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1.
Cell ; 185(11): 1905-1923.e25, 2022 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-35523183

RESUMO

Tumor evolution is driven by the progressive acquisition of genetic and epigenetic alterations that enable uncontrolled growth and expansion to neighboring and distal tissues. The study of phylogenetic relationships between cancer cells provides key insights into these processes. Here, we introduced an evolving lineage-tracing system with a single-cell RNA-seq readout into a mouse model of Kras;Trp53(KP)-driven lung adenocarcinoma and tracked tumor evolution from single-transformed cells to metastatic tumors at unprecedented resolution. We found that the loss of the initial, stable alveolar-type2-like state was accompanied by a transient increase in plasticity. This was followed by the adoption of distinct transcriptional programs that enable rapid expansion and, ultimately, clonal sweep of stable subclones capable of metastasizing. Finally, tumors develop through stereotypical evolutionary trajectories, and perturbing additional tumor suppressors accelerates progression by creating novel trajectories. Our study elucidates the hierarchical nature of tumor evolution and, more broadly, enables in-depth studies of tumor progression.


Assuntos
Neoplasias , Animais , Genes ras , Camundongos , Neoplasias/genética , Filogenia , Sequenciamento do Exoma
2.
Nature ; 570(7759): 77-82, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31086336

RESUMO

Ontogeny describes the emergence of complex multicellular organisms from single totipotent cells. This field is particularly challenging in mammals, owing to the indeterminate relationship between self-renewal and differentiation, variation in progenitor field sizes, and internal gestation in these animals. Here we present a flexible, high-information, multi-channel molecular recorder with a single-cell readout and apply it as an evolving lineage tracer to assemble mouse cell-fate maps from fertilization through gastrulation. By combining lineage information with single-cell RNA sequencing profiles, we recapitulate canonical developmental relationships between different tissue types and reveal the nearly complete transcriptional convergence of endodermal cells of extra-embryonic and embryonic origins. Finally, we apply our cell-fate maps to estimate the number of embryonic progenitor cells and their degree of asymmetric partitioning during specification. Our approach enables massively parallel, high-resolution recording of lineage and other information in mammalian systems, which will facilitate the construction of a quantitative framework for understanding developmental processes.


Assuntos
Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Endoderma/embriologia , Endoderma/metabolismo , Feminino , Fertilização , Gastrulação , Regulação da Expressão Gênica no Desenvolvimento/genética , Masculino , Camundongos , Especificidade de Órgãos/genética , Fenótipo , Análise de Sequência de RNA , Análise de Célula Única
3.
Genes Dev ; 30(2): 191-207, 2016 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-26773003

RESUMO

Many long noncoding RNAs (lncRNAs) can regulate chromatin states, but the evolutionary origin and dynamics driving lncRNA-genome interactions are unclear. We adapted an integrative strategy that identifies lncRNA orthologs in different species despite limited sequence similarity, which is applicable to mammalian and insect lncRNAs. Analysis of the roX lncRNAs, which are essential for dosage compensation of the single X chromosome in Drosophila males, revealed 47 new roX orthologs in diverse Drosophilid species across ∼40 million years of evolution. Genetic rescue by roX orthologs and engineered synthetic lncRNAs showed that altering the number of focal, repetitive RNA structures determines roX ortholog function. Genomic occupancy maps of roX RNAs in four species revealed conserved targeting of X chromosome neighborhoods but rapid turnover of individual binding sites. Many new roX-binding sites evolved from DNA encoding a pre-existing RNA splicing signal, effectively linking dosage compensation to transcribed genes. Thus, dynamic change in lncRNAs and their genomic targets underlies conserved and essential lncRNA-genome interactions.


Assuntos
Evolução Biológica , Drosophila melanogaster/fisiologia , Genoma de Inseto/genética , RNA Longo não Codificante/metabolismo , Animais , Sítios de Ligação , Cromossomos de Insetos/genética , Cromossomos de Insetos/metabolismo , Mecanismo Genético de Compensação de Dose/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Masculino , Ligação Proteica
4.
Nat Rev Genet ; 17(1): 47-62, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26666209

RESUMO

Long non-coding RNAs (lncRNAs) are a diverse class of RNAs that engage in numerous biological processes across every branch of life. Although initially discovered as mRNA-like transcripts that do not encode proteins, recent studies have revealed features of lncRNAs that further distinguish them from mRNAs. In this Review, we describe special events in the lifetimes of lncRNAs - before, during and after transcription - and discuss how these events ultimately shape the unique characteristics and functional roles of lncRNAs.


Assuntos
RNA Longo não Codificante/biossíntese , Animais , Cromatina/genética , Cromatina/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Impressão Genômica , Humanos , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , Transporte de RNA , RNA Longo não Codificante/genética
5.
Mol Cell ; 51(2): 156-73, 2013 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-23870142

RESUMO

Dosage compensation in Drosophila is an epigenetic phenomenon utilizing proteins and long noncoding RNAs (lncRNAs) for transcriptional upregulation of the male X chromosome. Here, by using UV crosslinking followed by deep sequencing, we show that two enzymes in the Male-Specific Lethal complex, MLE RNA helicase and MSL2 ubiquitin ligase, bind evolutionarily conserved domains containing tandem stem-loops in roX1 and roX2 RNAs in vivo. These domains constitute the minimal RNA unit present in multiple copies in diverse arrangements for nucleation of the MSL complex. MLE binds to these domains with distinct ATP-independent and ATP-dependent behavior. Importantly, we show that different roX RNA domains have overlapping function, since only combinatorial mutations in the tandem stem-loops result in severe loss of dosage compensation and consequently male-specific lethality. We propose that repetitive structural motifs in lncRNAs could provide plasticity during multiprotein complex assemblies to ensure efficient targeting in cis or in trans along chromosomes.


Assuntos
Mecanismo Genético de Compensação de Dose/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas de Ligação a RNA/genética , RNA/genética , Fatores de Transcrição/genética , Cromossomo X/genética , Animais , Animais Geneticamente Modificados , Pareamento de Bases , Western Blotting , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Imunoprecipitação , Masculino , Mutação/genética , Conformação de Ácido Nucleico , RNA/química , RNA/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Sequências de Repetição em Tandem/genética , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Cromossomo X/metabolismo
6.
PLoS Genet ; 11(12): e1005680, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26633036

RESUMO

Gene expression profiling in E 11 mouse embryos identified high expression of the long noncoding RNA (lncRNA), LNCRNA-HIT in the undifferentiated limb mesenchyme, gut, and developing genital tubercle. In the limb mesenchyme, LncRNA-HIT was found to be retained in the nucleus, forming a complex with p100 and CBP. Analysis of the genome-wide distribution of LncRNA-HIT-p100/CBP complexes by ChIRP-seq revealed LncRNA-HIT associated peaks at multiple loci in the murine genome. Ontological analysis of the genes contacted by LncRNA-HIT-p100/CBP complexes indicate a primary role for these loci in chondrogenic differentiation. Functional analysis using siRNA-mediated reductions in LncRNA-HIT or p100 transcripts revealed a significant decrease in expression of many of the LncRNA-HIT-associated loci. LncRNA-HIT siRNA treatments also impacted the ability of the limb mesenchyme to form cartilage, reducing mesenchymal cell condensation and the formation of cartilage nodules. Mechanistically the LncRNA-HIT siRNA treatments impacted pro-chondrogenic gene expression by reducing H3K27ac or p100 activity, confirming that LncRNA-HIT is essential for chondrogenic differentiation in the limb mesenchyme. Taken together, these findings reveal a fundamental epigenetic mechanism functioning during early limb development, using LncRNA-HIT and its associated proteins to promote the expression of multiple genes whose products are necessary for the formation of cartilage.


Assuntos
Diferenciação Celular/genética , Condrogênese/genética , RNA Longo não Codificante/genética , Proteína p120 Ativadora de GTPase/genética , Animais , Epigênese Genética/genética , Extremidades/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Botões de Extremidades/crescimento & desenvolvimento , Mesoderma/crescimento & desenvolvimento , Camundongos , RNA Longo não Codificante/biossíntese , Proteína p120 Ativadora de GTPase/biossíntese
7.
Science ; 371(6532)2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33479121

RESUMO

Detailed phylogenies of tumor populations can recount the history and chronology of critical events during cancer progression, such as metastatic dissemination. We applied a Cas9-based, single-cell lineage tracer to study the rates, routes, and drivers of metastasis in a lung cancer xenograft mouse model. We report deeply resolved phylogenies for tens of thousands of cancer cells traced over months of growth and dissemination. This revealed stark heterogeneity in metastatic capacity, arising from preexisting and heritable differences in gene expression. We demonstrate that these identified genes can drive invasiveness and uncovered an unanticipated suppressive role for KRT17 We also show that metastases disseminated via multidirectional tissue routes and complex seeding topologies. Overall, we demonstrate the power of tracing cancer progression at subclonal resolution and vast scale.


Assuntos
Neoplasias Pulmonares/patologia , Metástase Neoplásica , Animais , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Linhagem da Célula , Células Clonais , Regulação Neoplásica da Expressão Gênica , Humanos , Queratina-17/genética , Neoplasias Pulmonares/genética , Camundongos , Invasividade Neoplásica/genética , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , Inoculação de Neoplasia , Transplante de Neoplasias , Fenótipo , RNA-Seq , Análise de Célula Única , Transcriptoma , Transplante Heterólogo
8.
Genome Biol ; 21(1): 92, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32290857

RESUMO

The pairing of CRISPR/Cas9-based gene editing with massively parallel single-cell readouts now enables large-scale lineage tracing. However, the rapid growth in complexity of data from these assays has outpaced our ability to accurately infer phylogenetic relationships. First, we introduce Cassiopeia-a suite of scalable maximum parsimony approaches for tree reconstruction. Second, we provide a simulation framework for evaluating algorithms and exploring lineage tracer design principles. Finally, we generate the most complex experimental lineage tracing dataset to date, 34,557 human cells continuously traced over 15 generations, and use it for benchmarking phylogenetic inference approaches. We show that Cassiopeia outperforms traditional methods by several metrics and under a wide variety of parameter regimes, and provide insight into the principles for the design of improved Cas9-enabled recorders. Together, these should broadly enable large-scale mammalian lineage tracing efforts. Cassiopeia and its benchmarking resources are publicly available at www.github.com/YosefLab/Cassiopeia.


Assuntos
Linhagem da Célula , Filogenia , Análise de Célula Única , Algoritmos , Sistemas CRISPR-Cas , Humanos , Mutação
9.
Methods Mol Biol ; 1262: 199-213, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25555583

RESUMO

Here we describe domain-specific chromatin isolation by RNA purification (dChIRP), a technique for dissecting the functional domains of a target RNA in situ. For an RNA of interest, dChIRP can identify domain-level intramolecular and intermolecular RNA-RNA, RNA-protein, and RNA-DNA interactions and maps the RNA's genomic binding sites with higher precision than domain-agnostic methods. We illustrate how this technique has been applied to the roX1 lncRNA to resolve its domain-level architecture, discover its protein- and chromatin-interacting domains, and map its occupancy on the X chromosome.


Assuntos
Cromatina/química , Cromatina/isolamento & purificação , Proteínas de Drosophila/isolamento & purificação , Drosophila melanogaster/genética , Fatores de Transcrição/isolamento & purificação , Animais , Sítios de Ligação , Cromatina/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , RNA Longo não Codificante/química , RNA Longo não Codificante/genética , RNA Longo não Codificante/isolamento & purificação , Análise de Sequência de DNA , Fatores de Transcrição/química , Fatores de Transcrição/genética
10.
Nat Genet ; 46(9): 929-31, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25162802

RESUMO

Polycomb/Trithorax response elements (PRE/TREs) are genetic elements that can stably silence or activate genes. A new study describes how long noncoding RNAs (lncRNAs) transcribed from opposite strands of the Drosophila melanogaster vestigial PRE/TRE throw the switch between these two opposing epigenetic states.


Assuntos
Proteínas Cromossômicas não Histona/genética , Proteínas de Drosophila/genética , Genes de Troca , Proteínas do Grupo Polycomb/genética , RNA não Traduzido , Elementos de Resposta , Transcrição Gênica , Animais
11.
Nat Biotechnol ; 32(9): 933-940, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24997788

RESUMO

Little is known about the functional domain architecture of long noncoding RNAs (lncRNAs) because of a relative paucity of suitable methods to analyze RNA function at a domain level. Here we describe domain-specific chromatin isolation by RNA purification (dChIRP), a scalable technique to dissect pairwise RNA-RNA, RNA-protein and RNA-chromatin interactions at the level of individual RNA domains in living cells. dChIRP of roX1, a lncRNA essential for Drosophila melanogaster X-chromosome dosage compensation, reveals a 'three-fingered hand' ribonucleoprotein topology. Each RNA finger binds chromatin and the male-specific lethal (MSL) protein complex and can individually rescue male lethality in roX-null flies, thus defining a minimal RNA domain for chromosome-wide dosage compensation. dChIRP improves the RNA genomic localization signal by >20-fold relative to previous techniques, and these binding sites are correlated with chromosome conformation data, indicating that most roX-bound loci cluster in a nuclear territory. These results suggest dChIRP can reveal lncRNA architecture and function with high precision and sensitivity.


Assuntos
Cromatina/genética , RNA Longo não Codificante/genética , RNA/isolamento & purificação , Animais , Sítios de Ligação , Cromatina/isolamento & purificação , Mecanismo Genético de Compensação de Dose , Feminino , Masculino
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