RESUMO
The effect of exertional heat stroke (EHS) exposure on skeletal muscles is incompletely understood. Muscle weakness is an early symptom of EHS but is not considered a major target of multiorgan injury. Previously, in a preclinical mouse model of EHS, we observed the vulnerability of limb muscles to a second EHS exposure, suggesting hidden processes contributing to declines in muscle resilience. Here, we evaluated the possible molecular origins of EHS-induced declines in muscle resilience. Female C57BL/6 mice [total n = 56; 28/condition, i.e., EHS and exercise control (EXC)] underwent forced wheel running at 37.5°C/40% relative humidity until symptom limitation (unconsciousness). EXC mice exercised identically at room temperature (22-23°C). After 1 mo of recovery, the following were assessed: 1) specific force and caffeine-induced contracture in soleus (SOL) and extensor digitorum longus (EDL) muscles; 2) transcriptome and DNA methylome responses in gastrocnemius (GAST); and 3) primary satellite cell function (proliferation and differentiation). There were no differences in specific force in either SOL or EDL from EXC. Only EHS solei exhibited lower caffeine sensitivity. EHS GAST exhibited higher RNA expression of genes encoding structural proteins of slow fibers, heat shock proteins, and myogenesis. A total of â¼2,500 differentially methylated regions of DNA that could potentially affect many cell functions were identified. Primary satellite cells exhibited suppressed proliferation rates but normal differentiation responses. Results demonstrate long-term changes in skeletal muscles 1 mo after EHS that could contribute to declines in muscle resilience. Skeletal muscle may join other, more recognized tissues considered vulnerable to long-term effects of EHS.NEW & NOTEWORTHY Exertional heat stroke (EHS) in mice induces long-term molecular and functional changes in limb muscle that could reflect a loss of "resilience" to further stress. The phenotype was characterized by altered caffeine sensitivity and suppressed satellite cell proliferative potential. This was accompanied by changes in gene expression and DNA methylation consistent with ongoing muscle remodeling and stress adaptation. We propose that EHS may induce a prolonged vulnerability of skeletal muscle to further stress or injury.
Assuntos
Cafeína , Golpe de Calor , Camundongos , Feminino , Animais , Atividade Motora , Camundongos Endogâmicos C57BL , Músculo Esquelético/fisiologia , Golpe de Calor/genética , Transcriptoma , Epigênese GenéticaRESUMO
Evidence from human epidemiological studies suggests that exertional heat stroke (EHS) results in an elevated risk of long-term cardiovascular and systemic disease. Previous results using a preclinical mouse model of EHS demonstrated severe metabolic imbalances in ventricular myocardium developing at 9-14 days of recovery. Whether this resolves over time is unknown. We hypothesized that the long-term effects of EHS on the heart reflect retained maladaptive epigenetic responses. In this study, we evaluated genome-wide DNA methylation, RNA-Seq, and metabolomic profiles of the left ventricular myocardium in female C57BL/6 mice, 30 days after EHS (exercise in 37.5°C; n = 7-8), compared with exercise controls. EHS mice ran to loss of consciousness, reaching core temperatures of 42.4 ± 0.2°C. All mice recovered quickly. After 30 days, the left ventricles were rapidly frozen for DNA methyl sequencing, RNA-Seq, and untargeted metabolomics. Ventricular DNA from EHS mice revealed >13,000 differentially methylated cytosines (DMCs) and >900 differentially methylated regions (DMRs; ≥5 DMCs with ≤300 bp between each CpG). Pathway analysis using DMRs revealed alterations in genes regulating basic cell functions, DNA binding, transcription, and metabolism. Metabolomics and mRNA expression revealed modest changes that are consistent with a return to homeostasis. Methylation status did not predict RNA expression or metabolic state at 30 days. We conclude that EHS induces a sustained DNA methylation memory lasting over 30 days of recovery, but ventricular gene expression and metabolism return to a relative homeostasis at rest. Such long-lasting alterations to the DNA methylation landscape could alter responsiveness to environmental or clinical challenges later in life.
Assuntos
Ventrículos do Coração , Golpe de Calor , Humanos , Animais , Camundongos , Feminino , Camundongos Endogâmicos C57BL , Golpe de Calor/genética , Golpe de Calor/metabolismo , Miocárdio/metabolismo , Epigênese GenéticaRESUMO
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.
Assuntos
Aplicativos Móveis , Nucleossomos , Metilação de DNA , Cromatina , Epigênese Genética , DNARESUMO
KEY POINTS: Exposure to exertional heat stroke (EHS) has been linked to increased long-term decrements of health. Epigenetic reprogramming is involved in the response to heat acclimation; however, whether the long-term effects of EHS are mediated by epigenetic reprogramming is unknown. In female mice, we observed DNA methylation reprogramming in bone marrow-derived (BMD) monocytes as early as 4 days of recovery from EHS and as late as 30 days compared with sham exercise controls. Whole blood, collected after 30 days of recovery from EHS, exhibited an immunosuppressive phenotype when challenged in vitro by lipopolysaccharide. After 30 days of recovery from EHS, BMD monocytes exhibited an altered in vitro heat shock response. The location of differentially methylated CpGs are predictive of both the immunosuppressive phenotype and altered heat shock responses. ABSTRACT: Exposure to exertional heat stroke (EHS) has been linked to increased susceptibility to a second heat stroke, infection and cardiovascular disease. Whether these clinical outcomes are mediated by an epigenetic memory is unknown. Using a preclinical mouse model of EHS, we investigated whether EHS exposure produces a lasting epigenetic memory in monocytes and whether there are phenotypic alterations that may be consistent with these epigenetic changes. Female mice underwent forced wheel running at 37.5°C/40% relative humidity until symptom limitation, characterized by CNS dysfunction. Results were compared with matched exercise controls at 22.5°C. Monocytes were isolated from bone marrow after 4 or 30 days of recovery to extract DNA and analyse methylation. Broad-ranging alterations to the DNA methylome were observed at both time points. At 30 days, very specific alterations were observed to the promoter regions of genes involved with immune responsiveness. To test whether these changes might be related to phenotype, whole blood at 30 days was challenged with lipopolysaccharide (LPS) to measure cytokine secretion; monocytes were also challenged with heat shock to quantify mRNA expression. Whole blood collected from EHS mice showed markedly attenuated inflammatory responses to LPS challenge. Furthermore, monocyte mRNA from EHS mice showed significantly altered responses to heat shock challenge. These results demonstrate that EHS leads to a unique DNA methylation pattern in monocytes and altered immune and heat shock responsiveness after 30 days. These data support the hypothesis that EHS exposure can induce long-term physiological changes that may be linked to altered epigenetic profiles.
Assuntos
Golpe de Calor , Atividade Motora , Animais , Epigênese Genética , Feminino , Golpe de Calor/genética , Resposta ao Choque Térmico/genética , Terapia de Imunossupressão , CamundongosRESUMO
The ATP-dependent chromatin remodeling complex SWI/SNF regulates transcription and has been implicated in promoter nucleosome eviction. Efficient nucleosome disassembly by SWI/SNF alone in biochemical assays, however, has not been directly observed. Employing a model system of dinucleosomes rather than mononucleosomes, we demonstrate that remodeling leads to ordered and efficient disassembly of one of the two nucleosomes. An H2A/H2B dimer is first rapidly displaced, and then, in a slower reaction, an entire histone octamer is lost. Nucleosome disassembly by SWI/SNF did not require additional factors such as chaperones or acceptors of histones. Observations in single molecules as well as bulk measurement suggest that a key intermediate in this process is one in which a nucleosome is moved toward the adjacent nucleosome. SWI/SNF recruited by the transcriptional activator Gal4-VP16 preferentially mobilizes the proximal nucleosome and destabilizes the adjacent nucleosome.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Nucleossomos/metabolismo , Fatores de Transcrição/metabolismo , Mapeamento Cromossômico , Modelos Moleculares , Nucleossomos/genética , Regiões Promotoras Genéticas/genética , Transcrição Gênica/genéticaRESUMO
Human tumors are comprised of heterogeneous cell populations that display diverse molecular and phenotypic features. To examine the extent to which epigenetic differences contribute to intratumoral cellular heterogeneity, we have developed a high-throughput method, termed MAPit-patch. The method uses multiplexed amplification of targeted sequences from submicrogram quantities of genomic DNA followed by next generation bisulfite sequencing. This provides highly scalable and simultaneous mapping of chromatin accessibility and DNA methylation on single molecules at high resolution. Long sequencing reads from targeted regions maintain the structural integrity of epigenetic information and provide substantial depth of coverage, detecting for the first time minority subpopulations of epigenetic configurations formerly obscured by existing genome-wide and population-ensemble methodologies. Analyzing a cohort of 71 promoters of genes with exons commonly mutated in cancer, MAPit-patch uncovered several differentially accessible and methylated promoters that are associated with altered gene expression between neural stem cell (NSC) and glioblastoma (GBM) cell populations. In addition, considering each promoter individually, substantial epigenetic heterogeneity was observed across the sequenced molecules, indicating the presence of epigenetically distinct cellular subpopulations. At the divergent MLH1/EPM2AIP1 promoter, a locus with three well-defined, nucleosome-depleted regions (NDRs), a fraction of promoter copies with inaccessible chromatin was detected and enriched upon selection of temozolomide-tolerant GBM cells. These results illustrate the biological relevance of epigenetically distinct subpopulations that in part underlie the phenotypic heterogeneity of tumor cell populations. Furthermore, these findings show that alterations in chromatin accessibility without accompanying changes in DNA methylation may constitute a novel class of epigenetic biomarker.
Assuntos
Metilação de DNA/genética , Epigênese Genética/genética , Glioblastoma/genética , Células-Tronco Neurais , Linhagem Celular Tumoral , Cromatina/genética , Mapeamento Cromossômico , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Nucleossomos/genética , Regiões Promotoras Genéticas , Análise de Sequência de DNARESUMO
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.
Assuntos
Cromatina/metabolismo , Epigênese Genética , Regulação Viral da Expressão Gênica , Herpesvirus Humano 8/fisiologia , Sequência de Bases , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/virologia , Montagem e Desmontagem da Cromatina , Mapeamento Cromossômico , Ilhas de CpG , Metilação de DNA , Loci Gênicos , Interações Hospedeiro-Patógeno , Humanos , Proteínas Imediatamente Precoces/biossíntese , Proteínas Imediatamente Precoces/genética , Regiões Promotoras Genéticas , Transativadores/biossíntese , Transativadores/genética , Latência ViralRESUMO
Genome-scale mapping suggests that the function of DNA methylation varies with genomic context beyond transcriptional repression. However, the use of DNA-demethylating agents (e.g. 5-aza-2'-deoxycytidine (5aza-dC)) to study epigenetic regulation often focuses on gene activation and ignores repression elicited by 5aza-dC. Here, we show that repression of NEK2, which encodes the never in mitosis A (NIMA)-related kinase, by 5aza-dC is context-specific as NEK2 transcript levels were reduced in HCT116 colon cancer cells but not in isogenic p53(-/-) cells. Bisulfite sequencing showed that DNA methylation was restricted to the distal region of the NEK2 promoter. Demethylation by 5aza-dC was associated with increased accessibility to micrococcal nuclease, i.e. nucleosome depletion. Conversely, methyltransferase accessibility protocol for individual templates (MAPit) methylation footprinting showed that nucleosome occupancy and DNA methylation at the distal promoter were significantly increased in p53(-/-) cells, suggesting dynamic regulation of chromatin structure at this region by p53 in HCT116 cells. Stabilization of endogenous p53 by doxorubicin or ectopic expression of p53, but not a p53 DNA-binding mutant, decreased NEK2 expression. Chromatin immunoprecipitation demonstrated direct and specific association of p53 with the distal NEK2 promoter, which was enhanced by doxorubicin. Luciferase reporters confirmed that this region is required for p53-mediated repression of NEK2 promoter activity. Lastly, modulation of p53 abundance altered nucleosome occupancy and DNA methylation at its binding region. These results identify NEK2 as a novel p53-repressed gene, illustrate that its repression by 5aza-dC is specific and associated with nucleosome reorganization, and provide evidence that identification of partially methylated regions can reveal novel p53 target genes.
Assuntos
Metilação de DNA , Regiões Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinases/genética , Proteína Supressora de Tumor p53/metabolismo , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Sequência de Bases , Sítios de Ligação , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Proliferação de Células/efeitos dos fármacos , DNA/metabolismo , Metilação de DNA/efeitos dos fármacos , Decitabina , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Células HCT116 , Humanos , Quinases Relacionadas a NIMA , Nucleossomos/efeitos dos fármacos , Nucleossomos/genética , Nucleossomos/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genéticaRESUMO
Angiogenesis is meticulously controlled by a fine balance between positive and negative regulatory activities. Vascular endothelial growth factor (VEGF) is a predominant angiogenic factor and its dosage is precisely regulated during normal vascular formation. In cancer, VEGF is commonly overproduced, resulting in abnormal neovascularization. VEGF is induced in response to various stimuli including hypoxia; however, very little is known about the mechanisms that confine its induction to ensure proper angiogenesis. Chromatin insulation is a key transcription mechanism that prevents promiscuous gene activation by interfering with the action of enhancers. Here we show that the chromatin insulator-binding factor CTCF binds to the proximal promoter of VEGF. Consistent with the enhancer-blocking mode of chromatin insulators, CTCF has little effect on basal expression of VEGF but specifically affects its activation by enhancers. CTCF knockdown cells are sensitized for induction of VEGF and exhibit elevated proangiogenic potential. Cancer-derived CTCF missense mutants are mostly defective in blocking enhancers at the VEGF locus. Moreover, during mouse retinal development, depletion of CTCF causes excess angiogenesis. Therefore, CTCF-mediated chromatin insulation acts as a crucial safeguard against hyperactivation of angiogenesis.
Assuntos
Cromatina/metabolismo , Elementos Isolantes/genética , Neovascularização Patológica/genética , Proteínas Repressoras/metabolismo , Dedos de Zinco/genética , Animais , Fator de Ligação a CCCTC , Linhagem Celular , Elementos Facilitadores Genéticos/genética , Genes Reporter/genética , Humanos , Camundongos , Neoplasias/irrigação sanguínea , Neoplasias/patologia , Neovascularização Patológica/patologia , Regiões Promotoras Genéticas/genética , Ligação Proteica , Retina/crescimento & desenvolvimento , Retina/patologia , Transcrição Gênica , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: The Advanced Plant Experiment-04 - Epigenetic Expression (APEX-04-EpEx) experiment onboard the International Space Station examined the spaceflight-altered cytosine methylation in two genetic lines of Arabidopsis thaliana, wild-type Col-0 and the mutant elp2-5, which is deficient in an epigenetic regulator Elongator Complex Subunit 2 (ELP2). Whole-genome bisulfite sequencing (WGBS) revealed distinct spaceflight associated methylation differences, presenting the need to explore specific space-altered methylation at single-molecule resolution to associate specific changes over large regions of spaceflight related genes. To date, tools of multiplexed targeted DNA methylation sequencing remain limited for plant genomes. RESULTS: To provide methylation data at single-molecule resolution, Flap-enabled next-generation capture (FENGC), a novel targeted multiplexed DNA capture and enrichment technique allowing cleavage at any specified sites, was applied to survey spaceflight-altered DNA methylation in genic regions of interest. The FENGC capture panel contained 108 targets ranging from 509 to 704 nt within the promoter or gene body regions of gene targets derived from spaceflight whole-genome data sets. In addition to genes with significant changes in expression and average methylation levels between spaceflight and ground control, targets with space-altered distributions of the proportion of methylated cytosines per molecule were identified. Moreover, trends of co-methylation of different cytosine contexts were exhibited in the same DNA molecules. We further identified significant DNA methylation changes in three previously biological process-unknown genes, and loss-of-function mutants of two of these genes (named as EMO1 and EMO2 for ELP2-regulated Methylation in Orbit 1 and 2) showed enhanced root growth rate. CONCLUSIONS: FENGC simplifies and reduces the cost of multiplexed, targeted, single-molecule profiling of methylation in plants, providing additional resolution along each DNA molecule that is not seen in population-based short-read data such as WGBS. This case study has revealed spaceflight-altered regional modification of cytosine methylation occurring within single DNA molecules of cell subpopulations, which were not identified by WGBS. The single-molecule survey by FENGC can lead to identification of novel functional genes. The newly identified EMO1 and EMO2 are root growth regulators which may be epigenetically involved in plant adaptation to spaceflight.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Metilação de DNA , Raízes de Plantas , Voo Espacial , Arabidopsis/genética , Raízes de Plantas/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Epigênese GenéticaRESUMO
ABSTRACT: Postsepsis early mortality is being replaced by survivors who experience either a rapid recovery and favorable hospital discharge or the development of chronic critical illness with suboptimal outcomes. The underlying immunological response that determines these clinical trajectories remains poorly defined at the transcriptomic level. As classical and nonclassical monocytes are key leukocytes in both the innate and adaptive immune systems, we sought to delineate the transcriptomic response of these cell types. Using single-cell RNA sequencing and pathway analyses, we identified gene expression patterns between these two groups that are consistent with differences in TNF-α production based on clinical outcome. This may provide therapeutic targets for those at risk for chronic critical illness in order to improve their phenotype/endotype, morbidity, and long-term mortality.
Assuntos
Monócitos , Sepse , Transcriptoma , Humanos , Monócitos/metabolismo , Monócitos/imunologia , Sepse/imunologia , Sepse/genética , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Introduction: Sepsis engenders distinct host immunologic changes that include the expansion of myeloid-derived suppressor cells (MDSCs). These cells play a physiologic role in tempering acute inflammatory responses but can persist in patients who develop chronic critical illness. Methods: Cellular Indexing of Transcriptomes and Epitopes by Sequencing and transcriptomic analysis are used to describe MDSC subpopulations based on differential gene expression, RNA velocities, and biologic process clustering. Results: We identify a unique lineage and differentiation pathway for MDSCs after sepsis and describe a novel MDSC subpopulation. Additionally, we report that the heterogeneous response of the myeloid compartment of blood to sepsis is dependent on clinical outcome. Discussion: The origins and lineage of these MDSC subpopulations were previously assumed to be discrete and unidirectional; however, these cells exhibit a dynamic phenotype with considerable plasticity.
Assuntos
Células Supressoras Mieloides , Sepse , Células Supressoras Mieloides/imunologia , Células Supressoras Mieloides/metabolismo , Humanos , Sepse/imunologia , Transcriptoma , Masculino , Feminino , Diferenciação Celular/imunologia , Perfilação da Expressão GênicaRESUMO
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.
Assuntos
Células-Tronco Embrionárias/metabolismo , Proteínas de Homeodomínio/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Multimerização Proteica/fisiologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Transcrição Gênica/fisiologia , Animais , Linhagem Celular , Metilação de DNA/fisiologia , Células-Tronco Embrionárias/citologia , Endoderma/citologia , Endoderma/embriologia , Proteínas de Homeodomínio/genética , Camundongos , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismoRESUMO
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.
Assuntos
Ilhas de CpG , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Análise de Sequência de DNA/métodos , Software , Sulfitos/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Linhagem Celular Tumoral , Cromatina/metabolismo , Biologia Computacional , Citidina/análise , Citidina/metabolismo , Citosina/metabolismo , DNA/química , Humanos , Aumento da Imagem , Proteína 1 Homóloga a MutL , Proteínas Nucleares/genética , Alinhamento de SequênciaRESUMO
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.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Carcinoma de Células Escamosas/genética , Metilação de DNA , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Proteínas Repressoras/genética , Neoplasias do Colo do Útero/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Western Blotting , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular , Colo do Útero/metabolismo , Ilhas de CpG/genética , Decitabina , Feminino , Inativação Gênica , Humanos , Técnicas Imunoenzimáticas , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologiaRESUMO
Transcriptional activation is often associated with chromatin remodeling. However, little is known about the dynamics of remodeling of nucleosome arrays in vivo. Upon induction of Saccharomyces cerevisiae PHO5, a novel kinetic assay of DNA methyltransferase accessibility showed that nucleosomes adjacent to the histone-free upstream activating sequence (UASp1) are disrupted earlier and at higher frequency in the cell population than are those more distal. Individually cloned molecules, each representing the chromatin state of a full promoter from a single cell, revealed multiple promoter classes with either no remodeling or variable numbers of disrupted nucleosomes. Individual promoters in the remodeled fraction were highly enriched for contiguous blocks of disrupted nucleosomes, the majority of which overlapped the UAS region. These results support a probabilistic model in which chromatin remodeling at PHO5 spreads from sites of transactivator association with DNA and attenuates with distance.
Assuntos
Nucleossomos/genética , Nucleossomos/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Fosfatase Ácida , Montagem e Desmontagem da Cromatina , Regulação Fúngica da Expressão Gênica , Cinética , Metiltransferases/metabolismo , Sequências Reguladoras de Ácido Nucleico/genéticaRESUMO
Non-invasive methods for mapping chromatin structure are necessary for creating an accurate view of genome function and dynamics in vivo. Ectopic induction of cytosine-5 DNA methyltransferases (C5 MTases) in Saccharomyces cerevisiae is a powerful technique for probing chromatin structure with minimal disruption to yeast physiology. Accessibility of MTases to their cognate sites is impaired based on the strength and span of the protein-DNA interaction to be probed. Methylated cytosines that resist chemical deamination are detected positively by the PCR-based technique of bisulfite genomic sequencing. PCR amplicons can be sequenced directly yielding an average m(5)C frequency or accessibility of each target site within the population, a technique termed methyltransferase accessibility protocol (MAP). More recently, the sequencing of cloned molecules in MAP for individual templates (MAPit) enables assignment of the methylation status of each target site along a continuous DNA strand from a single cell. The unique capability to score methylation at multiple sites in single molecules permits detection of inherent structural variability in chromatin. Here, MAPit analysis of the repressed and induced PHO5 promoter of budding yeast, using a C5 MTase with dinucleotide recognition specificity, reveals considerable cell-to-cell heterogeneity in chromatin structure. Substantial variation is observed in the extent to which the MTase gains entry to each of the nucleosomes positioned at PHO5, suggesting differences in their intrinsic thermodynamic stability in vivo. MAPit should be readily adaptable to the analysis of chromatin structure and non-histone protein-DNA interactions in a variety of model systems.
Assuntos
Cromatina/química , Cromatina/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Biologia Molecular/métodos , Sequência de Bases , Cromatina/genética , Metilação de DNA , DNA Fúngico/metabolismo , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Análise de Sequência de DNARESUMO
Wrapping DNA into chromatin provides a wealth of regulatory mechanisms that ensure normal growth and development in eukaryotes. Our understanding of chromatin structure, including nucleosomes and non-histone protein-DNA interactions, has benefited immensely from nuclease and chemical digestion techniques. DNA-bound proteins, such as histones or site-specific factors, protect DNA against nuclease cleavage and generate large nucleosomal or small regulatory factor footprints. Chromatin subject to distinct modes of regulation often coincides with sites of nuclease hypersensitivity or nucleosome positioning. An inherent limitation of cleavage-based analyses has been the inability to reliably analyze regions of interest when levels of digestion depart from single-hit kinetics. Moreover, cleavage-based techniques provide views that are averaged over all the molecules in a sample population. Therefore, in cases of occupancy of multiple regulatory elements by factors, one cannot define whether the factors are bound to the same or different molecules in the population. The recent development of DNA methyltransferase-based, single-molecule MAP-IT technology overcomes limitations of ensemble approaches and has opened numerous new avenues in chromatin research. Here, we review the strengths, limitations, applications and future prospects of MAP-IT ranging from structural issues to mechanistic questions in eukaryotic chromatin regulation.
Assuntos
Cromatina/química , Técnicas de Sonda Molecular , Metilases de Modificação do DNA , GenomaRESUMO
Chromatin creates transcriptional barriers that are overcome by coactivator activities such as histone acetylation by Gcn5 and ATP-dependent chromatin remodeling by SWI/SNF. Factors defining the differential coactivator requirements in the transactivation of various promoters remain elusive. Induction of the Saccharomyces cerevisiae PHO5 promoter does not require Gcn5 or SWI/SNF under fully inducing conditions of no phosphate. We show that PHO5 activation is highly dependent on both coactivators at intermediate phosphate concentrations, conditions that reduce the nuclear concentration of the Pho4 transactivator and severely diminish its association with PHO5 in the absence of Gcn5 or SWI/SNF. Conversely, physiological increases in Pho4 nuclear concentration and binding at PHO5 suppress the need for both Gcn5 and SWI/SNF, suggesting that coactivator redundancy is established at high Pho4 binding site occupancy. Consistent with this, we demonstrate, using chromatin immunoprecipitation, that Gcn5 and SWI/SNF are directly recruited to PHO5 and other strongly transcribed promoters, including GAL1-10, RPL19B, RPS22B, PYK1, and EFT2, which do not require either coactivator for expression. These results show that activator concentration and binding site occupancy play crucial roles in defining the extent to which transcription requires individual chromatin remodeling enzymes. In addition, Gcn5 and SWI/SNF associate with many more genomic targets than previously appreciated.
Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/genética , Regiões Promotoras Genéticas/genética , Saccharomyces cerevisiae/enzimologia , Fosfatase Ácida , Sítios de Ligação , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/metabolismo , Regulação Fúngica da Expressão Gênica , Histona Acetiltransferases , Proteínas Nucleares/metabolismo , Fosfatos/farmacologia , Ligação Proteica , Proteínas Quinases/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Tempo , Transativadores/metabolismo , Fatores de Transcrição/metabolismoRESUMO
In the originally published version of this Article, the affiliation details for Dorina Avram incorrectly included "Department of Infectious Diseases and Immunology, College of Veterinary Medicine, University of Florida, 2015 SW 16th Ave, Gainesville, FL, 32608, USA", instead of "UF Health Cancer Center, University of Florida, Gainesville, FL 32610, USA". This has now been corrected in both the PDF and HTML versions of the Article.