RESUMEN
SUMMARY: Differential DNA methylation and chromatin accessibility are associated with disease development, particularly cancer. Methods that allow profiling of these epigenetic mechanisms in the same reaction and at the single-molecule or single-cell level continue to emerge. However, a challenge lies in jointly visualizing and analyzing the heterogeneous nature of the data and extracting regulatory insight. Here, we present methylscaper, a visualization framework for simultaneous analysis of DNA methylation and chromatin accessibility landscapes. Methylscaper implements a weighted principal component analysis that orders DNA molecules, each providing a record of the chromatin state of one epiallele, and reveals patterns of nucleosome positioning, transcription factor occupancy, and DNA methylation. We demonstrate methylscaper's utility on a long-read, single-molecule methyltransferase accessibility protocol for individual templates (MAPit-BGS) dataset and a single-cell nucleosome, methylation, and transcription sequencing (scNMT-seq) dataset. In comparison to other procedures, methylscaper is able to readily identify chromatin features that are biologically relevant to transcriptional status while scaling to larger datasets. AVAILABILITY AND IMPLEMENTATION: Methylscaper, is implemented in R (version > 4.1) and available on Bioconductor: https://bioconductor.org/packages/methylscaper/, GitHub: https://github.com/rhondabacher/methylscaper/, and Web: https://methylscaper.com. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Asunto(s)
Aplicaciones Móviles , Nucleosomas , Metilación de ADN , Cromatina , Epigénesis Genética , ADNRESUMEN
Spontaneous lytic reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) occurs at a low rate in latently infected cells in disease and culture. This suggests imperfect epigenetic maintenance of viral transcription programs, perhaps due to variability in chromatin structure at specific loci across the population of KSHV episomal genomes. To characterize this locus-specific chromatin structural diversity, we used MAPit single-molecule footprinting, which simultaneously maps endogenous CG methylation and accessibility to M.CviPI at GC sites. Diverse chromatin structures were detected at the LANA, RTA and vIL6 promoters. At each locus, chromatin ranged from fully closed to fully open across the population. This diversity has not previously been reported in a virus. Phorbol ester and RTA transgene induction were used to identify chromatin conformations associated with reactivation of lytic transcription, which only a fraction of episomes had. Moreover, certain chromatin conformations correlated with CG methylation patterns at the RTA and vIL6 promoters. This indicated that some of the diverse chromatin conformations at these loci were epigenetically distinct. Finally, by comparing chromatin structures from a cell line infected with constitutively latent virus, we identified products of lytic replication. Our findings show that epigenetic drift can restrict viral propagation by chromatin compaction at latent and lytic promoters.
Asunto(s)
Cromatina/metabolismo , Epigénesis Genética , Regulación Viral de la Expresión Génica , Herpesvirus Humano 8/fisiología , Secuencia de Bases , Línea Celular Tumoral , Cromatina/genética , Cromatina/virología , Ensamble y Desensamble de Cromatina , Mapeo Cromosómico , Islas de CpG , Metilación de ADN , Sitios Genéticos , Interacciones Huésped-Patógeno , Humanos , Proteínas Inmediatas-Precoces/biosíntesis , Proteínas Inmediatas-Precoces/genética , Regiones Promotoras Genéticas , Transactivadores/biosíntesis , Transactivadores/genética , Latencia del VirusRESUMEN
Nanog or Gata6-positive cells co-exist and are convertible within the inner cell mass of murine blastocysts and embryonic stem (ES) cells. Previous studies demonstrate fibroblast growth factor receptor 2 (FGFR2) triggers Nanog gene down-regulation and differentiation to primitive endoderm (PE); however, the underlying mechanisms responsible for reversible and fluctuating cell fate are poorly understood. Using an inducible FGFR2 dimerization system in ES cells, we demonstrate that FGFR2 activation rapidly down-regulated Nanog gene transcription through activation of the Mek pathway and subsequently differentiated ES cells into PE cells. FGFR2 rather selectively repressed the Nanog gene with minimal effect on other pluripotency genes, including Oct4 and Sox2. We determined the Nanog promoter region containing minimum Oct4/Sox2 binding sites was sufficient for this transcriptional down-regulation by FGFR2, when the reporter transgenes were integrated with insulators. Of interest, FGFR2-mediated Nanog transcriptional reduction occurred without dissociation of RNA polymerase II, p300, Oct4, Sox2, and Tet1 from the Nanog proximal promoter region and with no increase in repressive histone methylation marks or DNA methylation, implying the gene repression is in the early and transient phase. Furthermore, addition of a specific FGFR inhibitor readily reversed this Nanog repression status. These findings illustrate well how FGFR2 induces rapid but reversible Nanog repression within ES cells.
Asunto(s)
Células Madre Embrionarias/metabolismo , Proteínas de Homeodominio/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Multimerización de Proteína/fisiología , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Transcripción Genética/fisiología , Animales , Línea Celular , Metilación de ADN/fisiología , Células Madre Embrionarias/citología , Endodermo/citología , Endodermo/embriología , Proteínas de Homeodominio/genética , Ratones , Proteína Homeótica Nanog , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismoRESUMEN
Bisulfite sequencing is a widely-used technique for examining cytosine DNA methylation at nucleotide resolution along single DNA strands. Probing with cytosine DNA methyltransferases followed by bisulfite sequencing (MAPit) is an effective technique for mapping protein-DNA interactions. Here, MAPit methylation footprinting with M.CviPI, a GC methyltransferase we previously cloned and characterized, was used to probe hMLH1 chromatin in HCT116 and RKO colorectal cancer cells. Because M.CviPI-probed samples contain both CG and GC methylation, we developed a versatile, visually-intuitive program, called MethylViewer, for evaluating the bisulfite sequencing results. Uniquely, MethylViewer can simultaneously query cytosine methylation status in bisulfite-converted sequences at as many as four different user-defined motifs, e.g. CG, GC, etc., including motifs with degenerate bases. Data can also be exported for statistical analysis and as publication-quality images. Analysis of hMLH1 MAPit data with MethylViewer showed that endogenous CG methylation and accessible GC sites were both mapped on single molecules at high resolution. Disruption of positioned nucleosomes on single molecules of the PHO5 promoter was detected in budding yeast using M.CviPII, increasing the number of enzymes available for probing protein-DNA interactions. MethylViewer provides an integrated solution for primer design and rapid, accurate and detailed analysis of bisulfite sequencing or MAPit datasets from virtually any biological or biochemical system.
Asunto(s)
Islas de CpG , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , Análisis de Secuencia de ADN/métodos , Programas Informáticos , Sulfitos/química , Proteínas Adaptadoras Transductoras de Señales/genética , Línea Celular Tumoral , Cromatina/metabolismo , Biología Computacional , Citidina/análisis , Citidina/metabolismo , Citosina/metabolismo , ADN/química , Humanos , Aumento de la Imagen , Homólogo 1 de la Proteína MutL , Proteínas Nucleares/genética , Alineación de SecuenciaRESUMEN
Aberrant activation of the Wnt/ß-catenin signaling axis is a prominent oncogenic mechanism in numerous cancers including cervical cancer. Wnt inhibitory factor-1 (WIF1) is a secreted protein that binds Wnt and antagonizes Wnt activity. While the WIF1 gene is characterized as a target for epigenetic silencing in some tumor types, WIF1 expression has not been examined in human cervical tissue and cervical cancer. Here, we show that WIF1 is unmethylated and its gene product is expressed in normal cervical epithelium and some cultured cervical tumor lines. In contrast, several cervical cancer lines contained dense CpG methylation within the WIF1 gene, and expression of both WIF1 transcript and protein was restored by culturing cells in the presence of the global DNA demethylating agent 5-aza-2'-deoxycytidine. Using single-molecule MAPit methylation footprinting, we observed differences in chromatin structure within the WIF1 promoter region between cell lines that express and those that do not express WIF1, consistent with transcriptional activity and repression, respectively. The WIF1 promoter was aberrantly methylated in â¼60% (10 of 17) high-grade highly undifferentiated squamous cell cervical tumors examined, whereas paired normal tissue showed significantly lower levels of CpG methylation. WIF1 protein was not detectable by immunohistochemistry in tumors with quantitatively high levels of WIF1 methylation. Of note, WIF1 protein was not detectable in two of the seven unmethylated cervical tumors examined, suggesting other mechanisms may contribute WIF1 repression. Our findings establish the WIF1 gene as a frequent target for epigenetic silencing in squamous cell carcinoma of the cervix.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Carcinoma de Células Escamosas/genética , Metilación de ADN , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Proteínas Represoras/genética , Neoplasias del Cuello Uterino/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Azacitidina/análogos & derivados , Azacitidina/farmacología , Western Blotting , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Línea Celular , Cuello del Útero/metabolismo , Islas de CpG/genética , Decitabina , Femenino , Silenciador del Gen , Humanos , Técnicas para Inmunoenzimas , Regiones Promotoras Genéticas , ARN Mensajero/genética , Proteínas Represoras/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/patologíaRESUMEN
Enhancers and promoters assemble protein complexes that ultimately regulate the recruitment and activity of RNA polymerases. Previous work has shown that at least some enhancers form stable protein complexes, leading to the formation of enhanceosomes. We analyzed protein-DNA interactions in the murine ß-globin gene locus using the methyltransferase accessibility protocol for individual templates (MAPit). The data show that a tandem Maf recognition element (MARE) in locus control region (LCR) hypersensitive site 2 (HS2) reveals a remarkably high degree of occupancy during differentiation of mouse erythroleukemia cells. Most of the other transcription factor binding sites in LCR HS2 or in the adult ß-globin gene promoter regions exhibit low fractional occupancy, suggesting highly dynamic protein-DNA interactions. Targeting of an artificial zinc finger DNA-binding domain (ZF-DBD) to the HS2 tandem MARE caused a reduction in the association of MARE-binding proteins and transcription complexes at LCR HS2 and the adult ßmajor-globin gene promoter but did not affect expression of the ßminor-globin gene. The data demonstrate that a stable MARE-associated footprint in LCR HS2 is important for the recruitment of transcription complexes to the adult ßmajor-globin gene promoter during erythroid cell differentiation.
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Regulación Leucémica de la Expresión Génica , Leucemia Eritroblástica Aguda/genética , Región de Control de Posición , Activación Transcripcional , Globinas beta/genética , Animales , Diferenciación Celular , Línea Celular Tumoral , ADN/genética , ADN/metabolismo , Células Eritroides/citología , Células Eritroides/metabolismo , Regulación de la Expresión Génica , Leucemia Eritroblástica Aguda/metabolismo , Ratones , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , Factores de Transcripción/metabolismo , Dedos de ZincRESUMEN
Chromatin limits the accessibility of DNA to trans-acting factors in transcription, replication, and repair. Although transcriptional variation between cells in a population may contribute to survival and disease, most assays of chromatin structure recover only population averages. We have developed DNA methyltransferases (MTases) as probing agents of DNA accessibility in chromatin, either expressed in vivo in budding yeast or as recombinant enzymatic probes of nuclei isolated from mammalian cells. In this chapter, we focus on the use of recombinant MTase (M) M.CviPI to probe chromatin accessibility in nuclei isolated from mammalian cell lines and animal tissue. This technique, named methylation accessibility protocol for individual templates (MAPit), reports protein-DNA interactions at single-molecule resolution. The single-molecule readout allows identification of chromatin subpopulations and rare epigenetic variants within a cell population. Furthermore, the use of M.CviPI in mammalian systems gives a comprehensive view of both chromatin structure and endogenous DNA methylation in a single assay.
Asunto(s)
Cromatina/genética , Cromatina/metabolismo , Metilación de ADN , Animales , Cromatina/química , Islas de CpG , ADN-Citosina Metilasas/metabolismo , Humanos , Unión Proteica , Análisis de Secuencia de ADN/métodosRESUMEN
Chromosomal translocations are a hallmark of hematopoietic malignancies. CG motifs within translocation fragile zones (typically 20 to 600 bp in size) are prone to chromosomal translocation in lymphomas. Here we demonstrate that the CG motifs in human translocation fragile zones are hypomethylated relative to the adjacent DNA. Using a methyltransferase footprinting assay on isolated nuclei (in vitro), we find that the chromatin at these fragile zones is accessible. We also examined in vivo accessibility using cellular expression of a prokaryotic methylase. Based on this assay, which measures accessibility over a much longer time interval than is possible with in vitro methods, these fragile zones were found to be more accessible than the adjacent DNA. Because DNA within the fragile zones can be methylated by both cellular and exogenous methyltransferases, the fragile zones are predominantly in a duplex DNA conformation. These observations permit more-refined models for why these zones are 100- to 1,000-fold more prone to undergo chromosomal translocation than the adjacent regions.
Asunto(s)
Sitios Frágiles del Cromosoma , Linfoma/genética , Translocación Genética , Línea Celular Tumoral , Células Cultivadas , Cromatina/genética , ADN/genética , Metilación de ADN , HumanosRESUMEN
TRIM29 (ATDC) exhibits a contextual function in cancer, but seems to exert a tumor-suppressor role in breast cancer. Here, we show that TRIM29 is often silenced in primary breast tumors and cultured tumor cells as a result of aberrant gene hypermethylation. RNAi-mediated silencing of TRIM29 in breast tumor cells increased their motility, invasiveness, and proliferation in a manner associated with increased expression of mesenchymal markers (N-cadherin and vimentin), decreased expression of epithelial markers (E-cadherin and EpCAM), and increased expression and activity of the oncogenic transcription factor TWIST1, an important driver of the epithelial-mesenchymal transition (EMT). Functional investigations revealed an inverse relationship in the expression of TRIM29 and TWIST1, suggesting the existence of a negative regulatory feedback loop. In support of this relationship, we found that TWIST1 inhibited TRIM29 promoter activity through direct binding to a region containing a cluster of consensus E-box elements, arguing that TWIST1 transcriptionally represses TRIM29 expression. Analysis of a public breast cancer gene-expression database indicated that reduced TRIM29 expression was associated with reduced relapse-free survival, increased tumor size, grade, and metastatic characteristics. Taken together, our results suggest that TRIM29 acts as a tumor suppressor in breast cancer through its ability to inhibit TWIST1 and suppress EMT.
Asunto(s)
Neoplasias de la Mama/genética , Proteínas de Unión al ADN/genética , Invasividad Neoplásica/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Proteína 1 Relacionada con Twist/genética , Antígenos de Neoplasias/genética , Cadherinas/genética , Moléculas de Adhesión Celular/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Metilación de ADN/genética , Elementos E-Box/genética , Molécula de Adhesión Celular Epitelial , Transición Epitelial-Mesenquimal/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Recurrencia Local de Neoplasia/genética , Regiones Promotoras Genéticas/genética , Transcripción Genética/genética , Vimentina/genéticaRESUMEN
A single-molecule probe of chromatin structure can uncover dynamic chromatin states and rare epigenetic variants of biological importance that bulk measures of chromatin structure miss. In bisulfite genomic sequencing, each sequenced clone records the methylation status of multiple sites on an individual molecule of DNA. An exogenous DNA methyltransferase can thus be used to image nucleosomes and other protein-DNA complexes. In this chapter, we describe the adaptation of this technique, termed Methylation Accessibility Protocol for individual templates, to modern high-throughput sequencing, which both simplifies the workflow and extends its utility.
Asunto(s)
Biología Computacional/métodos , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , ADN/metabolismo , Neurofibromina 1/genética , Nucleosomas/metabolismo , Islas de CpG , ADN/genética , Biblioteca de Genes , Genoma Humano , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Neurofibromina 1/metabolismo , Nucleosomas/genética , Reacción en Cadena de la Polimerasa/métodos , Regiones Promotoras Genéticas , Análisis de Secuencia de ADN , Sulfitos/metabolismo , Moldes GenéticosRESUMEN
Bisulfite genomic sequencing provides a single-molecule view of cytosine methylation states. After deamination, each cloned molecule contains a record of methylation within its sequence. The full power of this technique is harnessed by treating nuclei with an exogenous DNMT prior to DNA extraction. This exogenous methylation marks regions of accessibility and footprints nucleosomes, as well as other DNA-binding proteins. Thus, each cloned molecule records not only the endogenous methylation present (at CG sites, in mammals), but also the exogenous (GC, when using the Chlorella virus protein M.CviPI). We term this technique MAPit, methylation accessibility protocol for individual templates.
Asunto(s)
5-Metilcitosina/metabolismo , Cromatina/metabolismo , Metilación de ADN/genética , Biología Molecular/métodos , Secuencia de Bases , Ensamble y Desensamble de Cromatina , Clonación Molecular , Humanos , Células K562 , Datos de Secuencia Molecular , Sulfitos , Sitio de Iniciación de la TranscripciónRESUMEN
Sites of protein binding to DNA are inferred from footprints or spans of protection against a probing reagent. In most protocols, sites of accessibility to a probe are detected by mapping breaks in DNA strands. As discussed in this unit, such methods obscure molecular heterogeneity by averaging cuts at a given site over all DNA strands in a sample population. The DNA methyltransferase accessibility protocol for individual templates (MAPit), an alternative method described in this unit, localizes protein-DNA interactions by probing with cytosine-modifying DNA methyltransferases followed by bisulfite sequencing. Sequencing individual DNA products after amplification of bisulfite-converted sequences permits assignment of the methylation status of every enzyme target site along a single DNA strand. Use of the GC-methylating enzyme M.CviPI allows simultaneous mapping of chromatin accessibility and endogenous CpG methylation. MAPit is therefore the only footprinting method that can detect subpopulations of molecules with distinct patterns of protein binding or chromatin architecture and correlate them directly with the occurrence of endogenous methylation. Additional advantages of MAPit methylation footprinting as well as considerations for experimental design and potential sources of error are discussed.
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Huella de ADN/métodos , Metilación de ADN , ADN/química , Proteínas/química , Análisis de Secuencia de ADN/métodos , Animales , Secuencia de Bases , Cromatina , Islas de CpG , Citosina/química , ADN (Citosina-5-)-Metiltransferasas/química , Humanos , Datos de Secuencia Molecular , Regiones Promotoras GenéticasRESUMEN
Exact positions of 5-methylcytosine (m(5)C) on a single strand of DNA can be determined by bisulfite genomic sequencing (BGS). Treatment with bisulfite ion preferentially deaminates unmethylated cytosines, which are then converted to uracil upon desulfonation. Amplifying regions of interest from deaminated DNA and sequencing products cloned from amplicons permits determination of methylation at single-nucleotide resolution along single DNA molecules, which is not possible with other methylation analysis techniques. This unit describes a BGS technique suitable for most DNA sources, including formaldehyde-fixed tissue. Considerations for experimental design and common sources of error are discussed.