RESUMO
Triple-negative breast cancer (TNBC) is an aggressive subtype that frequently develops resistance to chemotherapy. An unresolved question is whether resistance is caused by the selection of rare pre-existing clones or alternatively through the acquisition of new genomic aberrations. To investigate this question, we applied single-cell DNA and RNA sequencing in addition to bulk exome sequencing to profile longitudinal samples from 20 TNBC patients during neoadjuvant chemotherapy (NAC). Deep-exome sequencing identified 10 patients in which NAC led to clonal extinction and 10 patients in which clones persisted after treatment. In 8 patients, we performed a more detailed study using single-cell DNA sequencing to analyze 900 cells and single-cell RNA sequencing to analyze 6,862 cells. Our data showed that resistant genotypes were pre-existing and adaptively selected by NAC, while transcriptional profiles were acquired by reprogramming in response to chemotherapy in TNBC patients.
Assuntos
Antineoplásicos/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Sequenciamento de Nucleotídeos em Larga Escala , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Estudos de Casos e Controles , Análise por Conglomerados , Variações do Número de Cópias de DNA , Exoma/genética , Feminino , Frequência do Gene , Genótipo , Humanos , Terapia Neoadjuvante , Análise de Sequência de DNA , Análise de Sequência de RNA , Análise de Célula Única , Análise de Sobrevida , Transcriptoma , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/mortalidade , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
A recent publication in Nature by Arnould et al.1 describes a novel chromatin compartment, termed "damaged" or "D compartment," that facilitates the repair of DNA double-strand breaks but also increases the risk of potentially oncogenic translocation formation.
Assuntos
Cromatina , Quebras de DNA de Cadeia Dupla , Humanos , Cromatina/genética , Dano ao DNA , Translocação Genética , DNA/genéticaRESUMO
As cells enter mitosis, chromatin compacts to facilitate chromosome segregation yet remains transcribed. Transcription supercoils DNA to levels that can impede further progression of RNA polymerase II (RNAPII) unless it is removed by DNA topoisomerase 1 (TOP1). Using ChIP-seq on mitotic cells, we found that TOP1 is required for RNAPII translocation along genes. The stimulation of TOP1 activity by RNAPII during elongation allowed RNAPII clearance from genes in prometaphase and enabled chromosomal segregation. Disruption of the TOP1-RNAPII interaction impaired RNAPII spiking at promoters and triggered defects in the post-mitotic transcription program. This program includes factors necessary for cell growth, and cells with impaired TOP1-RNAPII interaction are more sensitive to inhibitors of mTOR signaling. We conclude that TOP1 is necessary for assisting transcription during mitosis with consequences for growth and gene expression long after mitosis is completed. In this sense, TOP1 ensures that cellular memory is preserved in subsequent generations.
Assuntos
Proliferação de Células , Montagem e Desmontagem da Cromatina , Neoplasias Colorretais/enzimologia , DNA Topoisomerases Tipo I/metabolismo , Fase G1 , Mitose , RNA Polimerase II/metabolismo , Transcrição Gênica , Proliferação de Células/efeitos dos fármacos , Sequenciamento de Cromatina por Imunoprecipitação , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , DNA Topoisomerases Tipo I/genética , Fase G1/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Inibidores de MTOR/farmacologia , Mitose/efeitos dos fármacos , RNA Polimerase II/genéticaRESUMO
Oncogene-induced senescence (OIS) is an inherent and important tumor suppressor mechanism. However, if not removed timely via immune surveillance, senescent cells also have detrimental effects. Although this has mostly been attributed to the senescence-associated secretory phenotype (SASP) of these cells, we recently proposed that "escape" from the senescent state is another unfavorable outcome. The mechanism underlying this phenomenon remains elusive. Here, we exploit genomic and functional data from a prototypical human epithelial cell model carrying an inducible CDC6 oncogene to identify an early-acquired recurrent chromosomal inversion that harbors a locus encoding the circadian transcription factor BHLHE40. This inversion alone suffices for BHLHE40 activation upon CDC6 induction and driving cell cycle re-entry of senescent cells, and malignant transformation. Ectopic overexpression of BHLHE40 prevented induction of CDC6-triggered senescence. We provide strong evidence in support of replication stress-induced genomic instability being a causative factor underlying "escape" from oncogene-induced senescence.
Assuntos
Senescência Celular , Inversão Cromossômica , Cromossomos/ultraestrutura , Transição Epitelial-Mesenquimal , Neoplasias/genética , Oncogenes , Recombinação Genética , Animais , Brônquios/metabolismo , Sistemas CRISPR-Cas , Ciclo Celular , Transformação Celular Neoplásica , Ritmo Circadiano , Biologia Computacional , Células Epiteliais/metabolismo , Citometria de Fluxo , Genômica , Humanos , Cariotipagem , Camundongos , Camundongos SCID , Neoplasias/metabolismo , Fenótipo , Ligação Proteica , Domínios Proteicos , Fenótipo Secretor Associado à SenescênciaRESUMO
Microscopy-based spatially resolved omic methods are transforming the life sciences. However, these methods rely on high numerical aperture objectives and cannot resolve crowded molecular targets, limiting the amount of extractable biological information. To overcome these limitations, here we develop Deconwolf, an open-source, user-friendly software for high-performance deconvolution of widefield fluorescence microscopy images, which efficiently runs on laptop computers. Deconwolf enables accurate quantification of crowded diffraction limited fluorescence dots in DNA and RNA fluorescence in situ hybridization images and allows robust detection of individual transcripts in tissue sections imaged with ×20 air objectives. Deconvolution of in situ spatial transcriptomics images with Deconwolf increased the number of transcripts identified more than threefold, while the application of Deconwolf to images obtained by fluorescence in situ sequencing of barcoded Oligopaint probes drastically improved chromosome tracing. Deconwolf greatly facilitates the use of deconvolution in many bioimaging applications.
Assuntos
Processamento de Imagem Assistida por Computador , Hibridização in Situ Fluorescente , Microscopia de Fluorescência , Software , Microscopia de Fluorescência/métodos , Hibridização in Situ Fluorescente/métodos , Processamento de Imagem Assistida por Computador/métodos , Animais , Camundongos , HumanosRESUMO
How spatial chromosome organization influences genome integrity is still poorly understood. Here, we show that DNA double-strand breaks (DSBs) mediated by topoisomerase 2 (TOP2) activities are enriched at chromatin loop anchors with high transcriptional activity. Recurrent DSBs occur at CCCTC-binding factor (CTCF) and cohesin-bound sites at the bases of chromatin loops, and their frequency positively correlates with transcriptional output and directionality. The physiological relevance of this preferential positioning is indicated by the finding that genes recurrently translocating to drive leukemias are highly transcribed and are enriched at loop anchors. These genes accumulate DSBs at recurrent hotspots that give rise to chromosomal fusions relying on the activity of both TOP2 isoforms and on transcriptional elongation. We propose that transcription and 3D chromosome folding jointly pose a threat to genomic stability and are key contributors to the occurrence of genome rearrangements that drive cancer.
Assuntos
DNA Topoisomerases Tipo II/genética , Instabilidade Genômica/genética , Histona-Lisina N-Metiltransferase/genética , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Translocação Genética/genética , Fator de Ligação a CCCTC/genética , Carcinogênese/genética , Linhagem Celular Tumoral , Cromatina/química , Cromatina/genética , Cromossomos/química , Cromossomos/genética , DNA/genética , Quebras de DNA de Cadeia Dupla , Humanos , Leucemia/genética , Leucemia/patologiaRESUMO
Over a decade ago the advent of high-throughput chromosome conformation capture (Hi-C) sparked a new era of 3D genomics. Since then the number of methods for mapping the 3D genome has flourished, enabling an ever-increasing understanding of how DNA is packaged in the nucleus and how the spatiotemporal organization of the genome orchestrates its vital functions. More recently, the next generation of spatial genomics technologies has begun to reveal how genome sequence and 3D genome organization vary between cells in their tissue context. We summarize how the toolkit for charting genome topology has evolved over the past decade and discuss how new technological developments are advancing the field of 3D and spatial genomics.
Assuntos
Genoma , Genômica , Núcleo Celular , Cromatina/genética , Cromossomos/genética , Genoma/genética , Genômica/métodos , Conformação MolecularRESUMO
Ubiquitin signals and ubiquitin-binding domains are implicated in almost every cellular process, but how is their functionality achieved in cells? We assess recent advances in monitoring the dynamics and specificity of ubiquitin networks in vivo and discuss challenges ahead.
Assuntos
Transdução de Sinais , Ubiquitina/metabolismo , Animais , Humanos , Domínios e Motivos de Interação entre Proteínas , Proteínas/química , Proteínas/metabolismo , Ubiquitina/química , UbiquitinaçãoRESUMO
BACKGROUND: Despite the fact that tumor microenvironment (TME) and gene mutations are the main determinants of progression of the deadliest cancer in the world - lung cancer, their interrelations are not well understood. Digital pathology data provides a unique insight into the spatial composition of the TME. Various spatial metrics and machine learning approaches were proposed for prediction of either patient survival or gene mutations from this data. Still, these approaches are limited in the scope of analyzed features and in their explainability, and as such fail to transfer to clinical practice. METHODS: Here, we generated 23,199 image patches from 26 hematoxylin-and-eosin (H&E)-stained lung cancer tissue sections and annotated them into 9 different tissue classes. Using this dataset, we trained a deep neural network ARA-CNN. Next, we applied the trained network to segment 467 lung cancer H&E images from The Cancer Genome Atlas (TCGA) database. We used the segmented images to compute human-interpretable features reflecting the heterogeneous composition of the TME, and successfully utilized them to predict patient survival and cancer gene mutations. RESULTS: We achieved per-class AUC ranging from 0.72 to 0.99 for classifying tissue types in lung cancer with ARA-CNN. Machine learning models trained on the proposed human-interpretable features achieved a c-index of 0.723 in the task of survival prediction and AUC up to 73.5% for PDGFRB in the task of mutation classification. CONCLUSIONS: We presented a framework that accurately predicted survival and gene mutations in lung adenocarcinoma patients based on human-interpretable features extracted from H&E slides. Our approach can provide important insights for designing novel cancer treatments, by linking the spatial structure of the TME in lung adenocarcinoma to gene mutations and patient survival. It can also expand our understanding of the effects that the TME has on tumor evolutionary processes. Our approach can be generalized to different cancer types to inform precision medicine strategies.
Assuntos
Adenocarcinoma de Pulmão , Carcinoma Pulmonar de Células não Pequenas , Aprendizado Profundo , Neoplasias Pulmonares , Adenocarcinoma de Pulmão/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Amarelo de Eosina-(YS) , Hematoxilina , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mutação , Receptor beta de Fator de Crescimento Derivado de Plaquetas , Microambiente Tumoral/genéticaRESUMO
Considerable progress in sequencing technologies makes it now possible to study the genomic and transcriptomic landscape of single cells. However, to better understand the complexity of multicellular organisms, we must devise ways to perform high-throughput measurements while preserving spatial information about the tissue context or subcellular localization of analysed nucleic acids. In this Innovation article, we summarize pioneering technologies that enable spatially resolved transcriptomics and discuss how these methods have the potential to extend beyond transcriptomics to encompass spatially resolved genomics, proteomics and possibly other omic disciplines.
Assuntos
Perfilação da Expressão Gênica/métodos , Ensaios de Triagem em Larga Escala/métodos , Imagem Molecular/métodos , Análise de Sequência/métodos , Análise de Célula Única/métodos , Análise EspacialRESUMO
Hematopoietic stem and progenitor cells (HSPCs) are vulnerable to endogenous damage and defects in DNA repair can limit their function. The 2 single-stranded DNA (ssDNA) binding proteins SSB1 and SSB2 are crucial regulators of the DNA damage response; however, their overlapping roles during normal physiology are incompletely understood. We generated mice in which both Ssb1 and Ssb2 were constitutively or conditionally deleted. Constitutive Ssb1/Ssb2 double knockout (DKO) caused early embryonic lethality, whereas conditional Ssb1/Ssb2 double knockout (cDKO) in adult mice resulted in acute lethality due to bone marrow failure and intestinal atrophy featuring stem and progenitor cell depletion, a phenotype unexpected from the previously reported single knockout models of Ssb1 or Ssb2 Mechanistically, cDKO HSPCs showed altered replication fork dynamics, massive accumulation of DNA damage, genome-wide double-strand breaks enriched at Ssb-binding regions and CpG islands, together with the accumulation of R-loops and cytosolic ssDNA. Transcriptional profiling of cDKO HSPCs revealed the activation of p53 and interferon (IFN) pathways, which enforced cell cycling in quiescent HSPCs, resulting in their apoptotic death. The rapid cell death phenotype was reproducible in in vitro cultured cDKO-hematopoietic stem cells, which were significantly rescued by nucleotide supplementation or after depletion of p53. Collectively, Ssb1 and Ssb2 control crucial aspects of HSPC function, including proliferation and survival in vivo by resolving replicative stress to maintain genomic stability.
Assuntos
Proliferação de Células/fisiologia , Quebras de DNA de Cadeia Dupla , Instabilidade Genômica/fisiologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Animais , Sobrevivência Celular/fisiologia , Ilhas de CpG/fisiologia , Células-Tronco Hematopoéticas/citologia , Camundongos , Camundongos Knockout , Proteínas Supressoras da Sinalização de Citocina/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
DNA polymerase eta is a Y family polymerase involved in translesion synthesis (TLS). Its action is initiated by simultaneous interaction between the PIP box in pol eta and PCNA and between the UBZ in pol eta and monoubiquitin attached to PCNA. Whereas monoubiquitination of PCNA is required for its interaction with pol eta during TLS, we now show that monoubiquitination of pol eta inhibits this interaction, preventing its functions in undamaged cells. Identification of monoubiquitination sites within pol eta nuclear localization signal (NLS) led to the discovery that pol eta NLS directly contacts PCNA, forming an extended pol eta-PCNA interaction surface. We name this the PCNA-interacting region (PIR) and show that its monoubiquitination is downregulated by various DNA-damaging agents. We propose that this mechanism ensures optimal availability of nonubiquitinated, TLS-competent pol eta after DNA damage. Our work shows how monoubiquitination can either positively or negatively regulate the assembly of a protein complex, depending on which substrates are targeted by ubiquitin.
Assuntos
DNA Polimerase Dirigida por DNA/fisiologia , Sequência de Aminoácidos , Linhagem Celular , Dano ao DNA , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Humanos , Dados de Sequência Molecular , Mutagênicos/farmacologia , Sinais de Localização Nuclear , Antígeno Nuclear de Célula em Proliferação/metabolismo , Alinhamento de Sequência , UbiquitinaçãoRESUMO
We developed a cost-effective genome-scale PCR-based method for high-definition DNA FISH (HD-FISH). We visualized gene loci with diffraction-limited resolution, chromosomes as spot clusters and single genes together with transcripts by combining HD-FISH with single-molecule RNA FISH. We provide a database of over 4.3 million primer pairs targeting the human and mouse genomes that is readily usable for rapid and flexible generation of probes.
Assuntos
Primers do DNA , Biblioteca Genômica , Hibridização in Situ Fluorescente/métodos , Análise de Sequência de DNA/métodos , Animais , Humanos , Camundongos , Reação em Cadeia da PolimeraseRESUMO
We present a genome-wide approach to map DNA double-strand breaks (DSBs) at nucleotide resolution by a method we termed BLESS (direct in situ breaks labeling, enrichment on streptavidin and next-generation sequencing). We validated and tested BLESS using human and mouse cells and different DSBs-inducing agents and sequencing platforms. BLESS was able to detect telomere ends, Sce endonuclease-induced DSBs and complex genome-wide DSB landscapes. As a proof of principle, we characterized the genomic landscape of sensitivity to replication stress in human cells, and we identified >2,000 nonuniformly distributed aphidicolin-sensitive regions (ASRs) overrepresented in genes and enriched in satellite repeats. ASRs were also enriched in regions rearranged in human cancers, with many cancer-associated genes exhibiting high sensitivity to replication stress. Our method is suitable for genome-wide mapping of DSBs in various cells and experimental conditions, with a specificity and resolution unachievable by current techniques.
Assuntos
Quebras de DNA de Cadeia Dupla , Genômica/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Animais , Afidicolina , Sequência de Bases , Linhagem Celular Tumoral , Clonagem Molecular , Replicação do DNA , Fibroblastos/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Repetições de Microssatélites , Mapeamento Físico do Cromossomo/métodos , Análise de Sequência de DNA , Baço , Testículo , Replicação ViralRESUMO
Since the advent of Hi-C in 2009, a plethora of high-throughput sequencing methods have emerged to profile the three-dimensional (3D) organization of eukaryotic genomes, igniting the era of 3D genomics. In recent years, the genomic resolution achievable by these approaches has dramatically increased and several single-cell versions of Hi-C have been developed. Moreover, a new repertoire of tools not based on proximity ligation of digested chromatin has emerged, enabling the investigation of the higher-order organization of chromatin in the nucleus. In this review, we summarize the expanding portfolio of 3D genomic technologies, highlighting recent developments and applications from the past three years. Lastly, we present an outlook of where this technology-driven field might be headed.
RESUMO
The identification of genes involved in replicative stress is key to understanding cancer evolution and to identify therapeutic targets. Here, we show that CDK12 prevents transcription-replication conflicts (TRCs) and the activation of cytotoxic replicative stress upon deregulation of the MYC oncogene. CDK12 was recruited at damaged genes by PARP-dependent DDR-signaling and elongation-competent RNAPII, to repress transcription. Either loss or chemical inhibition of CDK12 led to DDR-resistant transcription of damaged genes. Loss of CDK12 exacerbated TRCs in MYC-overexpressing cells and led to the accumulation of double-strand DNA breaks, occurring between co-directional early-replicating regions and transcribed genes. Overall, our data demonstrate that CDK12 protects genome integrity by repressing transcription of damaged genes, which is required for proper resolution of DSBs at oncogene-induced TRCs. This provides a rationale that explains both how CDK12 deficiency can promote tandem duplications of early-replicated regions during tumor evolution, and how CDK12 targeting can exacerbate replicative-stress in tumors.
Assuntos
Quinases Ciclina-Dependentes , Replicação do DNA , Transcrição Gênica , Humanos , Quinases Ciclina-Dependentes/metabolismo , Quinases Ciclina-Dependentes/genética , Quebras de DNA de Cadeia Dupla , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Linhagem Celular Tumoral , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Dano ao DNARESUMO
Extrachromosomal circular DNAs (eccDNAs) have emerged as important intra-cellular mobile genetic elements that affect gene copy number and exert in trans regulatory roles within the cell nucleus. Here, we describe scCircle-seq, a method for profiling eccDNAs and unraveling their diversity and complexity in single cells. We implement and validate scCircle-seq in normal and cancer cell lines, demonstrating that most eccDNAs vary largely between cells and are stochastically inherited during cell division, although their genomic landscape is cell type-specific and can be used to accurately cluster cells of the same origin. eccDNAs are preferentially produced from chromatin regions enriched in H3K9me3 and H3K27me3 histone marks and are induced during replication stress conditions. Concomitant sequencing of eccDNAs and RNA from the same cell uncovers the absence of correlation between eccDNA copy number and gene expression levels, except for a few oncogenes, including MYC, contained within a large eccDNA in colorectal cancer cells. Lastly, we apply scCircle-seq to one prostate cancer and two breast cancer specimens, revealing cancer-specific eccDNA landscapes and a higher propensity of eccDNAs to form in amplified genomic regions. scCircle-seq is a scalable tool that can be used to dissect the complexity of eccDNAs across different cell and tissue types, and further expands the potential of eccDNAs for cancer diagnostics.
Assuntos
DNA Circular , DNA , Masculino , Humanos , DNA Circular/genética , Cromossomos , Linhagem Celular , OncogenesRESUMO
Somatic copy number alterations (SCNAs) are pervasive in advanced human cancers, but their prevalence and spatial distribution in early-stage, localized tumors and their surrounding normal tissues are poorly characterized. Here, we perform multi-region, single-cell DNA sequencing to characterize the SCNA landscape across tumor-rich and normal tissue in two male patients with localized prostate cancer. We identify two distinct karyotypes: 'pseudo-diploid' cells harboring few SCNAs and highly aneuploid cells. Pseudo-diploid cells form numerous small-sized subclones ranging from highly spatially localized to broadly spread subclones. In contrast, aneuploid cells do not form subclones and are detected throughout the prostate, including normal tissue regions. Highly localized pseudo-diploid subclones are confined within tumor-rich regions and carry deletions in multiple tumor-suppressor genes. Our study reveals that SCNAs are widespread in normal and tumor regions across the prostate in localized prostate cancer patients and suggests that a subset of pseudo-diploid cells drive tumorigenesis in the aging prostate.
Assuntos
Variações do Número de Cópias de DNA , Neoplasias da Próstata , Análise de Célula Única , Humanos , Masculino , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Aneuploidia , Próstata/patologia , Próstata/metabolismo , Células Clonais , Diploide , IdosoRESUMO
Proteins containing ubiquitin-binding domains (UBDs) interact with ubiquitinated targets and regulate diverse biological processes, including endocytosis, signal transduction, transcription and DNA repair. Many of the UBD-containing proteins are also themselves monoubiquitinated, but the functional role and the mechanisms that underlie this modification are less well understood. Here, we demonstrate that monoubiquitination of the endocytic proteins Sts1, Sts2, Eps15 and Hrs results in intramolecular interactions between ubiquitin and their UBDs, thereby preventing them from binding in trans to ubiquitinated targets. Permanent monoubiquitination of these proteins, mimicked by the fusion of ubiquitin to their carboxyl termini, impairs their ability to regulate trafficking of ubiquitinated receptors. Moreover, we mapped the in vivo monoubiquitination site in Sts2 and demonstrated that its mutation enhances the Sts2-mediated effects of epidermal-growth-factor-receptor downregulation. We propose that monoubiquitination of ubiquitin-binding proteins inhibits their capacity to bind to and control the functions of ubiquitinated targets in vivo.