0.1-1%) or no mutations were detectable. The majority of mosaic mutations were located outside the somatic mutation cluster region of the gene. CONCLUSIONS: The present data indicate a high prevalence of pathogenic mosaic APC mutations below the detection thresholds of routine diagnostics in adenomatous polyposis, even if high-coverage sequencing of leucocyte DNA alone is taken into account. This has important implications for both routine work-up and strategies to identify new causative genes in this patient group.
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Poliposis Adenomatosa del Colon/genética , Genes APC , Mutación , Adolescente , Adulto , Neoplasias Colorrectales/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Persona de Mediana Edad , Tasa de MutaciónRESUMEN
BACKGROUND: Legionella pneumophila is a causative agent of severe pneumonia. Infection leads to a broad host cell response, as evident, for example, on the transcriptional level. Chromatin modifications, which control gene expression, play a central role in the transcriptional response to L. pneumophila METHODS: We infected human-blood-derived macrophages (BDMs) with L. pneumophila and used chromatin immunoprecipitation followed by sequencing to screen for gene promoters with the activating histone 4 acetylation mark. RESULTS: We found the promoter of tumor necrosis factor α-induced protein 2 (TNFAIP2) to be acetylated at histone H4. This factor has not been characterized in the pathology of L. pneumophila TNFAIP2 messenger RNA and protein were upregulated in response to L. pneumophila infection of human-BDMs and human alveolar epithelial (A549) cells. We showed that L. pneumophila-induced TNFAIP2 expression is dependent on the NF-κB transcription factor. Importantly, knock down of TNFAIP2 led to reduced intracellular replication of L. pneumophila Corby in A549 cells. CONCLUSIONS: Taken together, genome-wide chromatin analysis of L. pneumophila-infected macrophages demonstrated induction of TNFAIP2, a NF-κB-dependent factor relevant for bacterial replication.
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Citocinas/análisis , Interacciones Huésped-Patógeno , Legionella pneumophila/patogenicidad , Macrófagos/química , Macrófagos/microbiología , Acetilación , Línea Celular , Cromatina/química , Inmunoprecipitación de Cromatina , Citocinas/genética , Células Epiteliales/química , Células Epiteliales/microbiología , Histonas/análisis , HumanosRESUMEN
Genome-wide association studies have identified genomic loci, whose single-nucleotide polymorphisms (SNPs) predispose to prostate cancer (PCa). However, the mechanisms of most of these variants are largely unknown. We integrated chromatin-immunoprecipitation-coupled sequencing and microarray expression profiling in TMPRSS2-ERG gene rearrangement positive DUCaP cells with the GWAS PCa risk SNPs catalog to identify disease susceptibility SNPs localized within functional androgen receptor-binding sites (ARBSs). Among the 48 GWAS index risk SNPs and 3,917 linked SNPs, 80 were found located in ARBSs. Of these, rs11891426:T>G in an intron of the melanophilin gene (MLPH) was within a novel putative auxiliary AR-binding motif, which is enriched in the neighborhood of canonical androgen-responsive elements. TâG exchange attenuated the transcriptional activity of the ARBS in an AR reporter gene assay. The expression of MLPH in primary prostate tumors was significantly lower in those with the G compared with the T allele and correlated significantly with AR protein. Higher melanophilin level in prostate tissue of patients with a favorable PCa risk profile points out a tumor-suppressive effect. These results unravel a hidden link between AR and a functional putative PCa risk SNP, whose allele alteration affects androgen regulation of its host gene MLPH.
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Proteínas Adaptadoras Transductoras de Señales/genética , Sitios de Unión , Polimorfismo de Nucleótido Simple , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/metabolismo , Elementos de Respuesta , Adulto , Anciano , Alelos , Secuencia de Bases , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Regulación Neoplásica de la Expresión Génica , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Estadificación de Neoplasias , Motivos de Nucleótidos , Posición Específica de Matrices de Puntuación , Neoplasias de la Próstata/patología , Unión Proteica , Carga TumoralRESUMEN
HPMRS or Mabry syndrome is a heterogeneous glycosylphosphatidylinositol (GPI) anchor deficiency that is caused by an impairment of synthesis or maturation of the GPI-anchor. The expressivity of the clinical features in HPMRS varies from severe syndromic forms with multiple organ malformations to mild nonsyndromic intellectual disability. In about half of the patients with the clinical diagnosis of HPMRS, pathogenic mutations can be identified in the coding region in one of the six genes, one among them is PGAP3. In this work, we describe a screening approach with sequence specific baits for transcripts of genes of the GPI pathway that allows the detection of functionally relevant mutations also including introns and the 5' and 3' UTR. By this means, we also identified pathogenic noncoding mutations, which increases the diagnostic yield for HPMRS on the basis of intellectual disability and elevated serum alkaline phosphatase. In eight affected individuals from different ethnicities, we found seven novel pathogenic mutations in PGAP3. Besides five missense mutations, we identified an intronic mutation, c.558-10G>A, that causes an aberrant splice product and a mutation in the 3'UTR, c.*559C>T, that is associated with substantially lower mRNA levels. We show that our novel screening approach is a useful rapid detection tool for alterations in genes coding for key components of the GPI pathway.
Asunto(s)
Anomalías Múltiples/genética , Anomalías Múltiples/patología , Discapacidad Intelectual/genética , Discapacidad Intelectual/patología , Mutación Missense , Trastornos del Metabolismo del Fósforo/genética , Trastornos del Metabolismo del Fósforo/patología , Polimorfismo de Nucleótido Simple , ARN Mensajero/genética , Receptores de Superficie Celular/genética , Regiones no Traducidas 3' , Adolescente , Adulto , Hidrolasas de Éster Carboxílico , Células Cultivadas , Niño , Preescolar , Femenino , Predisposición Genética a la Enfermedad , Humanos , Intrones , Masculino , Linaje , Análisis de Secuencia de ADN/métodos , Adulto JovenRESUMEN
Aberrant CpG methylation is a universal epigenetic trait of cancer cell genomes. However, human cancer samples or cell lines preclude the investigation of epigenetic changes occurring early during tumour development. Here, we have used MeDIP-seq to analyse the DNA methylome of APC(Min) adenoma as a model for intestinal cancer initiation, and we present a list of more than 13,000 recurring differentially methylated regions (DMRs) characterizing intestinal adenoma of the mouse. We show that Polycomb Repressive Complex (PRC) targets are strongly enriched among hypermethylated DMRs, and several PRC2 components and DNA methyltransferases were up-regulated in adenoma. We further demonstrate by bisulfite pyrosequencing of purified cell populations that the DMR signature arises de novo in adenoma cells rather than by expansion of a pre-existing pattern in intestinal stem cells or undifferentiated crypt cells. We found that epigenetic silencing of tumour suppressors, which occurs frequently in colon cancer, was rare in adenoma. Quite strikingly, we identified a core set of DMRs, which is conserved between mouse adenoma and human colon cancer, thus possibly revealing a global panel of epigenetically modified genes for intestinal tumours. Our data allow a distinction between early conserved epigenetic alterations occurring in intestinal adenoma and late stochastic events promoting colon cancer progression, and may facilitate the selection of more specific clinical epigenetic biomarkers.
Asunto(s)
Adenoma/genética , Neoplasias del Colon/genética , Metilación de ADN/genética , Neoplasias Intestinales/genética , Proteínas del Grupo Polycomb/genética , Adenoma/patología , Animales , Secuencia de Bases , Islas de CpG/genética , Epigenómica , Genoma , Humanos , Neoplasias Intestinales/patología , Ratones , SinteníaRESUMEN
BACKGROUND: High androgen receptor (AR) level in primary tumour predicts increased prostate cancer (PCa)-specific mortality. Furthermore, activations of the AR, PI3K, mTOR, NFκB and Hedgehog (Hh) signaling pathways are involved in the fatal development of castration-resistant prostate cancer during androgen ablation therapy. MID1, a negative regulator of the tumor-suppressor PP2A, is known to promote PI3K, mTOR, NFκB and Hh signaling. Here we investigate the interaction of MID1 and AR. METHODS: AR and MID1 mRNA and protein levels were measured by qPCR, Western blot and immunohistochemistry. Co-immunoprecipitation followed by PCR and RNA-pull-down followed by Western blot was used to investigate protein-mRNA interaction, chromatin-immunoprecipitation followed by next-generation sequencing for identification of AR chromatin binding sites. AR transcriptional activity and activity of promoter binding sites for AR were analyzed by reporter gene assays. For knockdown or overexpression of proteins of interest prostate cancer cells were transfected with siRNA or expression plasmids, respectively. RESULTS: The microtubule-associated MID1 protein complex associates with AR mRNA via purine-rich trinucleotide repeats, expansions of which are known to correlate with ataxia and cancer. The level of MID1 directly correlates with the AR protein level in PCa cells. Overexpression of MID1 results in a several fold increase in AR protein and activity without major changes in mRNA-levels, whereas siRNA-triggered knockdown of MID1 mRNA reduces AR-protein levels significantly. Upregulation of AR protein by MID1 occurs via increased translation as no major changes in AR protein stability could be observed. AR on the other hand, regulates MID1 via several functional AR binding sites in the MID1 gene, and, in the presence of androgens, exerts a negative feedback loop on MID1 transcription. Thus, androgen withdrawal increases MID1 and concomitantly AR-protein levels. In line with this, MID1 is significantly over-expressed in PCa in a stage-dependent manner. CONCLUSION: Promotion of AR, in addition to enhancement of the Akt-, NFκB-, and Hh-pathways by sustained MID1-upregulation during androgen deprivation therapy provides a powerful proliferative scenario for PCa progression into castration resistance. Thus MID1 represents a novel, multi-faceted player in PCa and a promising target to treat castration resistant prostate cancer.