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1.
Mol Cell ; 81(8): 1666-1681.e6, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33823140

RESUMO

Nuclear speckles are prominent nuclear bodies that contain proteins and RNA involved in gene expression. Although links between nuclear speckles and gene activation are emerging, the mechanisms regulating association of genes with speckles are unclear. We find that speckle association of p53 target genes is driven by the p53 transcription factor. Focusing on p21, a key p53 target, we demonstrate that speckle association boosts expression by elevating nascent RNA amounts. p53-regulated speckle association did not depend on p53 transactivation functions but required an intact proline-rich domain and direct DNA binding, providing mechanisms within p53 for regulating gene-speckle association. Beyond p21, a substantial subset of p53 targets have p53-regulated speckle association. Strikingly, speckle-associating p53 targets are more robustly activated and occupy a distinct niche of p53 biology compared with non-speckle-associating p53 targets. Together, our findings illuminate regulated speckle association as a mechanism used by a transcription factor to boost gene expression.


Assuntos
Núcleo Celular/genética , Regulação da Expressão Gênica/genética , Proteínas Nucleares/genética , RNA/genética , Ativação Transcricional/genética , Proteína Supressora de Tumor p53/genética , DNA/genética , Células HEK293 , Humanos , Corpos de Inclusão Intranuclear/genética , Ligação Proteica/genética , Fatores de Transcrição/genética , Transcrição Genética/genética
2.
Nat Biotechnol ; 2021 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-33619394

RESUMO

Molecular differences between individual cells can lead to dramatic differences in cell fate, such as death versus survival of cancer cells upon drug treatment. These originating differences remain largely hidden due to difficulties in determining precisely what variable molecular features lead to which cellular fates. Thus, we developed Rewind, a methodology that combines genetic barcoding with RNA fluorescence in situ hybridization to directly capture rare cells that give rise to cellular behaviors of interest. Applying Rewind to BRAFV600E melanoma, we trace drug-resistant cell fates back to single-cell gene expression differences in their drug-naive precursors (initial frequency of ~1:1,000-1:10,000 cells) and relative persistence of MAP kinase signaling soon after drug treatment. Within this rare subpopulation, we uncover a rich substructure in which molecular differences among several distinct subpopulations predict future differences in phenotypic behavior, such as proliferative capacity of distinct resistant clones after drug treatment. Our results reveal hidden, rare-cell variability that underlies a range of latent phenotypic outcomes upon drug exposure.

3.
Nat Genet ; 53(1): 76-85, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33398196

RESUMO

Cellular plasticity describes the ability of cells to transition from one set of phenotypes to another. In melanoma, transient fluctuations in the molecular state of tumor cells mark the formation of rare cells primed to survive BRAF inhibition and reprogram into a stably drug-resistant fate. However, the biological processes governing cellular priming remain unknown. We used CRISPR-Cas9 genetic screens to identify genes that affect cell fate decisions by altering cellular plasticity. We found that many factors can independently affect cellular priming and fate decisions. We discovered a new plasticity-based mode of increasing resistance to BRAF inhibition that pushes cells towards a more differentiated state. Manipulating cellular plasticity through inhibition of DOT1L before the addition of the BRAF inhibitor resulted in more therapy resistance than concurrent administration. Our results indicate that modulating cellular plasticity can alter cell fate decisions and may prove useful for treating drug resistance in other cancers.


Assuntos
Plasticidade Celular/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Testes Genéticos , Neoplasias/genética , Neoplasias/patologia , Animais , Sistemas CRISPR-Cas/genética , Diferenciação Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Histona-Lisina N-Metiltransferase/genética , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Camundongos Endogâmicos NOD , Camundongos SCID , Modelos Biológicos , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Proteínas Proto-Oncogênicas B-raf/genética , Transcrição Genética
4.
Elife ; 92020 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-33284110

RESUMO

Two different cell signals often affect transcription of the same gene. In such cases, it is natural to ask how the combined transcriptional response compares to the individual responses. The most commonly used mechanistic models predict additive or multiplicative combined responses, but a systematic genome-wide evaluation of these predictions is not available. Here, we analyzed the transcriptional response of human MCF-7 cells to retinoic acid and TGF-ß, applied individually and in combination. The combined transcriptional responses of induced genes exhibited a range of behaviors, but clearly favored both additive and multiplicative outcomes. We performed paired chromatin accessibility measurements and found that increases in accessibility were largely additive. There was some association between super-additivity of accessibility and multiplicative or super-multiplicative combined transcriptional responses, while sub-additivity of accessibility associated with additive transcriptional responses. Our findings suggest that mechanistic models of combined transcriptional regulation must be able to reproduce a range of behaviors.

5.
Sci Adv ; 6(34): eaba9869, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32875108

RESUMO

In social insects, workers and queens arise from the same genome but display profound differences in behavior and longevity. In Harpegnathos saltator ants, adult workers can transition to a queen-like state called gamergate, which results in reprogramming of social behavior and life-span extension. Using single-cell RNA sequencing, we compared the distribution of neuronal and glial populations before and after the social transition. We found that the conversion of workers into gamergates resulted in the expansion of neuroprotective ensheathing glia. Brain injury assays revealed that activation of the damage response gene Mmp1 was weaker in old workers, where the relative frequency of ensheathing glia also declined. On the other hand, long-lived gamergates retained a larger fraction of ensheathing glia and the ability to mount a strong Mmp1 response to brain injury into old age. We also observed molecular and cellular changes suggestive of age-associated decline in ensheathing glia in Drosophila.

6.
Cell ; 182(4): 947-959.e17, 2020 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-32735851

RESUMO

Non-genetic factors can cause individual cells to fluctuate substantially in gene expression levels over time. It remains unclear whether these fluctuations can persist for much longer than the time of one cell division. Current methods for measuring gene expression in single cells mostly rely on single time point measurements, making the duration of gene expression fluctuations or cellular memory difficult to measure. Here, we combined Luria and Delbrück's fluctuation analysis with population-based RNA sequencing (MemorySeq) for identifying genes transcriptome-wide whose fluctuations persist for several divisions. MemorySeq revealed multiple gene modules that expressed together in rare cells within otherwise homogeneous clonal populations. These rare cell subpopulations were associated with biologically distinct behaviors like proliferation in the face of anti-cancer therapeutics. The identification of non-genetic, multigenerational fluctuations can reveal new forms of biological memory in single cells and suggests that non-genetic heritability of cellular state may be a quantitative property.

7.
Cell Syst ; 10(4): 363-378.e12, 2020 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-32325034

RESUMO

Non-genetic transcriptional variability is a potential mechanism for therapy resistance in melanoma. Specifically, rare subpopulations of cells occupy a transient pre-resistant state characterized by coordinated high expression of several genes and survive therapy. How might these rare states arise and disappear within the population? It is unclear whether the canonical models of probabilistic transcriptional pulsing can explain this behavior, or if it requires special, hitherto unidentified mechanisms. We show that a minimal model of transcriptional bursting and gene interactions can give rise to rare coordinated high expression states. These states occur more frequently in networks with low connectivity and depend on three parameters. While entry into these states is initiated by a long transcriptional burst that also triggers entry of other genes, the exit occurs through independent inactivation of individual genes. Together, we demonstrate that established principles of gene regulation are sufficient to describe this behavior and argue for its more general existence. A record of this paper's transparent peer review process is included in the Supplemental Information.

8.
Immunity ; 52(2): 257-274.e11, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32049053

RESUMO

Genetics is a major determinant of susceptibility to autoimmune disorders. Here, we examined whether genome organization provides resilience or susceptibility to sequence variations, and how this would contribute to the molecular etiology of an autoimmune disease. We generated high-resolution maps of linear and 3D genome organization in thymocytes of NOD mice, a model of type 1 diabetes (T1D), and the diabetes-resistant C57BL/6 mice. Multi-enhancer interactions formed at genomic regions harboring genes with prominent roles in T cell development in both strains. However, diabetes risk-conferring loci coalesced enhancers and promoters in NOD, but not C57BL/6 thymocytes. 3D genome mapping of NODxC57BL/6 F1 thymocytes revealed that genomic misfolding in NOD mice is mediated in cis. Moreover, immune cells infiltrating the pancreas of humans with T1D exhibited increased expression of genes located on misfolded loci in mice. Thus, genetic variation leads to altered 3D chromatin architecture and associated changes in gene expression that may underlie autoimmune pathology.


Assuntos
Cromatina/metabolismo , Diabetes Mellitus Tipo 1/genética , Predisposição Genética para Doença/genética , Timócitos/patologia , Animais , Fator de Ligação a CCCTC/metabolismo , Mapeamento Cromossômico , Diabetes Mellitus Tipo 1/patologia , Epigênese Genética , Expressão Gênica , Loci Gênicos/genética , Variação Genética , Genoma/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Pâncreas/patologia , Sequências Reguladoras de Ácido Nucleico
9.
Nat Methods ; 16(7): 633-639, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31235883

RESUMO

Mammalian genomes are folded into tens of thousands of long-range looping interactions. The cause-and-effect relationship between looping and genome function is poorly understood, and the extent to which loops are dynamic on short time scales remains an unanswered question. Here, we engineer a new class of synthetic architectural proteins for directed rearrangement of the three-dimensional genome using blue light. We target our light-activated-dynamic-looping (LADL) system to two genomic anchors with CRISPR guide RNAs and induce their spatial colocalization via light-induced heterodimerization of cryptochrome 2 and a dCas9-CIBN fusion protein. We apply LADL to redirect a stretch enhancer (SE) away from its endogenous Klf4 target gene and to the Zfp462 promoter. Using single-molecule RNA-FISH, we demonstrate that de novo formation of the Zfp462-SE loop correlates with a modest increase in Zfp462 expression. LADL facilitates colocalization of genomic loci without exogenous chemical cofactors and will enable future efforts to engineer reversible and oscillatory loops on short time scales.


Assuntos
Regulação da Expressão Gênica , Engenharia de Proteínas , Animais , Proteínas de Transporte/genética , Células Cultivadas , Proteínas de Ligação a DNA , Fatores de Transcrição Kruppel-Like/genética , Luz , Masculino , Camundongos , Proteínas do Tecido Nervoso/genética , Regiões Promotoras Genéticas , RNA Guia/genética
10.
Artigo em Inglês | MEDLINE | ID: mdl-31043413

RESUMO

In the postgenomic era, it is clear that the human genome encodes thousands of long noncoding RNAs (lncRNAs). Along the way, RNA imaging (e.g., RNA fluorescence in situ hybridization [RNA-FISH]) has been instrumental in identifying powerful roles for lncRNAs based on their subcellular localization patterns. Here, we explore how RNA imaging technologies have shed new light on how, when, and where lncRNAs may play functional roles. Specifically, we will synthesize the underlying principles of RNA imaging techniques by exploring several landmark lncRNA imaging studies that have illuminated key insights into lncRNA biology.


Assuntos
Genômica/métodos , RNA Longo não Codificante/genética , Análise de Sequência de RNA/métodos , Humanos , Reconhecimento Automatizado de Padrão
12.
PLoS Genet ; 15(1): e1007874, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30625149

RESUMO

Extensive cell-to-cell variation exists even among putatively identical cells, and there is great interest in understanding how the properties of transcription relate to this heterogeneity. Differential expression from the two gene copies in diploid cells could potentially contribute, yet our ability to measure from which gene copy individual RNAs originated remains limited, particularly in the context of tissues. Here, we demonstrate quantitative, single molecule allele-specific RNA FISH adapted for use on tissue sections, allowing us to determine the chromosome of origin of individual RNA molecules in formaldehyde-fixed tissues. We used this method to visualize the allele-specific expression of Xist and multiple autosomal genes in mouse kidney. By combining these data with mathematical modeling, we evaluated models for allele-specific heterogeneity, in particular demonstrating that apparent expression from only one of the alleles in single cells can arise as a consequence of low-level mRNA abundance and transcriptional bursting.


Assuntos
Desequilíbrio Alélico/genética , Hibridização in Situ Fluorescente/métodos , Rim/metabolismo , RNA Longo não Codificante/genética , Alelos , Animais , Regulação da Expressão Gênica no Desenvolvimento/genética , Camundongos , Especificidade de Órgãos , RNA Longo não Codificante/isolamento & purificação
13.
Cancer Discov ; 9(1): 64-81, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30279173

RESUMO

Physical changes in skin are among the most visible signs of aging. We found that young dermal fibroblasts secrete high levels of extracellular matrix (ECM) constituents, including proteoglycans, glycoproteins, and cartilage-linking proteins. The most abundantly secreted was HAPLN1, a hyaluronic and proteoglycan link protein. HAPLN1 was lost in aged fibroblasts, resulting in a more aligned ECM that promoted metastasis of melanoma cells. Reconstituting HAPLN1 inhibited metastasis in an aged microenvironment, in 3-D skin reconstruction models, and in vivo. Intriguingly, aged fibroblast-derived matrices had the opposite effect on the migration of T cells, inhibiting their motility. HAPLN1 treatment of aged fibroblasts restored motility of mononuclear immune cells, while impeding that of polymorphonuclear immune cells, which in turn affected regulatory T-cell recruitment. These data suggest that although age-related physical changes in the ECM can promote tumor cell motility, they may adversely affect the motility of some immune cells, resulting in an overall change in the immune microenvironment. Understanding the physical changes in aging skin may provide avenues for more effective therapy for older patients with melanoma. SIGNIFICANCE: These data shed light on the mechanochemical interactions that occur between aged skin, tumor, and immune cell populations, which may affect tumor metastasis and immune cell infiltration, with implications for the efficacy of current therapies for melanoma.See related commentary by Marie and Merlino, p. 19.This article is highlighted in the In This Issue feature, p. 1.


Assuntos
Envelhecimento , Colágeno/metabolismo , Melanoma/metabolismo , Pele/metabolismo , Animais , Células Cultivadas , Proteínas da Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Humanos , Sistema Imunitário , Melanoma/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Metástase Neoplásica , Proteoglicanas/metabolismo , Pele/fisiopatologia , Microambiente Tumoral
14.
Mol Cell ; 73(3): 519-532.e4, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30554946

RESUMO

Transcriptional regulation occurs via changes to rates of different biochemical steps of transcription, but it remains unclear which rates are subject to change upon biological perturbation. Biochemical studies have suggested that stimuli predominantly affect the rates of RNA polymerase II (Pol II) recruitment and polymerase release from promoter-proximal pausing. Single-cell studies revealed that transcription occurs in discontinuous bursts, suggesting that features of such bursts like frequency and intensity could also be regulated. We combined Pol II chromatin immunoprecipitation sequencing (ChIP-seq) and single-cell transcriptional measurements to show that an independently regulated burst initiation step is required before polymerase recruitment can occur. Using a number of global and targeted transcriptional regulatory perturbations, we showed that biological perturbations regulated both burst initiation and polymerase pause release rates but seemed not to regulate polymerase recruitment rate. Our results suggest that transcriptional regulation primarily acts by changing the rates of burst initiation and polymerase pause release.


Assuntos
Células-Tronco Embrionárias Murinas/enzimologia , RNA Polimerase II/metabolismo , RNA/biossíntese , Sítio de Iniciação de Transcrição , Iniciação da Transcrição Genética , Ativação Transcricional , Animais , Sítios de Ligação , Linhagem Celular , Simulação por Computador , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Modelos Genéticos , Ligação Proteica , RNA/genética , RNA Polimerase II/genética , Receptores Estrogênicos/genética , Receptores Estrogênicos/metabolismo , Fatores de Tempo
15.
Nat Biotechnol ; 2018 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-30418432

RESUMO

Methods for detecting single nucleic acids in cell and tissues, such as fluorescence in situ hybridization (FISH), are limited by relatively low signal intensity and nonspecific probe binding. Here we present click-amplifying FISH (clampFISH), a method for fluorescence detection of nucleic acids that achieves high specificity and high-gain (>400-fold) signal amplification. ClampFISH probes form a 'C' configuration upon hybridization to the sequence of interest in a double helical manner. The ends of the probes are ligated together using bio-orthogonal click chemistry, effectively locking the probes around the target. Iterative rounds of hybridization and click amplify the fluorescence intensity. We show that clampFISH enables the detection of RNA species with low-magnification microscopy and in RNA-based flow cytometry. Additionally, we show that the modular design of clampFISH probes allows multiplexing of RNA and DNA detection, that the locking mechanism prevents probe detachment in expansion microscopy, and that clampFISH can be applied in tissue samples.

16.
Nat Methods ; 15(8): 587-590, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30065368

RESUMO

We describe Quanti.us , a crowd-based image-annotation platform that provides an accurate alternative to computational algorithms for difficult image-analysis problems. We used Quanti.us for a variety of medium-throughput image-analysis tasks and achieved 10-50× savings in analysis time compared with that required for the same task by a single expert annotator. We show equivalent deep learning performance for Quanti.us-derived and expert-derived annotations, which should allow scalable integration with tailored machine learning algorithms.


Assuntos
Processamento de Imagem Assistida por Computador/métodos , Software , Algoritmos , Animais , Biologia Computacional/métodos , Crowdsourcing/métodos , Humanos , Imageamento Tridimensional/métodos , Internet , Aprendizado de Máquina
17.
Cancer Res ; 78(17): 4957-4970, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29976575

RESUMO

The tumor microenvironment (TME) plays a major role in the pathogenesis of multiple cancer types, including upper-gastrointestinal (GI) cancers that currently lack effective therapeutic options. Cancer-associated fibroblasts (CAF) are an essential component of the TME, contributing to tumorigenesis by secreting growth factors, modifying the extracellular matrix, supporting angiogenesis, and suppressing antitumor immune responses. Through an unbiased approach, we have established that IL-6 mediates cross-talk between tumor cells and CAF not only by supporting tumor cell growth, but also by promoting fibroblast activation. As a result, IL-6 receptor (IL6Rα) and downstream effectors offer opportunities for targeted therapy in upper-GI cancers. IL-6 loss suppressed tumorigenesis in physiologically relevant three-dimensional (3D) organotypic and 3D tumoroid models and murine models of esophageal cancer. Tocilizumab, an anti-IL6Rα antibody, suppressed tumor growth in vivo in part via inhibition of STAT3 and MEK/ERK signaling. Analysis of a pan-cancer TCGA dataset revealed an inverse correlation between IL-6 and IL6Rα overexpression and patient survival. Therefore, we expanded evaluation of tocilizumab to head and neck squamous cell carcinoma patient-derived xenografts and gastric adenocarcinoma xenografts, demonstrating suppression of tumor growth and altered STAT3 and ERK1/2 gene signatures. We used small-molecule inhibitors of STAT3 and MEK1/2 signaling to suppress tumorigenesis in the 3D organotypic model of esophageal cancer. We demonstrate that IL6 is a major contributor to the dynamic cross-talk between tumor cells and CAF in the TME. Our findings provide a translational rationale for inhibition of IL6Rα and downstream signaling pathways as a novel targeted therapy in oral-upper-GI cancers.Significance: These findings demonstrate the interaction of esophageal cancer and cancer-associated fibroblasts through IL-6 signaling, providing rationale for a novel therapeutic approach to target these cancers. Cancer Res; 78(17); 4957-70. ©2018 AACR.


Assuntos
Neoplasias Esofágicas/genética , Neoplasias Gastrointestinais/genética , Interleucina-6/genética , Receptores de Interleucina-6/genética , Animais , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Carcinogênese/genética , Linhagem Celular Tumoral , Neoplasias Esofágicas/patologia , Neoplasias Gastrointestinais/patologia , Humanos , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Fator de Transcrição STAT3/genética , Transdução de Sinais , Microambiente Tumoral/genética , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Nat Methods ; 15(7): 539-542, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29941873

RESUMO

In single-cell RNA sequencing (scRNA-seq) studies, only a small fraction of the transcripts present in each cell are sequenced. This leads to unreliable quantification of genes with low or moderate expression, which hinders downstream analysis. To address this challenge, we developed SAVER (single-cell analysis via expression recovery), an expression recovery method for unique molecule index (UMI)-based scRNA-seq data that borrows information across genes and cells to provide accurate expression estimates for all genes.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA/genética , Análise de Célula Única/métodos , Animais , Sequência de Bases , Córtex Cerebral/citologia , Perfilação da Expressão Gênica/métodos , Humanos , Camundongos , RNA/química , Análise de Sequência de RNA/métodos , Software
19.
Proc Natl Acad Sci U S A ; 115(28): E6437-E6446, 2018 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-29946020

RESUMO

Single-cell RNA sequencing (scRNA-seq) enables the quantification of each gene's expression distribution across cells, thus allowing the assessment of the dispersion, nonzero fraction, and other aspects of its distribution beyond the mean. These statistical characterizations of the gene expression distribution are critical for understanding expression variation and for selecting marker genes for population heterogeneity. However, scRNA-seq data are noisy, with each cell typically sequenced at low coverage, thus making it difficult to infer properties of the gene expression distribution from raw counts. Based on a reexamination of nine public datasets, we propose a simple technical noise model for scRNA-seq data with unique molecular identifiers (UMI). We develop deconvolution of single-cell expression distribution (DESCEND), a method that deconvolves the true cross-cell gene expression distribution from observed scRNA-seq counts, leading to improved estimates of properties of the distribution such as dispersion and nonzero fraction. DESCEND can adjust for cell-level covariates such as cell size, cell cycle, and batch effects. DESCEND's noise model and estimation accuracy are further evaluated through comparisons to RNA FISH data, through data splitting and simulations and through its effectiveness in removing known batch effects. We demonstrate how DESCEND can clarify and improve downstream analyses such as finding differentially expressed genes, identifying cell types, and selecting differentiation markers.


Assuntos
Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Modelos Genéticos , RNA/biossíntese , RNA/genética , Animais , Humanos
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