RESUMEN
In diabetes mellitus, ß cell destruction is largely silent and can be detected only after significant loss of insulin secretion capacity. We have developed a method for detecting ß cell death in vivo by amplifying and measuring the proportion of insulin 1 DNA from ß cells in the serum. By using primers that are specific for DNA methylation patterns in ß cells, we have detected circulating copies of ß cell-derived demethylated DNA in serum of mice by quantitative PCR. Accordingly, we have identified a relative increase of ß cell-derived DNA after induction of diabetes with streptozotocin and during development of diabetes in nonobese diabetic mice. We have extended the use of this assay to measure ß cell-derived insulin DNA in human tissues and serum. We found increased levels of demethylated insulin DNA in subjects with new-onset type 1 diabetes compared with age-matched control subjects. Our method provides a noninvasive approach for detecting ß cell death in vivo that may be used to track the progression of diabetes and guide its treatment.
Asunto(s)
Muerte Celular/fisiología , ADN/sangre , Diabetes Mellitus/patología , Células Secretoras de Insulina/patología , Insulina/genética , Animales , Secuencia de Bases , Clonación Molecular , ADN/genética , Cartilla de ADN/genética , Diabetes Mellitus/sangre , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Células Secretoras de Insulina/química , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Datos de Secuencia Molecular , Monitoreo Fisiológico/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: DNA methylation (DNAm) age metrics have been widely accepted as an epigenetic biomarker for biological aging and disease. The purpose of this study is to assess whether or not individuals carrying Lynch Syndrome-associated mutations are affected in their rate of biological aging, as measured by the epigenetic clock. METHODS: Genome-wide bisulfite DNA sequencing data were generated using DNA from CD4 + T-cells obtained from peripheral blood using 27 patient samples from Lynch syndrome families. Horvath's DNAm age model based on penalized linear regression was applied to estimate DNAm age from patient samples with distinct clinical and genetic characteristics to investigate cancer mutation-related aging effects. RESULTS: Both Lynch mutation carriers and controls exhibited high variability in their estimated DNAm age, but regression analysis showed steeper slope for the Lynch mutation carriers. Remarkably, six Lynch Syndrome-associated mutation carriers showed a strong correlation to the control group, and two sisters carrying Lynch Syndrome-associated mutations, with no significant difference in lifestyle and similar chronological age, were assigned very different DNAm age. CONCLUSIONS: Future studies will be required to explore, in larger patient populations, whether specific epigenetic age acceleration is predictive of time-to-cancer development, treatment response, and survival. Epigenetic clock DNAm metrics may be affected by the presence of cancer mutations in the germline, and thus show promise of potential clinical utility for stratified surveillance strategies based on the relative risk for imminent emergence of tumor lesions in otherwise healthy Lynch Syndrome-associated mutation carriers.
Asunto(s)
Neoplasias Colorrectales Hereditarias sin Poliposis , Metilación de ADN , Aceleración , Envejecimiento/genética , Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Epigénesis Genética , Humanos , MutaciónRESUMEN
Considering carcinogenesis as a microevolutionary process, best described in the context of metapopulation dynamics, provides the basis for theoretical and empirical studies that indicate it is possible to estimate the relative contribution of genetic instability and selection to the process of tumor formation. We show that mutational load distribution analysis (MLDA) of DNA found in pancreatic fluids yields biometrics that reflect the interplay of instability, selection, accident, and gene function that determines the eventual emergence of a tumor. An in silico simulation of carcinogenesis indicates that MLDA may be a suitable tool for early detection of pancreatic cancer. We also present evidence indicating that, when performed serially in individuals harboring a p16 germ-line mutation bestowing a high risk for pancreatic cancer, MLDA may be an effective tool for the longitudinal assessment of risk and early detection of pancreatic cancer.
Asunto(s)
Transformación Celular Neoplásica/genética , Neoplasias Pancreáticas/genética , Biología Computacional , Humanos , Modelos Genéticos , Mutación/genética , Análisis de Secuencia de ADNRESUMEN
Mammary serine protease inhibitor (maspin) is an important tumor suppressor gene whose expression is associated not only with tumor growth inhibition but also with decreased angiogenesis and metastasis. Maspin expression is down-regulated in metastatic tumors by epigenetic mechanisms, including aberrant promoter hypermethylation. We have constructed artificial transcription factors (ATFs) as novel therapeutic effectors able to bind 18-bp sites in the maspin promoter and reactivate maspin expression in cell lines that harbor an epigenetically silenced promoter. In this article, we have investigated the influence of epigenetic modifications on ATF-mediated regulation of maspin by challenging MDA-MB-231 breast cancer cells, comprising a methylated maspin promoter, with different doses of ATFs and chromatin remodeling drugs: the methyltransferase inhibitor 5-aza-2'-deoxycytidine and the histone deacetylase inhibitor suberoylanilide hydroxamic acid. We found that the ATFs synergized with both inhibitors in reactivating endogenous maspin expression. The strongest synergy was observed with the triple treatment ATF-126 + 5-aza-2'-deoxycytidine + suberoylanilide hydroxamic acid, in which the tumor suppressor was reactivated by 600-fold. Furthermore, this combination inhibited tumor cell proliferation by 95%. Our data suggest that ATFs enhance the efficiency of chromatin remodeling drugs in reactivating silenced tumor suppressors. Our results document the power of a novel therapeutic approach that combines both epigenetic and genetic (sequence-specific ATFs) strategies to reactivate specifically silenced regions of the genome and reprogram cellular phenotypes.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Silenciador del Gen , Genes Supresores de Tumor , Inhibidores de Serina Proteinasa/genética , Serpinas/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Azacitidina/análogos & derivados , Azacitidina/metabolismo , Decitabina , Humanos , Ácidos Hidroxámicos/metabolismo , Modelos Biológicos , Inhibidores de Serina Proteinasa/metabolismo , Serpinas/metabolismo , Células Tumorales Cultivadas , VorinostatRESUMEN
There is an emerging concept that acquired genetic instability in cancer cells can arise from the dysregulation of critical DNA repair pathways due to cell stresses such as inflammation and hypoxia. Here we report that hypoxia specifically down-regulates the expression of RAD51, a key mediator of homologous recombination in mammalian cells. Decreased levels of Rad51 were observed in multiple cancer cell types during hypoxic exposure and were not associated with the cell cycle profile or with expression of hypoxia-inducible factor. Analyses of RAD51 gene promoter activity, as well as mRNA and protein stability, indicate that the hypoxia-mediated regulation of this gene occurs via transcriptional repression. Decreased expression of Rad51 was also observed to persist in posthypoxic cells for as long as 48 h following reoxygenation. Correspondingly, we found reduced levels of homologous recombination in both hypoxic and posthypoxic cells, suggesting that the hypoxia-associated reduction in Rad51 expression has functional consequences for DNA repair. In addition, hypoxia-mediated down-regulation of Rad51 was confirmed in vivo via immunofluorescent image analysis of experimental tumors in mice. Based on these findings, we propose a novel mechanism of genetic instability in the tumor microenvironment mediated by hypoxia-induced suppression of the homologous recombination pathway in cancer cells. The aberrant regulation of Rad51 expression may also create heterogeneity in the DNA damage response among cells within tumors, with implications for the response to cancer therapies.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/fisiología , Hipoxia/metabolismo , Recombinación Genética/fisiología , Ciclo Celular/fisiología , Reparación del ADN/fisiología , Proteínas de Unión al ADN/genética , Regulación hacia Abajo , Femenino , Humanos , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Hierro/metabolismo , Masculino , Proteínas Nucleares/metabolismo , Neoplasias de la Próstata/metabolismo , ARN Mensajero/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética/fisiología , Neoplasias del Cuello Uterino/metabolismoRESUMEN
We describe a transcriptional analysis platform consisting of a universal micro-array system (UMAS) combined with an enzymatic manipulation step that is capable of generating expression profiles from any organism without requiring a priori species-specific knowledge of transcript sequences. The transcriptome is converted to cDNA and processed with restriction endonucleases to generate low-complexity pools (approximately 80-120) of equal length DNA fragments. The resulting material is amplified and detected with the UMAS system, comprising all possible 4,096 (4(6)) DNA hexamers. Ligation to the arrays yields thousands of 14-mer sequence tags. The compendium of signals from all pools in the array-of-universal arrays comprises a full-transcriptome expression profile. The technology was validated by analysis of the galactose response of Saccharomyces cerevisiae, and the resulting profiles showed excellent agreement with the literature and real-time PCR assays. The technology was also used to demonstrate expression profiling from a hybrid organism in a proof-of-concept experiment where a T-cell receptor gene was expressed in yeast.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones no Traducidas 3' , Algoritmos , Animales , Fragmentación del ADN , Enzimas de Restricción del ADN/metabolismo , ADN Complementario/metabolismo , Galactosa/metabolismo , Humanos , Procesamiento de Imagen Asistido por Computador , Ratones , Modelos Genéticos , Músculo Esquelético/metabolismo , Músculos/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Saccharomyces cerevisiae/metabolismo , Análisis de Secuencia de ADN , Linfocitos T/metabolismo , TransgenesRESUMEN
Methylation-specific polymerase chain reaction (MS-PCR) is a more rapid way to detect changes in DNA methylation than is bisulfite sequencing. In addition, by incorporating some basic automation, samples can be prepared and analyzed in a 96-well plate format. The method can be used either quantitatively (qRT-PCR-based MethyLight) or qualitatively (using agarose gels) to detect changes in DNA methylation; both are described in this protocol.
Asunto(s)
Metilación de ADN , ADN/química , ADN/genética , Reacción en Cadena de la Polimerasa/métodosRESUMEN
Methylation of DNA, the most experimentally accessible epigenetic alteration of eukaryotic cells, has generated an extensive literature and an abundance of analytical tools. The term "methylome" (referring to the complete set of cytosine modifications in a genome) is appearing with greater frequency in the literature, reflecting the growing number of researchers in the field. Here we introduce a set of robust protocols for methods that can be performed routinely for the elucidation of DNA chemical modifications involving methylation of cytosine. The strengths and limitations of each approach are also discussed.
Asunto(s)
Técnicas de Química Analítica , Técnicas Citológicas/métodos , Citosina/química , Metilación de ADN , ADN/química , Biología Molecular/métodos , Animales , MamíferosRESUMEN
In mammalian cells, DNA methylation at the 5-position of cytosine leads to recruitment of proteins that selectively recognize and bind 5-methylcytosine (5mC). Taking advantage of the structural identity of 5mC, various affinity purification-based protocols have been developed to enrich for either DNA that is modified by 5mC or proteins that recognize 5mC. In this protocol, an antibody against 5mC is used to immunoprecipitate the methylated DNA. The method can be scaled up to perform genome-wide DNA methylation analysis. Because immunoprecipitation is a straightforward procedure that does not require any prior modification of genomic DNA, we also describe several commercial kits available to perform the immunoprecipitation-based detection of DNA methylation.
Asunto(s)
Citosina/inmunología , Metilación de ADN , ADN/inmunología , Inmunoprecipitación/métodos , Citosina/metabolismo , ADN/metabolismo , Juego de Reactivos para DiagnósticoRESUMEN
Here we describe a standard MethylC-seq protocol using single-read sequencing on an Illumina Genome Analyzer II platform. The protocol involves ligation of methylated sequencing adaptors to sonicated genomic DNA, gel purification, sodium bisulfite conversion, polymerase chain reaction (PCR) amplification, and sequencing.
Asunto(s)
Análisis de Secuencia de ADN/métodos , ADN Ligasas/metabolismo , Cartilla de ADN/metabolismo , Reacción en Cadena de la Polimerasa , Sulfitos/metabolismoRESUMEN
This protocol describes methylation mapping analysis by paired-end sequencing (Methyl-MAPS). In addition to the sequence information, paired-end sequencing provides information about the physical distance between the two reads in the genome. Methyl-MAPS typically samples ~80% of the CpG dinucleotides in the genome and is also able to report the methylation status of individual genomic loci harboring repetitive elements. This is achieved by enzymatic fractionation of the genome into methylated and unmethylated compartments. Because the method avoids the use of bisulfite modification, DNA fragments of relatively large size are preserved, permitting the generation of paired-end libraries with DNA inserts of known size in the ranges of 0.8-1.5, 1.5-3, and 3-6 kb. The paired-end configurations can be uniquely mapped to the genome in most instances, because the paired reads will span most repetitive element sequences.
Asunto(s)
Metilación de ADN , ADN/análisis , ADN/química , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Fraccionamiento Químico/métodos , Mapeo Cromosómico/métodosRESUMEN
DNA methylation plays an important role in multiple biological processes. Therefore, methodologies that can detect changes in DNA methylation are of general importance. A popular and reliable method for measuring DNA methylation status is DNA bisulfite sequencing. This protocol details the steps required for bisulfite conversion and analysis of either genes or a specific genomic region. Denatured DNA (i.e., single-stranded DNA) is treated with sodium bisulfite under conditions that preferentially convert unmethylated cytosine (C) residues to uracil (U) residues while methylated cytosines remain unmodified. The converted DNA can then be amplified using a gene-specific primer. U is amplified as thymidine (T) in polymerase chain reaction (PCR) and detected as T during DNA sequencing.
Asunto(s)
Metilación de ADN , Análisis de Secuencia de ADN/métodos , ADN de Cadena Simple/metabolismo , Desnaturalización de Ácido Nucleico , Sulfitos/metabolismoRESUMEN
There are many instances in which it is desirable to generate profiles of the relative abundance of a multiplicity of protein species. Examples include studies in embryonic development, immunobiology, drug responses, cancer biology, biomarkers, and so on. Microarray formats provide a convenient, high-throughput vehicle for generating such profiles, and the repertoire of proteins that can be measured is growing continuously as larger panels of specific antibodies become available. Here we describe methods for the use of antibody microarrays, whereby the detection of specifically bound antigens is enhanced by rolling circle amplification (RCA). RCA-enhanced protein detection on antibody microarrays provides a means for rapid protein profiling at high sensitivity. The set of RCA reagents remains unchanged for different microarray formats and compositions, and signal readout is performed using standard fluorescent dyes and scanners. The method is sensitive enough for the most challenging applications, such as the detection of low-abundance components of human serum.
Asunto(s)
Anticuerpos/química , Análisis por Matrices de Proteínas/instrumentación , Análisis por Matrices de Proteínas/métodos , Proteínas/química , Animales , Colorantes Fluorescentes/farmacología , Haptenos/química , Humanos , Inmunoensayo , Oligonucleótidos/química , Proteómica/métodosRESUMEN
Hypoxia is a common feature of solid tumors and is associated with genetic instability and tumor progression. It has been shown previously that alterations in the expression of DNA repair genes in response to hypoxic stress may account for a proportion of such genetic instability. Here, we demonstrate that the expression of RAD51, a critical mediator of homologous recombination (HR), is repressed by hypoxia in numerous cell lines derived from a wide range of tissues. Repression of this gene by hypoxia occurs in a cell cycle- and hypoxia-inducible factor (HIF)-independent manner, and decreased RAD51 expression was observed to persist during the post-hypoxic period. In addition, decreases in Rad51 expression were correlated with functional impairments in HR repair in hypoxic and post-hypoxic cells. Based on these data, we propose a novel mechanism of hypoxia-induced genetic instability via suppression of the HR pathway in cancer cells within the tumor microenvironment.
Asunto(s)
Reparación del ADN , Regulación Neoplásica de la Expresión Génica , Hipoxia , Ciclo Celular , Línea Celular Tumoral , Separación Celular , Progresión de la Enfermedad , Citometría de Flujo , Humanos , Hipoxia/metabolismo , Luciferasas/metabolismo , Recombinasa Rad51/metabolismo , Recombinación Genética , TransfecciónRESUMEN
An accurate biomarker for the follow-up of women positive for human papillomavirus type 16 (HPV16) DNA may improve the efficiency of cervical cancer prevention. Previously, we analyzed all 113 HPV16 CpGs in cervical cytology samples and discovered differential methylation at different stages of premalignancy. In the current study, we identified a methylation biomarker consisting of a panel of 12 HPV16 CpG sites in the E5, L2, and L1 open reading frames, and tested whether it fulfilled three necessary conditions of a prospective biomarker. A total of 33 cytology samples from North American and West African women with all grades of cervical intraepithelial neoplasia (CIN) and invasive cervical cancer (ICC) were analyzed by using DNA bisulfite sequencing. The results showed (i) a highly significant trend for increasing HPV16 biomarker methylation with increasing histologic severity (P < 0.0001), (ii) 100% sensitivity for ICC over a wide range of methylation cutoff scores; 80% detection of CIN3 at cutoff scores up to 39% methylation, and (iii) substantially lower detection of CIN2, from 0% to 71%, depending on the cutoff score. Our results support the prognostic potential of the HPV16 methylation biomarker for the triage to colposcopy of women with HPV16-positive screening tests and, eventually, for the management of women with HPV16-positive CIN2.
Asunto(s)
Biomarcadores de Tumor/genética , Islas de CpG , Metilación de ADN , Papillomavirus Humano 16/genética , Infecciones por Papillomavirus/diagnóstico , Triaje/métodos , Displasia del Cuello del Útero/diagnóstico , Neoplasias del Cuello Uterino/diagnóstico , Adulto , Anciano , ADN Viral/genética , Femenino , Humanos , Persona de Mediana Edad , Infecciones por Papillomavirus/genética , Infecciones por Papillomavirus/virología , Pronóstico , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/virología , Adulto Joven , Displasia del Cuello del Útero/genética , Displasia del Cuello del Útero/virologíaRESUMEN
Treatment with demethylating drugs can induce demethylation and reactivation of abnormally silenced tumor suppressor genes in cancer cells, but it can also induce potentially deleterious loss of methylation of repetitive elements. To enable the observation of unwanted drug effects related to loss of methylation of repetitive DNA, we have developed a novel biosensor capable of reporting changes in DNA accessibility via luminescence, in living cells. The biosensor design comprises two independent modules, each with a polydactyl zinc finger domain fused to a half intein and to a split-luciferase domain that can be joined by conditional protein splicing after binding to adjacent DNA targets. We show that an artificial zinc finger design specifically targeting DNA sequences near the promoter region of the L1PA2 subfamily of Line-1 retroelements is able to generate luminescent signals, reporting loss of epigenetic silencing and increased DNA accessibility of retroelements in human cells treated with the demethylating drugs decitabine or 5-azacytidine.
Asunto(s)
Metilación de ADN/genética , ADN/genética , Luciferasas/genética , Regiones Promotoras Genéticas/genética , Dedos de Zinc/genética , Azacitidina/análogos & derivados , Azacitidina/farmacología , Secuencia de Bases , Sitios de Unión/genética , Western Blotting , ADN/metabolismo , Metilación de ADN/efectos de los fármacos , Decitabina , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Células HeLa , Humanos , Inteínas/genética , Luciferasas/metabolismo , Mediciones Luminiscentes/instrumentación , Mediciones Luminiscentes/métodos , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Reproducibilidad de los Resultados , Retroelementos/genética , TransfecciónRESUMEN
One of the central aims of cancer research is to identify and characterize cancer-causing alterations in cancer genomes. In recent years, unprecedented advances in genome-wide sequencing, functional genomics technologies for RNA interference screens and methods for evaluating three-dimensional chromatin organization in vivo have resulted in important discoveries regarding human cancer. The cancer-causing genes identified from these new genome-wide technologies have also provided opportunities for effective and personalized cancer therapy. In this review, we describe some of the most recent technologies for cancer gene discovery. We also provide specific examples in which these technologies have proven remarkably successful in uncovering important cancer-causing alterations.
Asunto(s)
Genes Relacionados con las Neoplasias , Genómica/métodos , Neoplasias/genética , Animales , Bases de Datos Genéticas , Perfilación de la Expresión Génica , Genoma/genética , Ensayos Analíticos de Alto Rendimiento , Humanos , Neoplasias/metabolismo , Interferencia de ARNRESUMEN
Maspin is a tumor and metastasis suppressor playing an essential role as gatekeeper of tumor progression. It is highly expressed in epithelial cells but is silenced in the onset of metastatic disease by epigenetic mechanisms. Reprogramming of Maspin epigenetic silencing offers a therapeutic potential to lock metastatic progression. Herein we have investigated the ability of the Artificial Transcription Factor 126 (ATF-126) designed to upregulate the Maspin promoter to inhibit tumor progression in pre-established breast tumors in immunodeficient mice. ATF-126 was transduced in the aggressive, mesenchymal-like and triple negative breast cancer line, MDA-MB-231. Induction of ATF expression in vivo by Doxycycline resulted in 50% reduction in tumor growth and totally abolished tumor cell colonization. Genome-wide transcriptional profiles of ATF-induced cells revealed a gene signature that was found over-represented in estrogen receptor positive (ER+) "Normal-like" intrinsic subtype of breast cancer and in poorly aggressive, ER+ luminal A breast cancer cell lines. The comparison transcriptional profiles of ATF-126 and Maspin cDNA defined an overlapping 19-gene signature, comprising novel targets downstream the Maspin signaling cascade. Our data suggest that Maspin up-regulates downstream tumor and metastasis suppressor genes that are silenced in breast cancers, and are normally expressed in the neural system, including CARNS1, SLC8A2 and DACT3. In addition, ATF-126 and Maspin cDNA induction led to the re-activation of tumor suppressive miRNAs also expressed in neural cells, such as miR-1 and miR-34, and to the down-regulation of potential oncogenic miRNAs, such as miR-10b, miR-124, and miR-363. As expected from its over-representation in ER+ tumors, the ATF-126-gene signature predicted favorable prognosis for breast cancer patients. Our results describe for the first time an ATF able to reduce tumor growth and metastatic colonization by epigenetic reactivation of a dormant, normal-like, and more differentiated gene program.
Asunto(s)
Neoplasias de la Mama/metabolismo , Regulación Neoplásica de la Expresión Génica , Serpinas/metabolismo , Factores de Transcripción/química , Animales , Línea Celular Tumoral , Epigénesis Genética , Femenino , Silenciador del Gen , Genoma Humano , Humanos , Ratones , Ratones SCID , Familia de Multigenes , Metástasis de la Neoplasia , Trasplante de Neoplasias , Ingeniería de Proteínas , Receptores de Estrógenos/metabolismo , Transcripción GenéticaRESUMEN
Close to 50% of the human genome harbors repetitive sequences originally derived from mobile DNA elements, and in normal cells, this sequence compartment is tightly regulated by epigenetic silencing mechanisms involving chromatin-mediated repression. In cancer cells, repetitive DNA elements suffer abnormal demethylation, with potential loss of silencing. We used a genome-wide microarray approach to measure DNA methylation changes in cancers of the head and neck and to compare these changes to alterations found in adjacent non-tumor tissues. We observed specific alterations at thousands of small clusters of CpG dinucleotides associated with DNA repeats. Among the 257,599 repetitive elements probed, 5% to 8% showed disease-related DNA methylation alterations. In dysplasia, a large number of local events of loss of methylation appear in apparently stochastic fashion. Loss of DNA methylation is most pronounced for certain members of the SVA, HERV, LINE-1P, AluY, and MaLR families. The methylation levels of retrotransposons are discretely stratified, with younger elements being highly methylated in healthy tissues, while in tumors, these young elements suffer the most dramatic loss of methylation. Wilcoxon test statistics reveals that a subset of primate LINE-1 elements is demethylated preferentially in tumors, as compared to non-tumoral adjacent tissue. Sequence analysis of these strongly demethylated elements reveals genomic loci harboring full length, as opposed to truncated elements, while possible enrichment for functional LINE-1 ORFs is weaker. Our analysis suggests that, in non-tumor adjacent tissues, there is generalized and highly variable disruption of epigenetic control across the repetitive DNA compartment, while in tumor cells, a specific subset of LINE-1 retrotransposons that arose during primate evolution suffers the most dramatic DNA methylation alterations.
Asunto(s)
Silenciador del Gen , Neoplasias/genética , Primates/genética , Retroelementos/genética , Elementos Alu/genética , Animales , Carcinoma de Células Escamosas/genética , Islas de CpG/genética , Metilación de ADN/fisiología , Retrovirus Endógenos/genética , Epigénesis Genética/genética , Neoplasias de Cabeza y Cuello/genética , Humanos , Elementos de Nucleótido Esparcido Largo/genética , Modelos Biológicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Especificidad de la Especie , Células Tumorales CultivadasRESUMEN
Human papillomavirus (HPV) gene expression is dramatically altered during cervical carcinogenesis. Because dysregulated genes frequently show abnormal patterns of DNA methylation, we hypothesized that comprehensive mapping of the HPV methylomes in cervical samples at different stages of progression would reveal patterns of clinical significance. To test this hypothesis, thirteen HPV16-positive samples were obtained from women undergoing routine cervical cancer screening. Complete methylation data were obtained for 98.7% of the HPV16 CpGs in all samples by bisulfite-sequencing. Most HPV16 CpGs were unmethylated or methylated in only one sample. The other CpGs were methylated at levels ranging from 11% to 100% of the HPV16 copies per sample. The results showed three major patterns and two variants of one pattern. The patterns showed minimal or no methylation (A), low level methylation in the E1 and E6 genes (B), and high level methylation at many CpGs in the E5/L2/L1 region (C). Generally, pattern A was associated with negative cytology, pattern B with low-grade lesions, and pattern C with high-grade lesions. The severity of the cervical lesions was then ranked by the HPV16 DNA methylation patterns and, independently, by the pathologic diagnoses. Statistical analysis of the two rating methods showed highly significant agreement. In conclusion, analysis of the HPV16 DNA methylomes in clinical samples of cervical cells led to the identification of distinct methylation patterns which, after validation in larger studies, could have potential utility as biomarkers of neoplastic cervical progression.