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1.
Methods Mol Biol ; 2351: 307-320, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382197

RESUMO

The transition from silenced heterochromatin to a biologically active state and vice versa is a fundamental part of the implementation of cell type-specific gene expression programs. To reveal structure-function relationships and dissect the underlying mechanisms, experiments that ectopically induce transcription are highly informative. In particular, the approach to perturb chromatin states by recruiting fusions of the catalytically inactive dCas9 protein in a sequence-specific manner to a locus of interest has been used in numerous applications. Here, we describe how this approach can be applied to activate pericentric heterochromatin (PCH) in mouse cells as a prototypic silenced state by providing protocols for the following workflow: (a) Recruitment of dCas9 fusion constructs with the strong transcriptional activator VPR to PCH. (b) Analysis of the resulting changes in chromatin compaction, epigenetic marks, and active transcription by fluorescence microscopy-based readouts. (c) Automated analysis of the resulting images with a set of scripts in the R programming language. Furthermore, we discuss how parameters for chromatin decondensation and active transcription are extracted from these experiments and can be combined with other readouts to gain insights into PCH activation.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Regulação da Expressão Gênica , Heterocromatina/genética , Ativação Transcricional , Animais , Proteína 9 Associada à CRISPR/genética , Cromatina/genética , Cromatina/metabolismo , Fibroblastos/metabolismo , Imunofluorescência/métodos , Expressão Gênica , Heterocromatina/metabolismo , Processamento de Imagem Assistida por Computador , Camundongos , Microscopia de Fluorescência , Ligação Proteica , Transfecção , Fluxo de Trabalho
3.
Artigo em Inglês | MEDLINE | ID: mdl-34127447

RESUMO

In eukaryotic cells, protein and RNA factors involved in genome activities like transcription, RNA processing, DNA replication, and repair accumulate in self-organizing membraneless chromatin subcompartments. These structures contribute to efficiently conduct chromatin-mediated reactions and to establish specific cellular programs. However, the underlying mechanisms for their formation are only partly understood. Recent studies invoke liquid-liquid phase separation (LLPS) of proteins and RNAs in the establishment of chromatin activity patterns. At the same time, the folding of chromatin in the nucleus can drive genome partitioning into spatially distinct domains. Here, the interplay between chromatin organization, chromatin binding, and LLPS is discussed by comparing and contrasting three prototypical chromatin subcompartments: the nucleolus, clusters of active RNA polymerase II, and pericentric heterochromatin domains. It is discussed how the different ways of chromatin compartmentalization are linked to transcription regulation, the targeting of soluble factors to certain parts of the genome, and to disease-causing genetic aberrations.

5.
Nat Commun ; 12(1): 1269, 2021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33627664

RESUMO

Telomere maintenance by telomerase activation or alternative lengthening of telomeres (ALT) is a major determinant of poor outcome in neuroblastoma. Here, we screen for ALT in primary and relapsed neuroblastomas (n = 760) and characterize its features using multi-omics profiling. ALT-positive tumors are molecularly distinct from other neuroblastoma subtypes and enriched in a population-based clinical sequencing study cohort for relapsed cases. They display reduced ATRX/DAXX complex abundance, due to either ATRX mutations (55%) or low protein expression. The heterochromatic histone mark H3K9me3 recognized by ATRX is enriched at the telomeres of ALT-positive tumors. Notably, we find a high frequency of telomeric repeat loci with a neuroblastoma ALT-specific hotspot on chr1q42.2 and loss of the adjacent chromosomal segment forming a neo-telomere. ALT-positive neuroblastomas proliferate slowly, which is reflected by a protracted clinical course of disease. Nevertheless, children with an ALT-positive neuroblastoma have dismal outcome.


Assuntos
Sequenciamento Completo do Genoma/métodos , Western Blotting , Éxons/genética , Citometria de Fluxo , Humanos , Proteoma/metabolismo , Estudos Retrospectivos , Análise de Sequência de RNA/métodos , Telômero/genética , Telômero/metabolismo , Homeostase do Telômero/genética , Proteína Nuclear Ligada ao X/genética
6.
Methods Mol Biol ; 2173: 171-188, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32651918

RESUMO

Blue light-induced chromatin recruitment (BLInCR) is a versatile optogenetic tool to enrich effector proteins at specific loci within the nucleus using illumination in the 400-500 nm range. The resulting chromatin binding reaction is reversible on the time scale of minutes. BLInCR is advantageous over ligand-binding induced methods since it does not require a change of growth medium for the relatively slow depletion of the inducer from the nucleus. However, applying BLInCR for reversibility experiments is challenging because of the need to spectrally separate light-induced activation from visualization of the chromatin locus and effector and/or reader proteins by light microscopy. Here, we describe an improved BLInCR protocol for light-dependent association and dissociation of effectors using the near-infrared fluorescent protein iRFP713. Due to its spectral properties, iRFP713 can be detected separately from the red fluorescent protein mCherry. Thus, it becomes possible to trace two proteins labeled with iRFP713 and mCherry independently of the light activation reaction. This approach largely facilitates applications of the BLInCR system for experiments that test the reversibility, persistence, and memory of chromatin states.


Assuntos
Cromatina/metabolismo , Luz , Optogenética/métodos , Humanos , Software , Ativação Transcricional/genética , Ativação Transcricional/fisiologia
7.
Nat Cell Biol ; 22(7): 896-906, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32541878

RESUMO

Tumour heterogeneity encompasses both the malignant cells and their microenvironment. While heterogeneity between individual patients is known to affect the efficacy of cancer therapy, most personalized treatment approaches do not account for intratumour heterogeneity. We addressed this issue by studying the heterogeneity of nodal B-cell lymphomas by single-cell RNA-sequencing and transcriptome-informed flow cytometry. We identified transcriptionally distinct malignant subpopulations and compared their drug-response and genomic profiles. Malignant subpopulations from the same patient responded strikingly differently to anti-cancer drugs ex vivo, which recapitulated subpopulation-specific drug sensitivity during in vivo treatment. Infiltrating T cells represented the majority of non-malignant cells, whose gene-expression signatures were similar across all donors, whereas the frequencies of T-cell subsets varied significantly between the donors. Our data provide insights into the heterogeneity of nodal B-cell lymphomas and highlight the relevance of intratumour heterogeneity for personalized cancer therapy.


Assuntos
Antineoplásicos/farmacologia , Biomarcadores Tumorais/genética , Regulação Neoplásica da Expressão Gênica , Linfoma de Células B/patologia , Linfócitos T/imunologia , Transcriptoma/efeitos dos fármacos , Microambiente Tumoral/imunologia , Feminino , Perfilação da Expressão Gênica , Humanos , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/genética , Linfoma de Células B/imunologia , Masculino , Pessoa de Meia-Idade , Análise de Sequência de RNA , Análise de Célula Única , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo
8.
J Mol Biol ; 432(15): 4270-4286, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32320688

RESUMO

Repetitive RNA (repRNA) sequences emerge as important regulators of the dynamic organization of genomic loci into membrane-less subcompartments with distinct nuclear functions. These domains include sites of active transcription like the nucleolus as well as (peri)centromeric and telomeric satellite repeats. Recent studies point to an important role of repRNAs in complex with proteins to promote a phase separation-driven formation of chromatin domains. We review how key features of the phase separation process can be revealed by different experimental approaches and discuss the associated structure-function relationships for chromatin subcompartments that involve repRNA.


Assuntos
Cromatina/metabolismo , RNA/metabolismo , Animais , Nucléolo Celular/metabolismo , Humanos , Sequências Repetitivas de Ácido Nucleico , Sítio de Iniciação de Transcrição
9.
Commun Biol ; 3(1): 188, 2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32327715

RESUMO

Fibroblasts are an essential cell population for human skin architecture and function. While fibroblast heterogeneity is well established, this phenomenon has not been analyzed systematically yet. We have used single-cell RNA sequencing to analyze the transcriptomes of more than 5,000 fibroblasts from a sun-protected area in healthy human donors. Our results define four main subpopulations that can be spatially localized and show differential secretory, mesenchymal and pro-inflammatory functional annotations. Importantly, we found that this fibroblast 'priming' becomes reduced with age. We also show that aging causes a substantial reduction in the predicted interactions between dermal fibroblasts and other skin cells, including undifferentiated keratinocytes at the dermal-epidermal junction. Our work thus provides evidence for a functional specialization of human dermal fibroblasts and identifies the partial loss of cellular identity as an important age-related change in the human dermis. These findings have important implications for understanding human skin aging and its associated phenotypes.


Assuntos
Senescência Celular/genética , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Análise de Célula Única , Envelhecimento da Pele/genética , Pele/metabolismo , Transcriptoma , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Comunicação Celular , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , RNA-Seq , Pele/citologia
10.
Genome Med ; 12(1): 29, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-32188505

RESUMO

BACKGROUND: In cancer, normal epigenetic patterns are disturbed and contribute to gene expression changes, disease onset, and progression. The cancer epigenome is composed of the epigenetic patterns present in the tumor-initiating cell at the time of transformation, and the tumor-specific epigenetic alterations that are acquired during tumor initiation and progression. The precise dissection of these two components of the tumor epigenome will facilitate a better understanding of the biological mechanisms underlying malignant transformation. Chronic lymphocytic leukemia (CLL) originates from differentiating B cells, which undergo extensive epigenetic programming. This poses the challenge to precisely determine the epigenomic ground state of the cell-of-origin in order to identify CLL-specific epigenetic aberrations. METHODS: We developed a linear regression model, methylome-based cell-of-origin modeling (Methyl-COOM), to map the cell-of-origin for individual CLL patients based on the continuum of epigenomic changes during normal B cell differentiation. RESULTS: Methyl-COOM accurately maps the cell-of-origin of CLL and identifies CLL-specific aberrant DNA methylation events that are not confounded by physiologic epigenetic B cell programming. Furthermore, Methyl-COOM unmasks abnormal action of transcription factors, altered super-enhancer activities, and aberrant transcript expression in CLL. Among the aberrantly regulated transcripts were many genes that have previously been implicated in T cell biology. Flow cytometry analysis of these markers confirmed their aberrant expression on malignant B cells at the protein level. CONCLUSIONS: Methyl-COOM analysis of CLL identified disease-specific aberrant gene regulation. The aberrantly expressed genes identified in this study might play a role in immune-evasion in CLL and might serve as novel targets for immunotherapy approaches. In summary, we propose a novel framework for in silico modeling of reference DNA methylomes and for the identification of cancer-specific epigenetic changes, a concept that can be broadly applied to other human malignancies.


Assuntos
Linhagem da Célula , Epigenoma , Leucemia Linfocítica Crônica de Células B/genética , Modelos Genéticos , Linfócitos B/citologia , Linfócitos B/metabolismo , Diferenciação Celular , Hematopoiese Clonal , Elementos Facilitadores Genéticos , Epigênese Genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Transcrição , Transcriptoma
11.
Mol Cell ; 78(2): 236-249.e7, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32101700

RESUMO

The formation of silenced and condensed heterochromatin foci involves enrichment of heterochromatin protein 1 (HP1). HP1 can bridge chromatin segments and form liquid droplets, but the biophysical principles underlying heterochromatin compartmentalization in the cell nucleus are elusive. Here, we assess mechanistically relevant features of pericentric heterochromatin compaction in mouse fibroblasts. We find that (1) HP1 has only a weak capacity to form liquid droplets in living cells; (2) the size, global accessibility, and compaction of heterochromatin foci are independent of HP1; (3) heterochromatin foci lack a separated liquid HP1 pool; and (4) heterochromatin compaction can toggle between two "digital" states depending on the presence of a strong transcriptional activator. These findings indicate that heterochromatin foci resemble collapsed polymer globules that are percolated with the same nucleoplasmic liquid as the surrounding euchromatin, which has implications for our understanding of chromatin compartmentalization and its functional consequences.


Assuntos
Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Eucromatina/genética , Heterocromatina/genética , Animais , Fibroblastos , Camundongos
12.
Nat Commun ; 11(1): 733, 2020 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-32024817

RESUMO

Cancers require telomere maintenance mechanisms for unlimited replicative potential. They achieve this through TERT activation or alternative telomere lengthening associated with ATRX or DAXX loss. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, we dissect whole-genome sequencing data of over 2500 matched tumor-control samples from 36 different tumor types aggregated within the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium to characterize the genomic footprints of these mechanisms. While the telomere content of tumors with ATRX or DAXX mutations (ATRX/DAXXtrunc) is increased, tumors with TERT modifications show a moderate decrease of telomere content. One quarter of all tumor samples contain somatic integrations of telomeric sequences into non-telomeric DNA. This fraction is increased to 80% prevalence in ATRX/DAXXtrunc tumors, which carry an aberrant telomere variant repeat (TVR) distribution as another genomic marker. The latter feature includes enrichment or depletion of the previously undescribed singleton TVRs TTCGGG and TTTGGG, respectively. Our systematic analysis provides new insight into the recurrent genomic alterations associated with telomere maintenance mechanisms in cancer.


Assuntos
Mutação , Neoplasias/genética , Telômero/genética , Estudos de Casos e Controles , Proteínas Correpressoras/genética , Genoma Humano , Humanos , Chaperonas Moleculares/genética , RNA Longo não Codificante , Sequências Repetitivas de Ácido Nucleico , Telomerase/genética , Sequenciamento Completo do Genoma , Proteína Nuclear Ligada ao X/genética
13.
Int J Cancer ; 146(5): 1281-1292, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31456217

RESUMO

Tumor-initiating cells are a subpopulation of cells that have self-renewal capacity to regenerate a tumor. Here, we identify stem cell-like chromatin features in human glioblastoma initiating cells (GICs) and link them to a loss of the repressive histone H3 lysine 9 trimethylation (H3K9me3) mark. Increasing H3K9me3 levels by histone demethylase inhibition led to cell death in GICs but not in their differentiated counterparts. The induction of apoptosis was accompanied by a loss of the activating H3 lysine 9 acetylation (H3K9ac) modification and accumulation of DNA damage and downregulation of DNA damage response genes. Upon knockdown of histone demethylases, KDM4C and KDM7A both differentiation and DNA damage were induced. Thus, the H3K9me3-H3K9ac equilibrium is crucial for GIC viability and represents a chromatin feature that can be exploited to specifically target this tumor subpopulation.


Assuntos
Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Células-Tronco Neoplásicas/metabolismo , Acetilação , Animais , Apoptose/genética , Linhagem Celular Tumoral , Autorrenovação Celular/genética , Cromatina/metabolismo , Metilação de DNA , Reparo do DNA/genética , Técnicas de Silenciamento de Genes , Glioblastoma/patologia , Células HEK293 , Histonas , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Lisina/metabolismo , Camundongos , Regiões Promotoras Genéticas/genética , RNA Interferente Pequeno/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Mol Oncol ; 14(1): 129-138, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31736271

RESUMO

The chromatin-organizing factor CCCTC-binding factor (CTCF) is involved in transcriptional regulation, DNA-loop formation, and telomere maintenance. To evaluate the clinical impact of CTCF in prostate cancer, we analyzed CTCF expression by immunohistochemistry on a tissue microarray containing 17 747 prostate cancers. Normal prostate tissue showed negative to low CTCF expression, while in prostate cancers, CTCF expression was seen in 7726 of our 12 555 (61.5%) tumors and was considered low in 44.6% and high in 17% of cancers. Particularly, high CTCF expression was significantly associated with the presence of the transmembrane protease, serine 2:ETS-related gene fusion: Only 10% of ERG-negative cancers, but 30% of ERG-positive cancers had high-level CTCF expression (P < 0.0001). CTCF expression was significantly associated with advanced pathological tumor stage, high Gleason grade (P < 0.0001 each), nodal metastasis (P = 0.0122), and early biochemical recurrence (P < 0.0001). Multivariable modeling revealed that the prognostic impact of CTCF was independent from established presurgical parameters such as clinical stage and Gleason grade of the biopsy. Comparison with key molecular alterations showed strong associations with the expression of the Ki-67 proliferation marker and presence of phosphatase and tensin homolog deletions (P < 0.0001 each). The results of our study identify CTCF expression as a candidate biomarker for prognosis assessment in prostate cancer.


Assuntos
Fator de Ligação a CCCTC/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Proteínas de Fusão Oncogênica/genética , Neoplasias da Próstata/metabolismo , Serina Endopeptidases/genética , Idoso , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Fator de Ligação a CCCTC/genética , Proliferação de Células/genética , Humanos , Imuno-Histoquímica , Antígeno Ki-67/metabolismo , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Prognóstico , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Deleção de Sequência , Serina Endopeptidases/metabolismo , Análise Serial de Tecidos , Regulador Transcricional ERG/genética , Regulador Transcricional ERG/metabolismo
15.
Sci Rep ; 9(1): 12367, 2019 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-31451731

RESUMO

Patient-derived 3D cell culture systems are currently advancing cancer research since they potentiate the molecular analysis of tissue-like properties and drug response under well-defined conditions. However, our understanding of the relationship between the heterogeneity of morphological phenotypes and the underlying transcriptome is still limited. To address this issue, we here introduce "pheno-seq" to directly link visual features of 3D cell culture systems with profiling their transcriptome. As prototypic applications breast and colorectal cancer (CRC) spheroids were analyzed by pheno-seq. We identified characteristic gene expression signatures of epithelial-to-mesenchymal transition that are associated with invasive growth behavior of clonal breast cancer spheroids. Furthermore, we linked long-term proliferative capacity in a patient-derived model of CRC to a lowly abundant PROX1-positive cancer stem cell subtype. We anticipate that the ability to integrate transcriptome analysis and morphological patho-phenotypes of cancer cells will provide novel insight on the molecular origins of intratumor heterogeneity.


Assuntos
Técnicas de Cultura de Células/métodos , Regulação Neoplásica da Expressão Gênica , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Linhagem da Célula/genética , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Feminino , Genes Neoplásicos , Humanos , Células-Tronco Neoplásicas/patologia , Fenótipo , Análise de Célula Única
16.
Methods Mol Biol ; 2038: 251-270, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31407290

RESUMO

Gene expression can be monitored in living cells via the binding of fluorescently tagged proteins to RNA repeats engineered into a reporter transcript. This approach makes it possible to trace temporal changes of RNA production in real time in living cells to dissect transcription regulation. For a mechanistic analysis of the underlying activation process, it is essential to induce gene expression with high accuracy. Here, we describe how this can be accomplished with an optogenetic approach termed blue light-induced chromatin recruitment (BLInCR). It employs the recruitment of an activator protein to a target promoter via the interaction between the PHR and CIBN plant protein domains. This process occurs within seconds after setting the light trigger and is reversible. Protocols for continuous activation as well as pulsed activation and reactivation with imaging either by laser scanning confocal microscopy or automated widefield microscopy are provided. For the semiautomated quantification of the resulting image series, an approach has been implemented in a set of scripts in the R programming language. Thus, the complete workflow of the BLInCR method is described for mechanistic studies of the transcription activation process as well as the persistence and memory of the activated state.


Assuntos
Luz , Microscopia Confocal , Microscopia de Fluorescência , Optogenética , Fatores de Transcrição/metabolismo , Transcrição Genética/efeitos da radiação , Ativação Transcricional/efeitos da radiação , Linhagem Celular Tumoral , Genes Reporter , Humanos , Fatores de Tempo , Fatores de Transcrição/genética
17.
Int J Comput Assist Radiol Surg ; 14(11): 1847-1857, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31177423

RESUMO

PURPOSE: Automated analysis of microscopy image data typically requires complex pipelines that involve multiple methods for different image analysis tasks. To achieve best results of the analysis pipelines, method-dependent hyperparameters need to be optimized. However, complex pipelines often suffer from the fact that calculation of the gradient of the loss function is analytically or computationally infeasible. Therefore, first- or higher-order optimization methods cannot be applied. METHODS: We developed a new framework for zero-order black-box hyperparameter optimization called HyperHyper, which has a modular architecture that separates hyperparameter sampling and optimization. We also developed a visualization of the loss function based on infimum projection to obtain further insights into the optimization problem. RESULTS: We applied HyperHyper in three different experiments with different imaging modalities, and evaluated in total more than 400.000 hyperparameter combinations. HyperHyper was used for optimizing two pipelines for cell nuclei segmentation in prostate tissue microscopy images and two pipelines for detection of hepatitis C virus proteins in live cell microscopy data. We evaluated the impact of separating the sampling and optimization strategy using different optimizers and employed an infimum projection for visualizing the hyperparameter space. CONCLUSIONS: The separation of sampling and optimization strategy of the proposed HyperHyper optimization framework improves the result of the investigated image analysis pipelines. Visualization of the loss function based on infimum projection enables gaining further insights on the optimization process.


Assuntos
Algoritmos , Hepacivirus/isolamento & purificação , Processamento de Imagem Assistida por Computador/métodos , Próstata/diagnóstico por imagem , Humanos , Masculino , Próstata/virologia
18.
Mol Syst Biol ; 15(5): e8339, 2019 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-31118277

RESUMO

In chronic lymphocytic leukemia (CLL), a diverse set of genetic mutations is embedded in a deregulated epigenetic landscape that drives cancerogenesis. To elucidate the role of aberrant chromatin features, we mapped DNA methylation, seven histone modifications, nucleosome positions, chromatin accessibility, binding of EBF1 and CTCF, as well as the transcriptome of B cells from CLL patients and healthy donors. A globally increased histone deacetylase activity was detected and half of the genome comprised transcriptionally downregulated partially DNA methylated domains demarcated by CTCF CLL samples displayed a H3K4me3 redistribution and nucleosome gain at promoters as well as changes of enhancer activity and enhancer linkage to target genes. A DNA binding motif analysis identified transcription factors that gained or lost binding in CLL at sites with aberrant chromatin features. These findings were integrated into a gene regulatory enhancer containing network enriched for B-cell receptor signaling pathway components. Our study predicts novel molecular links to targets of CLL therapies and provides a valuable resource for further studies on the epigenetic contribution to the disease.


Assuntos
Cromatina/química , Regulação Leucêmica da Expressão Gênica , Redes Reguladoras de Genes , Histonas/química , Leucemia Linfocítica Crônica de Células B/genética , Idoso , Motivos de Aminoácidos , Sítios de Ligação , Fator de Ligação a CCCTC/genética , DNA/química , Metilação de DNA , Regulação para Baixo , Elementos Facilitadores Genéticos , Histona Desacetilases/genética , Humanos , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , Ligação Proteica , Transativadores/genética
19.
BMC Bioinformatics ; 20(1): 272, 2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138115

RESUMO

BACKGROUND: Establishment of telomere maintenance mechanisms is a universal step in tumor development to achieve replicative immortality. These processes leave molecular footprints in cancer genomes in the form of altered telomere content and aberrations in telomere composition. To retrieve these telomere characteristics from high-throughput sequencing data the available computational approaches need to be extended and optimized to fully exploit the information provided by large scale cancer genome data sets. RESULTS: We here present TelomereHunter, a software for the detailed characterization of telomere maintenance mechanism footprints in the genome. The tool is implemented for the analysis of large cancer genome cohorts and provides a variety of diagnostic diagrams as well as machine-readable output for subsequent analysis. A novel key feature is the extraction of singleton telomere variant repeats, which improves the identification and subclassification of the alternative lengthening of telomeres phenotype. We find that whole genome sequencing-derived telomere content estimates strongly correlate with telomere qPCR measurements (r = 0.94). For the first time, we determine the correlation of in silico telomere content quantification from whole genome sequencing and whole genome bisulfite sequencing data derived from the same tumor sample (r = 0.78). An analogous comparison of whole exome sequencing data and whole genome sequencing data measured slightly lower correlation (r = 0.79). However, this is considerably improved by normalization with matched controls (r = 0.91). CONCLUSIONS: TelomereHunter provides new functionality for the analysis of the footprints of telomere maintenance mechanisms in cancer genomes. Besides whole genome sequencing, whole exome sequencing and whole genome bisulfite sequencing are suited for in silico telomere content quantification, especially if matched control samples are available. The software runs under a GPL license and is available at https://www.dkfz.de/en/applied-bioinformatics/telomerehunter/telomerehunter.html .


Assuntos
Simulação por Computador , Genoma , Neoplasias/genética , Software , Telômero/genética , Sequência de Bases , Glioblastoma/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Meduloblastoma/genética , Sequenciamento Completo do Exoma , Sequenciamento Completo do Genoma
20.
Genome Res ; 29(5): 750-761, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30948436

RESUMO

Coordinated changes of DNA (de)methylation, nucleosome positioning, and chromatin binding of the architectural protein CTCF play an important role for establishing cell-type-specific chromatin states during differentiation. To elucidate molecular mechanisms that link these processes, we studied the perturbed DNA modification landscape in mouse embryonic stem cells (ESCs) carrying a double knockout (DKO) of the Tet1 and Tet2 dioxygenases. These enzymes are responsible for the conversion of 5-methylcytosine (5mC) into its hydroxymethylated (5hmC), formylated (5fC), or carboxylated (5caC) forms. We determined changes in nucleosome positioning, CTCF binding, DNA methylation, and gene expression in DKO ESCs and developed biophysical models to predict differential CTCF binding. Methylation-sensitive nucleosome repositioning accounted for a significant portion of CTCF binding loss in DKO ESCs, whereas unmethylated and nucleosome-depleted CpG islands were enriched for CTCF sites that remained occupied. A number of CTCF sites also displayed direct correlations with the CpG modification state: CTCF was preferentially lost from sites that were marked with 5hmC in wild-type (WT) cells but not from 5fC-enriched sites. In addition, we found that some CTCF sites can act as bifurcation points defining the differential methylation landscape. CTCF loss from such sites, for example, at promoters, boundaries of chromatin loops, and topologically associated domains (TADs), was correlated with DNA methylation/demethylation spreading and can be linked to down-regulation of neighboring genes. Our results reveal a hierarchical interplay between cytosine modifications, nucleosome positions, and DNA sequence that determines differential CTCF binding and regulates gene expression.


Assuntos
Metilação de DNA , Proteínas de Ligação a DNA/genética , Epigênese Genética , Células-Tronco Embrionárias Murinas/enzimologia , Proteínas Proto-Oncogênicas/genética , 5-Metilcitosina/química , Animais , Fator de Ligação a CCCTC/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Elementos Isolantes/genética , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas/metabolismo , Nucleossomos/enzimologia , Proteínas Proto-Oncogênicas/metabolismo
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