RESUMO
Treatment failure for the lethal brain tumor glioblastoma (GBM) is attributed to intratumoral heterogeneity and tumor evolution. We utilized 3D neuronavigation during surgical resection to acquire samples representing the whole tumor mapped by 3D spatial coordinates. Integrative tissue and single-cell analysis revealed sources of genomic, epigenomic, and microenvironmental intratumoral heterogeneity and their spatial patterning. By distinguishing tumor-wide molecular features from those with regional specificity, we inferred GBM evolutionary trajectories from neurodevelopmental lineage origins and initiating events such as chromothripsis to emergence of genetic subclones and spatially restricted activation of differential tumor and microenvironmental programs in the core, periphery, and contrast-enhancing regions. Our work depicts GBM evolution and heterogeneity from a 3D whole-tumor perspective, highlights potential therapeutic targets that might circumvent heterogeneity-related failures, and establishes an interactive platform enabling 360° visualization and analysis of 3D spatial patterns for user-selected genes, programs, and other features across whole GBM tumors.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Modelos Biológicos , Humanos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Epigenômica , Genômica , Glioblastoma/genética , Glioblastoma/patologia , Análise de Célula Única , Microambiente Tumoral , Heterogeneidade GenéticaRESUMO
The cleavage of zygotes generates totipotent blastomeres. In human 8-cell blastomeres, zygotic genome activation (ZGA) occurs to initiate the ontogenesis program. However, capturing and maintaining totipotency in human cells pose significant challenges. Here, we realize culturing human totipotent blastomere-like cells (hTBLCs). We find that splicing inhibition can transiently reprogram human pluripotent stem cells into ZGA-like cells (ZLCs), which subsequently transition into stable hTBLCs after long-term passaging. Distinct from reported 8-cell-like cells (8CLCs), both ZLCs and hTBLCs widely silence pluripotent genes. Interestingly, ZLCs activate a particular group of ZGA-specific genes, and hTBLCs are enriched with pre-ZGA-specific genes. During spontaneous differentiation, hTBLCs re-enter the intermediate ZLC stage and further generate epiblast (EPI)-, primitive endoderm (PrE)-, and trophectoderm (TE)-like lineages, effectively recapitulating human pre-implantation development. Possessing both embryonic and extraembryonic developmental potency, hTBLCs can autonomously generate blastocyst-like structures in vitro without external cell signaling. In summary, our study provides key criteria and insights into human cell totipotency.
Assuntos
Diferenciação Celular , Spliceossomos , Animais , Humanos , Camundongos , Blastocisto/metabolismo , Blastocisto/citologia , Blastômeros/metabolismo , Blastômeros/citologia , Reprogramação Celular , Desenvolvimento Embrionário/genética , Camadas Germinativas/metabolismo , Camadas Germinativas/citologia , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Splicing de RNA , Spliceossomos/metabolismo , Células-Tronco Totipotentes/metabolismo , Células-Tronco Totipotentes/citologia , Zigoto/metabolismo , Células Cultivadas , Modelos Moleculares , Estrutura Terciária de Proteína , Genoma Humano , Análise de Célula Única , Fator 15 de Diferenciação de Crescimento/química , Fator 15 de Diferenciação de Crescimento/genética , Fator 15 de Diferenciação de Crescimento/metabolismo , Epigenômica , Linhagem da CélulaRESUMO
Progenitor cells are critical in preserving organismal homeostasis, yet their diversity and dynamics in the aged brain remain underexplored. We introduced TrackerSci, a single-cell genomic method that combines newborn cell labeling and combinatorial indexing to characterize the transcriptome and chromatin landscape of proliferating progenitor cells in vivo. Using TrackerSci, we investigated the dynamics of newborn cells in mouse brains across various ages and in a mouse model of Alzheimer's disease. Our dataset revealed diverse progenitor cell types in the brain and their epigenetic signatures. We further quantified aging-associated shifts in cell-type-specific proliferation and differentiation and deciphered the associated molecular programs. Extending our study to the progenitor cells in the aged human brain, we identified conserved genetic signatures across species and pinpointed region-specific cellular dynamics, such as the reduced oligodendrogenesis in the cerebellum. We anticipate that TrackerSci will be broadly applicable to unveil cell-type-specific temporal dynamics in diverse systems.
Assuntos
Encéfalo , Células-Tronco , Animais , Humanos , Camundongos , Encéfalo/metabolismo , Diferenciação Celular , Cromatina/metabolismo , Transcriptoma , Envelhecimento , EpigenômicaRESUMO
Cellular lineage histories and their molecular states encode fundamental principles of tissue development and homeostasis. Current lineage-recording mouse models have insufficient barcode diversity and single-cell lineage coverage for profiling tissues composed of millions of cells. Here, we developed DARLIN, an inducible Cas9 barcoding mouse line that utilizes terminal deoxynucleotidyl transferase (TdT) and 30 CRISPR target sites. DARLIN is inducible, generates massive lineage barcodes across tissues, and enables the detection of edited barcodes in â¼70% of profiled single cells. Using DARLIN, we examined fate bias within developing hematopoietic stem cells (HSCs) and revealed unique features of HSC migration. Additionally, we established a protocol for joint transcriptomic and epigenomic single-cell measurements with DARLIN and found that cellular clonal memory is associated with genome-wide DNA methylation rather than gene expression or chromatin accessibility. DARLIN will enable the high-resolution study of lineage relationships and their molecular signatures in diverse tissues and physiological contexts.
Assuntos
Epigenômica , Transcriptoma , Animais , Camundongos , Transcriptoma/genética , Linhagem da Célula/genética , Perfilação da Expressão Gênica , Modelos Animais de Doenças , DNARESUMO
Recent work has identified dozens of non-coding loci for Alzheimer's disease (AD) risk, but their mechanisms and AD transcriptional regulatory circuitry are poorly understood. Here, we profile epigenomic and transcriptomic landscapes of 850,000 nuclei from prefrontal cortexes of 92 individuals with and without AD to build a map of the brain regulome, including epigenomic profiles, transcriptional regulators, co-accessibility modules, and peak-to-gene links in a cell-type-specific manner. We develop methods for multimodal integration and detecting regulatory modules using peak-to-gene linking. We show AD risk loci are enriched in microglial enhancers and for specific TFs including SPI1, ELF2, and RUNX1. We detect 9,628 cell-type-specific ATAC-QTL loci, which we integrate alongside peak-to-gene links to prioritize AD variant regulatory circuits. We report differential accessibility of regulatory modules in late AD in glia and in early AD in neurons. Strikingly, late-stage AD brains show global epigenome dysregulation indicative of epigenome erosion and cell identity loss.
Assuntos
Doença de Alzheimer , Encéfalo , Regulação da Expressão Gênica , Humanos , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Encéfalo/patologia , Epigenoma , Epigenômica , Estudo de Associação Genômica AmplaRESUMO
In addition to acute hyperinflammatory responses, SARS-CoV-2 infections can have long-term effects on our immune system leading to, for example, post-acute sequelae of COVID-19 (PASC). In this issue of Cell, Cheong et al. show that severe infections via IL-6 induce persistent epigenetic signatures in hemopoietic stem cells and their myeloid progenitors associated with increased inflammatory potential.
Assuntos
COVID-19 , Síndrome de COVID-19 Pós-Aguda , Humanos , COVID-19/genética , COVID-19/imunologia , COVID-19/patologia , Epigenômica , Células-Tronco Hematopoéticas , Síndrome de COVID-19 Pós-Aguda/imunologia , Síndrome de COVID-19 Pós-Aguda/patologia , SARS-CoV-2 , Imunidade Treinada , Inflamação/patologiaRESUMO
Spatial omics techniques generate spatially resolved, comprehensive data about molecules that define the identity and function of cells in tissues. Epigenetic multiplexing approaches such as Multiplexed Error-robust FISH (MERFISH), introduced by Lu et al.1 in this issue of Cell, now allows researchers to study the epigenomic regulation of gene expression in a tissue-region specific manner.
Assuntos
Epigenômica , Análise de Célula Única , Epigenômica/métodos , Análise de Célula Única/métodos , Perfilação da Expressão Gênica/métodos , Hibridização in Situ Fluorescente/métodosRESUMO
The recent development of spatial omics methods has enabled single-cell profiling of the transcriptome and 3D genome organization with high spatial resolution. Expanding the repertoire of spatial omics tools, a spatially resolved single-cell epigenomics method will accelerate understanding of the spatial regulation of cell and tissue functions. Here, we report a method for spatially resolved epigenomic profiling of single cells using in situ tagmentation and transcription followed by multiplexed imaging. We demonstrated the ability to profile histone modifications marking active promoters, putative enhancers, and silent promoters in individual cells, and generated high-resolution spatial atlas of hundreds of active promoters and putative enhancers in embryonic and adult mouse brains. Our results suggested putative promoter-enhancer pairs and enhancer hubs regulating developmentally important genes. We envision this approach will be generally applicable to spatial profiling of epigenetic modifications and DNA-binding proteins, advancing our understanding of how gene expression is spatiotemporally regulated by the epigenome.
Assuntos
Epigenômica , Código das Histonas , Camundongos , Animais , Regiões Promotoras Genéticas , Epigênese Genética , Transcriptoma , Elementos Facilitadores Genéticos , CromatinaRESUMO
In 1961, Jacob and Monod proposed the operon model of gene regulation. At the model's core was the modular assembly of regulators, operators, and structural genes. To illustrate the composability of these elements, Jacob and Monod linked phenotypic diversity to the architectures of regulatory circuits. In this review, we examine how the circuit blueprints imagined by Jacob and Monod laid the foundation for the first synthetic gene networks that launched the field of synthetic biology in 2000. We discuss the influences of the operon model and its broader theoretical framework on the first generation of synthetic biological circuits, which were predominantly transcriptional and posttranscriptional circuits. We also describe how recent advances in molecular biology beyond the operon model-namely, programmable DNA- and RNA-binding molecules as well as models of epigenetic and posttranslational regulation-are expanding the synthetic biology toolkit and enabling the design of more complex biological circuits.
Assuntos
Epigenômica/métodos , Óperon , Proteínas/genética , Biologia Sintética/métodos , Sistemas CRISPR-Cas , Retroalimentação Fisiológica , Regulação da Expressão Gênica , Biologia Molecular/métodos , Proteínas/metabolismo , RNA Mensageiro/genética , Transcrição GênicaRESUMO
The field of epigenetics has exploded over the last two decades, revealing an astonishing level of complexity in the way genetic information is stored and accessed in eukaryotes. This expansion of knowledge, which is very much ongoing, has been made possible by the availability of evermore sensitive and precise molecular tools. This review focuses on the increasingly important role that chemistry plays in this burgeoning field. In an effort to make these contributions more accessible to the nonspecialist, we group available chemical approaches into those that allow the covalent structure of the protein and DNA components of chromatin to be manipulated, those that allow the activity of myriad factors that act on chromatin to be controlled, and those that allow the covalent structure and folding of chromatin to be characterized. The application of these tools is illustrated through a series of case studies that highlight how the molecular precision afforded by chemistry is being used to establish causal biochemical relationships at the heart of epigenetic regulation.
Assuntos
Bioquímica/métodos , Técnicas de Química Analítica/métodos , Epigenômica/métodos , Epigenoma , Transferência Ressonante de Energia de Fluorescência , Heterocromatina/genética , Histonas/metabolismo , Técnicas de Sonda Molecular , Biossíntese de Proteínas , Fatores de Transcrição/genética , UbiquitinaçãoRESUMO
Regulatory T (Treg) cells are critical for immune tolerance but also form a barrier to antitumor immunity. As therapeutic strategies involving Treg cell depletion are limited by concurrent autoimmune disorders, identification of intratumoral Treg cell-specific regulatory mechanisms is needed for selective targeting. Epigenetic modulators can be targeted with small compounds, but intratumoral Treg cell-specific epigenetic regulators have been unexplored. Here, we show that JMJD1C, a histone demethylase upregulated by cytokines in the tumor microenvironment, is essential for tumor Treg cell fitness but dispensable for systemic immune homeostasis. JMJD1C deletion enhanced AKT signals in a manner dependent on histone H3 lysine 9 dimethylation (H3K9me2) demethylase and STAT3 signals independently of H3K9me2 demethylase, leading to robust interferon-γ production and tumor Treg cell fragility. We have also developed an oral JMJD1C inhibitor that suppresses tumor growth by targeting intratumoral Treg cells. Overall, this study identifies JMJD1C as an epigenetic hub that can integrate signals to establish tumor Treg cell fitness, and we present a specific JMJD1C inhibitor that can target tumor Treg cells without affecting systemic immune homeostasis.
Assuntos
Doenças Autoimunes , Humanos , Citocinas , Epigenômica , Histona Desmetilases , Homeostase , Oxirredutases N-Desmetilantes , Histona Desmetilases com o Domínio Jumonji/genéticaRESUMO
Memory B cells (MBCs) are phenotypically and functionally diverse, but their developmental origins remain undefined. Murine MBCs can be divided into subsets by expression of CD80 and PD-L2. Upon re-immunization, CD80/PD-L2 double-negative (DN) MBCs spawn germinal center B cells (GCBCs), whereas CD80/PD-L2 double-positive (DP) MBCs generate plasmablasts but not GCBCs. Using multiple approaches, including generation of an inducible GCBC-lineage reporter mouse, we demonstrate in a T cell-dependent response that DN cells formed independently of the germinal center (GC), whereas DP cells exhibited either extrafollicular (DPEX) or GCBC (DPGC) origins. Chromatin and transcriptional profiling revealed similarity of DN cells with an early memory precursor. Reciprocally, GCBC-derived DP cells shared distinct genomic features with GCBCs, while DPEX cells had hybrid features. Upon restimulation, DPEX cells were more prone to divide, while DPGC cells differentiated toward IgG1+ plasmablasts. Thus, MBC functional diversity is generated through distinct developmental histories, which imprint characteristic epigenetic patterns onto their progeny, thereby programming them for divergent functional responses.
Assuntos
Subpopulações de Linfócitos B , Animais , Camundongos , Células B de Memória , Epigenômica , Linfócitos B , Epigênese GenéticaRESUMO
In this issue of Cell, Nuñez et al. develop CRISPRoff, a programmable epigenetic memory writer capable of establishing specific gene silencing programs that are stably maintained across cell division and differentiation. The singular dCas9 fusion offers a simple, reliable, and general tool for genome-wide screens, multiplexed editing, and potential therapeutics.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Epigenômica , Regiões Promotoras Genéticas , RedaçãoRESUMO
Emerging evidence indicates a fundamental role for the epigenome in immunity. Here, we mapped the epigenomic and transcriptional landscape of immunity to influenza vaccination in humans at the single-cell level. Vaccination against seasonal influenza induced persistently diminished H3K27ac in monocytes and myeloid dendritic cells (mDCs), which was associated with impaired cytokine responses to Toll-like receptor stimulation. Single-cell ATAC-seq analysis revealed an epigenomically distinct subcluster of monocytes with reduced chromatin accessibility at AP-1-targeted loci after vaccination. Similar effects were observed in response to vaccination with the AS03-adjuvanted H5N1 pandemic influenza vaccine. However, this vaccine also stimulated persistently increased chromatin accessibility at interferon response factor (IRF) loci in monocytes and mDCs. This was associated with elevated expression of antiviral genes and heightened resistance to the unrelated Zika and Dengue viruses. These results demonstrate that vaccination stimulates persistent epigenomic remodeling of the innate immune system and reveal AS03's potential as an epigenetic adjuvant.
Assuntos
Epigenômica , Imunidade/genética , Vacinas contra Influenza/genética , Vacinas contra Influenza/imunologia , Análise de Célula Única , Transcrição Gênica , Vacinação , Adolescente , Adulto , Antibacterianos/farmacologia , Antígenos CD34/metabolismo , Antivirais/farmacologia , Reprogramação Celular , Cromatina/metabolismo , Citocinas/biossíntese , Combinação de Medicamentos , Feminino , Regulação da Expressão Gênica , Histonas/metabolismo , Humanos , Imunidade Inata/genética , Virus da Influenza A Subtipo H5N1/efeitos dos fármacos , Virus da Influenza A Subtipo H5N1/imunologia , Interferon Tipo I/metabolismo , Masculino , Células Mieloides/metabolismo , Polissorbatos/farmacologia , Esqualeno/farmacologia , Receptores Toll-Like/metabolismo , Fator de Transcrição AP-1/metabolismo , Transcriptoma/genética , Adulto Jovem , alfa-Tocoferol/farmacologiaRESUMO
The ability to identify single-nucleotide mutations is critical for probing cell biology and for precise detection of disease. However, the small differences in hybridization energy provided by single-base changes makes identification of these mutations challenging in living cells and complex reaction environments. Here, we report a class of de novo-designed prokaryotic riboregulators that provide ultraspecific RNA detection capabilities in vivo and in cell-free transcription-translation reactions. These single-nucleotide-specific programmable riboregulators (SNIPRs) provide over 100-fold differences in gene expression in response to target RNAs differing by a single nucleotide in E. coli and resolve single epitranscriptomic marks in vitro. By exploiting the programmable SNIPR design, we implement an automated design algorithm to develop riboregulators for a range of mutations associated with cancer, drug resistance, and genetic disorders. Integrating SNIPRs with portable paper-based cell-free reactions enables convenient isothermal detection of cancer-associated mutations from clinical samples and identification of Zika strains through unambiguous colorimetric reactions.
Assuntos
Epigenômica , Polimorfismo de Nucleotídeo Único/genética , RNA/genética , Transcriptoma/genética , Resistência a Medicamentos/genética , Escherichia coli/genética , Regulação da Expressão Gênica/genética , Humanos , Mutação/genética , Neoplasias/genética , Conformação de Ácido Nucleico , Células Procarióticas/metabolismo , Biologia Sintética , Zika virus/genética , Zika virus/isolamento & purificação , Zika virus/patogenicidadeRESUMO
Cytosine methylation of DNA is a widespread modification of DNA that plays numerous critical roles. In the yeast Cryptococcus neoformans, CG methylation occurs in transposon-rich repeats and requires the DNA methyltransferase Dnmt5. We show that Dnmt5 displays exquisite maintenance-type specificity in vitro and in vivo and utilizes similar in vivo cofactors as the metazoan maintenance methylase Dnmt1. Remarkably, phylogenetic and functional analysis revealed that the ancestral species lost the gene for a de novo methylase, DnmtX, between 50-150 mya. We examined how methylation has persisted since the ancient loss of DnmtX. Experimental and comparative studies reveal efficient replication of methylation patterns in C. neoformans, rare stochastic methylation loss and gain events, and the action of natural selection. We propose that an epigenome has been propagated for >50 million years through a process analogous to Darwinian evolution of the genome.
Assuntos
Cryptococcus neoformans/genética , Metilação de DNA/genética , Metiltransferases/genética , Evolução Biológica , Cryptococcus neoformans/metabolismo , DNA/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/fisiologia , Metilases de Modificação do DNA/genética , Elementos de DNA Transponíveis/genética , Epigenômica/métodos , Evolução Molecular , Genoma/genética , Metiltransferases/metabolismo , FilogeniaRESUMO
Tazemetostat is the first epigenetic therapy to gain FDA approval in a solid tumor. This lysine methyltransferase inhibitor targets EZH2, the enzymatic subunit of the PRC2 transcriptional silencing complex. Tumors with mutations in subunits of the SWI/SNF chromatin remodeling complex, inclusive of most epithelioid sarcomas, are sensitive to EZH2 inhibition.
Assuntos
Benzamidas/uso terapêutico , Epigênese Genética/genética , Piridonas/uso terapêutico , Sarcoma/tratamento farmacológico , Compostos de Bifenilo , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina , DNA Helicases/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/efeitos dos fármacos , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Inibidores Enzimáticos/farmacologia , Epigenômica , Terapia Genética/métodos , Humanos , Morfolinas , Proteínas Nucleares/metabolismo , Sarcoma/genética , Fatores de Transcrição/metabolismoRESUMO
Posterior fossa A (PFA) ependymomas are lethal malignancies of the hindbrain in infants and toddlers. Lacking highly recurrent somatic mutations, PFA ependymomas are proposed to be epigenetically driven tumors for which model systems are lacking. Here we demonstrate that PFA ependymomas are maintained under hypoxia, associated with restricted availability of specific metabolites to diminish histone methylation, and increase histone demethylation and acetylation at histone 3 lysine 27 (H3K27). PFA ependymomas initiate from a cell lineage in the first trimester of human development that resides in restricted oxygen. Unlike other ependymomas, transient exposure of PFA cells to ambient oxygen induces irreversible cellular toxicity. PFA tumors exhibit a low basal level of H3K27me3, and, paradoxically, inhibition of H3K27 methylation specifically disrupts PFA tumor growth. Targeting metabolism and/or the epigenome presents a unique opportunity for rational therapy for infants with PFA ependymoma.
Assuntos
Ependimoma/genética , Ependimoma/metabolismo , Epigenoma/genética , Neoplasias Infratentoriais/genética , Neoplasias Infratentoriais/metabolismo , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Linhagem Celular , Proliferação de Células/genética , Metilação de DNA/genética , Epigenômica/métodos , Histonas/genética , Histonas/metabolismo , Humanos , Lactente , Lisina/genética , Lisina/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Mutação/genéticaRESUMO
Widespread changes to DNA methylation and chromatin are well documented in cancer, but the fate of higher-order chromosomal structure remains obscure. Here we integrated topological maps for colon tumors and normal colons with epigenetic, transcriptional, and imaging data to characterize alterations to chromatin loops, topologically associated domains, and large-scale compartments. We found that spatial partitioning of the open and closed genome compartments is profoundly compromised in tumors. This reorganization is accompanied by compartment-specific hypomethylation and chromatin changes. Additionally, we identify a compartment at the interface between the canonical A and B compartments that is reorganized in tumors. Remarkably, similar shifts were evident in non-malignant cells that have accumulated excess divisions. Our analyses suggest that these topological changes repress stemness and invasion programs while inducing anti-tumor immunity genes and may therefore restrain malignant progression. Our findings call into question the conventional view that tumor-associated epigenomic alterations are primarily oncogenic.
Assuntos
Cromatina/metabolismo , Cromossomos/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Metilação de DNA , Epigênese Genética , Regulação Neoplásica da Expressão Gênica/genética , Divisão Celular , Senescência Celular/genética , Sequenciamento de Cromatina por Imunoprecipitação , Cromossomos/genética , Estudos de Coortes , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Biologia Computacional , Metilação de DNA/genética , Epigenômica , Células HCT116 , Humanos , Hibridização in Situ Fluorescente , Microscopia Eletrônica de Transmissão , Simulação de Dinâmica Molecular , RNA-Seq , Análise Espacial , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
The gut microbiome has been implicated in multiple human chronic gastrointestinal (GI) disorders. Determining its mechanistic role in disease has been difficult due to apparent disconnects between animal and human studies and lack of an integrated multi-omics view of disease-specific physiological changes. We integrated longitudinal multi-omics data from the gut microbiome, metabolome, host epigenome, and transcriptome in the context of irritable bowel syndrome (IBS) host physiology. We identified IBS subtype-specific and symptom-related variation in microbial composition and function. A subset of identified changes in microbial metabolites correspond to host physiological mechanisms that are relevant to IBS. By integrating multiple data layers, we identified purine metabolism as a novel host-microbial metabolic pathway in IBS with translational potential. Our study highlights the importance of longitudinal sampling and integrating complementary multi-omics data to identify functional mechanisms that can serve as therapeutic targets in a comprehensive treatment strategy for chronic GI diseases. VIDEO ABSTRACT.