RESUMO
Lungs undergo mechanical strain during breathing, but how these biophysical forces affect cell fate and tissue homeostasis are unclear. We show that biophysical forces through normal respiratory motion actively maintain alveolar type 1 (AT1) cell identity and restrict these cells from reprogramming into AT2 cells in the adult lung. AT1 cell fate is maintained at homeostasis by Cdc42- and Ptk2-mediated actin remodeling and cytoskeletal strain, and inactivation of these pathways causes a rapid reprogramming into the AT2 cell fate. This plasticity induces chromatin reorganization and changes in nuclear lamina-chromatin interactions, which can discriminate AT1 and AT2 cell identity. Unloading the biophysical forces of breathing movements leads to AT1-AT2 cell reprogramming, revealing that normal respiration is essential to maintain alveolar epithelial cell fate. These data demonstrate the integral function of mechanotransduction in maintaining lung cell fate and identifies the AT1 cell as an important mechanosensor in the alveolar niche.
Assuntos
Células Epiteliais Alveolares , Mecanotransdução Celular , Células Epiteliais Alveolares/metabolismo , Células Cultivadas , Pulmão , Diferenciação Celular/fisiologia , RespiraçãoRESUMO
Progenitor cells differentiate into specialized cell types through coordinated expression of lineage-specific genes and modification of complex chromatin configurations. We demonstrate that a histone deacetylase (Hdac3) organizes heterochromatin at the nuclear lamina during cardiac progenitor lineage restriction. Specification of cardiomyocytes is associated with reorganization of peripheral heterochromatin, and independent of deacetylase activity, Hdac3 tethers peripheral heterochromatin containing lineage-relevant genes to the nuclear lamina. Deletion of Hdac3 in cardiac progenitor cells releases genomic regions from the nuclear periphery, leading to precocious cardiac gene expression and differentiation into cardiomyocytes; in contrast, restricting Hdac3 to the nuclear periphery rescues myogenesis in progenitors otherwise lacking Hdac3. Our results suggest that availability of genomic regions for activation by lineage-specific factors is regulated in part through dynamic chromatin-nuclear lamina interactions and that competence of a progenitor cell to respond to differentiation signals may depend upon coordinated movement of responding gene loci away from the nuclear periphery.
Assuntos
Cromatina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Histona Desacetilases/metabolismo , Lâmina Nuclear/metabolismo , Células-Tronco/citologia , Animais , Genoma , Camundongos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Células-Tronco/metabolismoRESUMO
Aging is an inevitable outcome of life, characterized by progressive decline in tissue and organ function and increased risk of mortality. Accumulating evidence links aging to genetic and epigenetic alterations. Given the reversible nature of epigenetic mechanisms, these pathways provide promising avenues for therapeutics against age-related decline and disease. In this review, we provide a comprehensive overview of epigenetic studies from invertebrate organisms, vertebrate models, tissues, and in vitro systems. We establish links between common operative aging pathways and hallmark chromatin signatures that can be used to identify "druggable" targets to counter human aging and age-related disease.
Assuntos
Envelhecimento/genética , Epigênese Genética , Longevidade , Animais , Montagem e Desmontagem da Cromatina , Metilação de DNA , Histonas/metabolismo , HumanosRESUMO
The human genome functions as a three-dimensional chromatin polymer, driven by a complex collection of chromosome interactions1-3. Although the molecular rules governing these interactions are being quickly elucidated, relatively few proteins regulating this process have been identified. Here, to address this gap, we developed high-throughput DNA or RNA labelling with optimized Oligopaints (HiDRO)-an automated imaging pipeline that enables the quantitative measurement of chromatin interactions in single cells across thousands of samples. By screening the human druggable genome, we identified more than 300 factors that influence genome folding during interphase. Among these, 43 genes were validated as either increasing or decreasing interactions between topologically associating domains. Our findings show that genetic or chemical inhibition of the ubiquitous kinase GSK3A leads to increased long-range chromatin looping interactions in a genome-wide and cohesin-dependent manner. These results demonstrate the importance of GSK3A signalling in nuclear architecture and the use of HiDRO for identifying mechanisms of spatial genome organization.
Assuntos
Cromatina , Posicionamento Cromossômico , Cromossomos Humanos , Genoma Humano , Quinases da Glicogênio Sintase , Ensaios de Triagem em Larga Escala , Análise de Célula Única , Humanos , Cromatina/efeitos dos fármacos , Cromatina/genética , Cromatina/metabolismo , Posicionamento Cromossômico/efeitos dos fármacos , Cromossomos Humanos/efeitos dos fármacos , Cromossomos Humanos/genética , Cromossomos Humanos/metabolismo , DNA/análise , DNA/metabolismo , Genoma Humano/efeitos dos fármacos , Genoma Humano/genética , Quinases da Glicogênio Sintase/antagonistas & inibidores , Quinases da Glicogênio Sintase/deficiência , Quinases da Glicogênio Sintase/genética , Ensaios de Triagem em Larga Escala/métodos , Interfase , Reprodutibilidade dos Testes , RNA/análise , RNA/metabolismo , Transdução de Sinais/efeitos dos fármacos , Análise de Célula Única/métodos , CoesinasRESUMO
Interactions between the genome and the nuclear pore complex (NPC) have been implicated in multiple gene regulatory processes, but the underlying logic of these interactions remains poorly defined. Here, we report high-resolution chromatin binding maps of two core components of the NPC, Nup107 and Nup93, in Drosophila cells. Our investigation uncovered differential binding of these NPC subunits, where Nup107 preferentially targets active genes while Nup93 associates primarily with Polycomb-silenced regions. Comparison to Lamin-associated domains (LADs) revealed that NPC binding sites can be found within LADs, demonstrating a linear binding of the genome along the nuclear envelope. Importantly, we identified a functional role of Nup93 in silencing of Polycomb target genes and in spatial folding of Polycomb domains. Our findings lend to a model where different nuclear pores bind different types of chromatin via interactions with specific NPC sub-complexes, and a subset of Polycomb domains is stabilized by interactions with Nup93.
Assuntos
Cromatina/metabolismo , Poro Nuclear/metabolismo , Proteínas do Grupo Polycomb/metabolismo , Animais , Aquaporinas/metabolismo , Sítios de Ligação/fisiologia , Linhagem Celular , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Feminino , Regulação da Expressão Gênica/fisiologia , Genoma/fisiologia , Masculino , Membrana Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismoRESUMO
The relationship between cohesin-mediated chromatin looping and gene expression remains unclear. NIPBL and WAPL are two opposing regulators of cohesin activity; depletion of either is associated with changes in both chromatin folding and transcription across a wide range of cell types. However, a direct comparison of their individual and combined effects on gene expression in the same cell type is lacking. We find that NIPBL or WAPL depletion in human HCT116 cells each alter the expression of ~2,000 genes, with only ~30% of the genes shared between the conditions. We find that clusters of differentially expressed genes within the same topologically associated domain (TAD) show coordinated misexpression, suggesting some genomic domains are especially sensitive to both more or less cohesin. Finally, co-depletion of NIPBL and WAPL restores the majority of gene misexpression as compared to either knockdown alone. A similar set of NIPBL-sensitive genes are rescued following CTCF co-depletion. Together, this indicates that altered transcription due to reduced cohesin activity can be functionally offset by removal of either its negative regulator (WAPL) or the physical barriers (CTCF) that restrict loop-extrusion events.
Assuntos
Proteínas de Ciclo Celular , Cromatina , Proteínas Cromossômicas não Histona , Regulação da Expressão Gênica , Humanos , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Genes cdc , Genoma , Células HCT116 , CoesinasRESUMO
Oncogene-induced senescence (OIS) and therapy-induced senescence (TIS), while tumor-suppressive, also promote procarcinogenic effects by activating the DNA damage response (DDR), which in turn induces inflammation. This inflammatory response prominently includes an array of cytokines known as the senescence-associated secretory phenotype (SASP). Previous observations link the transcription-associated methyltransferase and oncoprotein MLL1 to the DDR, leading us to investigate the role of MLL1 in SASP expression. Our findings reveal direct MLL1 epigenetic control over proproliferative cell cycle genes: MLL1 inhibition represses expression of proproliferative cell cycle regulators required for DNA replication and DDR activation, thus disabling SASP expression. Strikingly, however, these effects of MLL1 inhibition on SASP gene expression do not impair OIS and, furthermore, abolish the ability of the SASP to enhance cancer cell proliferation. More broadly, MLL1 inhibition also reduces "SASP-like" inflammatory gene expression from cancer cells in vitro and in vivo independently of senescence. Taken together, these data demonstrate that MLL1 inhibition may be a powerful and effective strategy for inducing cancerous growth arrest through the direct epigenetic regulation of proliferation-promoting genes and the avoidance of deleterious OIS- or TIS-related tumor secretomes, which can promote both drug resistance and tumor progression.
Assuntos
Senescência Celular/genética , Regulação Neoplásica da Expressão Gênica/genética , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Transdução de Sinais/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular , Proliferação de Células , Dano ao DNA , Técnicas de Silenciamento de Genes , Células HEK293 , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Humanos , Inflamação/genética , Células MCF-7 , Proteína de Leucina Linfoide-Mieloide/antagonistas & inibidores , NF-kappa B/metabolismo , Neoplasias/fisiopatologia , FenótipoRESUMO
PURPOSE OF REVIEW: In this review, we explore the chromatin-related consequences of laminopathy-linked mutations through the lens of mechanotransduction. RECENT FINDINGS: Multiple studies have highlighted the role of the nuclear lamina in maintaining the integrity of the nucleus. The lamina also has a critical role in 3D genome organization. Mutations in lamina proteins associated with various laminopathies result in the loss of organization of DNA at the nuclear periphery. However, it remains unclear if or how these two aspects of lamin function are connected. Recent data suggests that unlinking the cytoskeleton from the nuclear lamina may be beneficial to slow progress of deleterious phenotypes observed in laminopathies. In this review, we highlight emerging data that suggest interlinked chromatin- and mechanical biology-related pathways are interconnected in the pathogenesis of laminopathies.
Assuntos
Núcleo Celular , Mecanotransdução Celular , Humanos , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Lâmina Nuclear/genética , Lâmina Nuclear/metabolismo , Cromatina/genética , Cromatina/metabolismo , BiofísicaRESUMO
Rationale: Mechanical forces are transduced to nuclear responses via the linkers of the nucleoskeleton and cytoskeleton (LINC) complex, which couples the cytoskeleton to the nuclear lamina and associated chromatin. While disruption of the LINC complex can cause cardiomyopathy, the relevant interactions that bridge the nucleoskeleton to cytoskeleton are poorly understood in the cardiomyocyte, where cytoskeletal organization is unique. Furthermore, while microtubules and desmin intermediate filaments associate closely with cardiomyocyte nuclei, the importance of these interactions is unknown. Objective: Here, we sought to determine how cytoskeletal interactions with the LINC complex regulate nuclear homeostasis in the cardiomyocyte. Methods and Results: To this end, we acutely disrupted the LINC complex, microtubules, actin, and intermediate filaments and assessed the consequences on nuclear morphology and genome organization in rat ventricular cardiomyocytes via a combination of super-resolution imaging, biophysical, and genomic approaches. We find that a balance of dynamic microtubules and desmin intermediate filaments is required to maintain nuclear shape and the fidelity of the nuclear envelope and lamina. Upon depletion of desmin (or nesprin [nuclear envelope spectrin repeat protein]-3, its binding partner in the LINC complex), polymerizing microtubules collapse the nucleus and drive infolding of the nuclear membrane. This results in DNA damage, a loss of genome organization, and broad transcriptional changes. The collapse in nuclear integrity is concomitant with compromised contractile function and may contribute to the pathophysiological changes observed in desmin-related myopathies. Conclusions: Disrupting the tethering of desmin to the nucleus results in a loss of nuclear homeostasis and rapid alterations to cardiomyocyte function. Our data suggest that a balance of forces imposed by intermediate filaments and microtubules is required to maintain nuclear structure and genome organization in the cardiomyocyte.
Assuntos
Citoesqueleto de Actina/metabolismo , Microtúbulos/metabolismo , Miócitos Cardíacos/metabolismo , Matriz Nuclear/metabolismo , Citoesqueleto de Actina/ultraestrutura , Animais , Células Cultivadas , Desmina/genética , Desmina/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Microtúbulos/ultraestrutura , Miócitos Cardíacos/ultraestrutura , Matriz Nuclear/ultraestrutura , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Gene regulatory networks control tissue homeostasis and disease progression in a cell type-specific manner. Ubiquitously expressed chromatin regulators modulate these networks, yet the mechanisms governing how tissue specificity of their function is achieved are poorly understood. BRD4 (bromodomain-containing protein 4), a member of the BET (bromo- and extraterminal domain) family of ubiquitously expressed acetyl-lysine reader proteins, plays a pivotal role as a coactivator of enhancer signaling across diverse tissue types in both health and disease and has been implicated as a pharmacological target in heart failure. However, the cell-specific role of BRD4 in adult cardiomyocytes remains unknown. METHODS: We combined conditional mouse genetics, unbiased transcriptomic and epigenomic analyses, and classic molecular biology and biochemical approaches to understand the mechanism by which BRD4 in adult cardiomyocyte homeostasis. RESULTS: Here, we show that cardiomyocyte-specific deletion of Brd4 in adult mice leads to acute deterioration of cardiac contractile function with mutant animals demonstrating a transcriptomic signature characterized by decreased expression of genes critical for mitochondrial energy production. Genome-wide occupancy data show that BRD4 enriches at many downregulated genes (including the master coactivators Ppargc1a, Ppargc1b, and their downstream targets) and preferentially colocalizes with GATA4 (GATA binding protein 4), a lineage-determining cardiac transcription factor not previously implicated in regulation of adult cardiac metabolism. BRD4 and GATA4 form an endogenous complex in cardiomyocytes and interact in a bromodomain-independent manner, revealing a new functional interaction partner for BRD4 that can direct its locus and tissue specificity. CONCLUSIONS: These results highlight a novel role for a BRD4-GATA4 module in cooperative regulation of a cardiomyocyte-specific gene program governing bioenergetic homeostasis in the adult heart.
Assuntos
Metabolismo Energético , Fator de Transcrição GATA4/metabolismo , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Disfunção Ventricular Esquerda/metabolismo , Animais , Metabolismo Energético/genética , Fator de Transcrição GATA4/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Genótipo , Células HEK293 , Homeostase , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Cardíacas/genética , Mitocôndrias Cardíacas/ultraestrutura , Miócitos Cardíacos/ultraestrutura , Proteínas Nucleares/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Fenótipo , Ligação Proteica , Ratos Sprague-Dawley , Fatores de Transcrição/genética , Transcriptoma , Disfunção Ventricular Esquerda/genética , Disfunção Ventricular Esquerda/patologia , Disfunção Ventricular Esquerda/fisiopatologia , Função Ventricular EsquerdaRESUMO
Macroautophagy (hereafter referred to as autophagy) is a catabolic membrane trafficking process that degrades a variety of cellular constituents and is associated with human diseases. Although extensive studies have focused on autophagic turnover of cytoplasmic materials, little is known about the role of autophagy in degrading nuclear components. Here we report that the autophagy machinery mediates degradation of nuclear lamina components in mammals. The autophagy protein LC3/Atg8, which is involved in autophagy membrane trafficking and substrate delivery, is present in the nucleus and directly interacts with the nuclear lamina protein lamin B1, and binds to lamin-associated domains on chromatin. This LC3-lamin B1 interaction does not downregulate lamin B1 during starvation, but mediates its degradation upon oncogenic insults, such as by activated RAS. Lamin B1 degradation is achieved by nucleus-to-cytoplasm transport that delivers lamin B1 to the lysosome. Inhibiting autophagy or the LC3-lamin B1 interaction prevents activated RAS-induced lamin B1 loss and attenuates oncogene-induced senescence in primary human cells. Our study suggests that this new function of autophagy acts as a guarding mechanism protecting cells from tumorigenesis.
Assuntos
Autofagia , Lâmina Nuclear/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Família da Proteína 8 Relacionada à Autofagia , Transformação Celular Neoplásica , Células Cultivadas , Senescência Celular , Cromatina/química , Cromatina/metabolismo , Citoplasma/metabolismo , Fibroblastos , Células HEK293 , Humanos , Lamina Tipo B/genética , Lamina Tipo B/metabolismo , Lisossomos/metabolismo , Camundongos , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteína Oncogênica p21(ras)/metabolismo , Ligação Proteica , ProteóliseRESUMO
Senescence is a stable proliferation arrest, associated with an altered secretory pathway, thought to promote tumor suppression and tissue aging. While chromatin regulation and lamin B1 down-regulation have been implicated as senescence effectors, functional interactions between them are poorly understood. We compared genome-wide Lys4 trimethylation on histone H3 (H3K4me3) and H3K27me3 distributions between proliferating and senescent human cells and found dramatic differences in senescence, including large-scale domains of H3K4me3- and H3K27me3-enriched "mesas" and H3K27me3-depleted "canyons." Mesas form at lamin B1-associated domains (LADs) in replicative senescence and oncogene-induced senescence and overlap DNA hypomethylation regions in cancer, suggesting that pre-malignant senescent chromatin changes foreshadow epigenetic cancer changes. Hutchinson-Gilford progeria syndrome fibroblasts (mutant lamin A) also show evidence of H3K4me3 mesas, suggesting a link between premature chromatin changes and accelerated cell senescence. Canyons mostly form between LADs and are enriched in genes and enhancers. H3K27me3 loss is correlated with up-regulation of key senescence genes, indicating a link between global chromatin changes and local gene expression regulation. Lamin B1 reduction in proliferating cells triggers senescence and formation of mesas and canyons. Our data illustrate profound chromatin reorganization during senescence and suggest that lamin B1 down-regulation in senescence is a key trigger of global and local chromatin changes that impact gene expression, aging, and cancer.
Assuntos
Envelhecimento/genética , Envelhecimento/patologia , Cromatina/genética , Cromatina/metabolismo , Regulação da Expressão Gênica , Lamina Tipo B/genética , Lamina Tipo B/metabolismo , Linhagem Celular , Proliferação de Células , Células Cultivadas , Montagem e Desmontagem da Cromatina , Epigênese Genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Histonas/metabolismo , Humanos , Metilação , Progéria/patologia , Estrutura Terciária de ProteínaRESUMO
Purified DNA translocases Rdh54 and Rad54 can dissociate complexes formed by eukaryotic RecA-like recombinases on double-stranded DNA. Here, we show that Rad51 complexes are dissociated by these translocases in mitotic cells. Rad51 overexpression blocked growth of cells deficient in Rdh54 activity. This toxicity was associated with accumulation of Rad51 foci on undamaged chromatin. At normal Rad51 levels, rdh54 deficiency resulted in slight elevation of Rad51 foci. A triple mutant lacking Rdh54, Rad54, and a third Swi2/Snf2 homolog Uls1 accumulated Rad51 foci, grew slowly, and suffered chromosome loss. Thus, Uls1 and Rad54 can partially substitute for Rdh54 in the removal of toxic, nondamage-associated Rad51-DNA complexes. Additional data suggest that the function of Rdh54 and Rad54 in removal of Rad51 foci is significantly specialized; Rad54 predominates for removal of damage-associated foci, and Rdh54 predominates for removal of nondamage-associated foci.
Assuntos
DNA Helicases/genética , Enzimas Reparadoras do DNA/genética , DNA Topoisomerases/genética , Mitose/fisiologia , Rad51 Recombinase/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Adenosina Trifosfatases/genética , Núcleo Celular/metabolismo , Proliferação de Células , Cromatina/metabolismo , Instabilidade Cromossômica/genética , Reparo do DNA/genética , Diploide , Raios gama , Deleção de Genes , Expressão Gênica/genética , Haploidia , Proteínas de Homeodomínio/genética , Rad51 Recombinase/genética , Proteína de Replicação A/metabolismo , Proteínas Repressoras/genética , Saccharomyces cerevisiae/efeitos da radiação , Fatores de Transcrição , Transcrição Gênica/genética , TransfecçãoRESUMO
Human induced pluripotent stem cell (hiPSC) to cardiomyocyte (CM) differentiation has reshaped approaches to studying cardiac development and disease. In this study, we employed a genome-wide CRISPR screen in a hiPSC to CM differentiation system and reveal here that BRD4, a member of the bromodomain and extraterminal (BET) family, regulates CM differentiation. Chemical inhibition of BET proteins in mouse embryonic stem cell (mESC)-derived or hiPSC-derived cardiac progenitor cells (CPCs) results in decreased CM differentiation and persistence of cells expressing progenitor markers. In vivo, BRD4 deletion in second heart field (SHF) CPCs results in embryonic or early postnatal lethality, with mutants demonstrating myocardial hypoplasia and an increase in CPCs. Single-cell transcriptomics identified a subpopulation of SHF CPCs that is sensitive to BRD4 loss and associated with attenuated CM lineage-specific gene programs. These results highlight a previously unrecognized role for BRD4 in CM fate determination during development and a heterogenous requirement for BRD4 among SHF CPCs.
Assuntos
Sistemas CRISPR-Cas , Diferenciação Celular , Células-Tronco Pluripotentes Induzidas , Miócitos Cardíacos , Fatores de Transcrição , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/citologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Humanos , Sistemas CRISPR-Cas/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Linhagem da Célula/genética , Células Cultivadas , Análise de Célula Única , Proteínas que Contêm BromodomínioRESUMO
Mammalian aging is characterized by the progressive loss of tissue function and increased risk for disease. Accumulation of senescent cells in aging tissues partly contributes to this decline, and targeted depletion of senescent cells in vivo ameliorates many age-related phenotypes. The fundamental molecular mechanisms responsible for the decline of cellular health and fitness during senescence and aging are largely unknown. In this study, we investigated whether chromatin-mediated loss of transcriptional fidelity, known to contribute to fitness and survival in yeast and worms, also occurs during human cellular senescence and mouse aging. Our findings reveal aberrant transcription initiation inside genes during senescence and aging that co-occurs with changes in the chromatin landscape. Interventions that alter these spurious transcripts have profound consequences on cellular health, primarily affecting intracellular signal transduction pathways. We propose that age-related spurious transcription promotes a noisy transcriptome and degradation of coherent transcriptional networks.
Assuntos
Envelhecimento , Senescência Celular , Humanos , Animais , Camundongos , Envelhecimento/genética , Senescência Celular/genética , Cromatina/genética , Transcriptoma , Fenótipo , Mamíferos/genéticaRESUMO
BACKGROUND: Association of chromatin with lamin proteins at the nuclear periphery has emerged as a potential mechanism to coordinate cell type-specific gene expression and maintain cellular identity via gene silencing. Unlike many histone modifications and chromatin-associated proteins, lamina-associated domains (LADs) are mapped genome-wide in relatively few genetically normal human cell types, which limits our understanding of the role peripheral chromatin plays in development and disease. RESULTS: To address this gap, we map LAMIN B1 occupancy across twelve human cell types encompassing pluripotent stem cells, intermediate progenitors, and differentiated cells from all three germ layers. Integrative analyses of this atlas with gene expression and repressive histone modification maps reveal that lamina-associated chromatin in all twelve cell types is organized into at least two subtypes defined by differences in LAMIN B1 occupancy, gene expression, chromatin accessibility, transposable elements, replication timing, and radial positioning. Imaging of fluorescently labeled DNA in single cells validates these subtypes and shows radial positioning of LADs with higher LAMIN B1 occupancy and heterochromatic histone modifications primarily embedded within the lamina. In contrast, the second subtype of lamina-associated chromatin is relatively gene dense, accessible, dynamic across development, and positioned adjacent to the lamina. Most genes gain or lose LAMIN B1 occupancy consistent with cell types along developmental trajectories; however, we also identify examples where the enhancer, but not the gene body and promoter, changes LAD state. CONCLUSIONS: Altogether, this atlas represents the largest resource to date for peripheral chromatin organization studies and reveals an intermediate chromatin subtype.
Assuntos
Cromatina , Lâmina Nuclear , Humanos , Cromatina/metabolismo , Lâmina Nuclear/genética , Núcleo Celular/genética , Montagem e Desmontagem da Cromatina , Diferenciação CelularRESUMO
The COVID-19 pandemic has created an urgent need for rapid, effective, and low-cost SARS-CoV-2 diagnostic testing. Here, we describe COV-ID, an approach that combines RT-LAMP with deep sequencing to detect SARS-CoV-2 in unprocessed human saliva with a low limit of detection (5-10 virions). Based on a multi-dimensional barcoding strategy, COV-ID can be used to test thousands of samples overnight in a single sequencing run with limited labor and laboratory equipment. The sequencing-based readout allows COV-ID to detect multiple amplicons simultaneously, including key controls such as host transcripts and artificial spike-ins, as well as multiple pathogens. Here, we demonstrate this flexibility by simultaneous detection of 4 amplicons in contrived saliva samples: SARS-CoV-2, influenza A, human STATHERIN, and an artificial SARS calibration standard. The approach was validated on clinical saliva samples, where it showed excellent agreement with RT-qPCR. COV-ID can also be performed directly on saliva absorbed on filter paper, simplifying collection logistics and sample handling.
Assuntos
COVID-19 , Orthomyxoviridae , COVID-19/diagnóstico , Humanos , Pandemias , RNA Viral/análise , SARS-CoV-2/genética , Saliva , Sensibilidade e EspecificidadeRESUMO
Identifying the particular transcription factors that maintain cell type in vitro is important for manipulating cell type. Identifying such transcription factors by their cell-type-specific expression or their involvement in developmental regulation has had limited success. We hypothesized that because cell type is often resilient to perturbations, the transcriptional response to perturbations would reveal identity-maintaining transcription factors. We developed perturbation panel profiling (P3) as a framework for perturbing cells across many conditions and measuring gene expression responsiveness transcriptome-wide. In human iPSC-derived cardiac myocytes, P3 showed that transcription factors important for cardiac myocyte differentiation and maintenance were among the most frequently upregulated (most responsive). We reasoned that one function of responsive genes may be to maintain cellular identity. We identified responsive transcription factors in fibroblasts using P3 and found that suppressing their expression led to enhanced reprogramming. We propose that responsiveness to perturbations is a property of transcription factors that help maintain cellular identity in vitro. A record of this paper's transparent peer review process is included in the supplemental information.
Assuntos
Células-Tronco Pluripotentes Induzidas , Fatores de Transcrição , Diferenciação Celular/genética , Fibroblastos/metabolismo , Humanos , Miócitos Cardíacos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Higher-order chromatin structure regulates gene expression, and mutations in proteins mediating genome folding underlie developmental disorders known as cohesinopathies. However, the relationship between three-dimensional genome organization and embryonic development remains unclear. Here we define a role for bromodomain-containing protein 4 (BRD4) in genome folding, and leverage it to understand the importance of genome folding in neural crest progenitor differentiation. Brd4 deletion in neural crest results in cohesinopathy-like phenotypes. BRD4 interacts with NIPBL, a cohesin agonist, and BRD4 depletion or loss of the BRD4-NIPBL interaction reduces NIPBL occupancy, suggesting that BRD4 stabilizes NIPBL on chromatin. Chromatin interaction mapping and imaging experiments demonstrate that BRD4 depletion results in compromised genome folding and loop extrusion. Finally, mutation of individual BRD4 amino acids that mediate an interaction with NIPBL impedes neural crest differentiation into smooth muscle. Remarkably, loss of WAPL, a cohesin antagonist, rescues attenuated smooth muscle differentiation resulting from BRD4 loss. Collectively, our data reveal that BRD4 choreographs genome folding and illustrates the relevance of balancing cohesin activity for progenitor differentiation.
Assuntos
Diferenciação Celular , Genoma , Crista Neural/citologia , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Integrases/metabolismo , Camundongos , Modelos Biológicos , Células-Tronco Embrionárias Murinas/metabolismo , Células Musculares/citologia , Crista Neural/metabolismo , Ligação Proteica , Domínios Proteicos , Proteólise , Fatores de Transcrição/química , Transcrição Gênica , CoesinasRESUMO
Pathogenic mutations in LAMIN A/C (LMNA) cause abnormal nuclear structure and laminopathies. These diseases have myriad tissue-specific phenotypes, including dilated cardiomyopathy (DCM), but how LMNA mutations result in tissue-restricted disease phenotypes remains unclear. We introduced LMNA mutations from individuals with DCM into human induced pluripotent stem cells (hiPSCs) and found that hiPSC-derived cardiomyocytes, in contrast to hepatocytes or adipocytes, exhibit aberrant nuclear morphology and specific disruptions in peripheral chromatin. Disrupted regions were enriched for transcriptionally active genes and regions with lower LAMIN B1 contact frequency. The lamina-chromatin interactions disrupted in mutant cardiomyocytes were enriched for genes associated with non-myocyte lineages and correlated with higher expression of those genes. Myocardium from individuals with LMNA variants similarly showed aberrant expression of non-myocyte pathways. We propose that the lamina network safeguards cellular identity and that pathogenic LMNA variants disrupt peripheral chromatin with specific epigenetic and molecular characteristics, causing misexpression of genes normally expressed in other cell types.