RESUMO
Recent advances in single-cell technologies have led to the discovery of thousands of brain cell types; however, our understanding of the gene regulatory programs in these cell types is far from complete1-4. Here we report a comprehensive atlas of candidate cis-regulatory DNA elements (cCREs) in the adult mouse brain, generated by analysing chromatin accessibility in 2.3 million individual brain cells from 117 anatomical dissections. The atlas includes approximately 1 million cCREs and their chromatin accessibility across 1,482 distinct brain cell populations, adding over 446,000 cCREs to the most recent such annotation in the mouse genome. The mouse brain cCREs are moderately conserved in the human brain. The mouse-specific cCREs-specifically, those identified from a subset of cortical excitatory neurons-are strongly enriched for transposable elements, suggesting a potential role for transposable elements in the emergence of new regulatory programs and neuronal diversity. Finally, we infer the gene regulatory networks in over 260 subclasses of mouse brain cells and develop deep-learning models to predict the activities of gene regulatory elements in different brain cell types from the DNA sequence alone. Our results provide a resource for the analysis of cell-type-specific gene regulation programs in both mouse and human brains.
Assuntos
Encéfalo , Cromatina , Análise de Célula Única , Animais , Humanos , Camundongos , Encéfalo/citologia , Encéfalo/metabolismo , Córtex Cerebral/citologia , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Aprendizado Profundo , Elementos de DNA Transponíveis/genética , Redes Reguladoras de Genes/genética , Neurônios/metabolismoRESUMO
A major factor in the progression to heart failure in humans is the inability of the adult heart to repair itself after injury. We recently demonstrated that the early postnatal mammalian heart is capable of regeneration following injury through proliferation of preexisting cardiomyocytes1,2 and that Meis1, a three amino acid loop extension (TALE) family homeodomain transcription factor, translocates to cardiomyocyte nuclei shortly after birth and mediates postnatal cell cycle arrest3. Here we report that Hoxb13 acts as a cofactor of Meis1 in postnatal cardiomyocytes. Cardiomyocyte-specific deletion of Hoxb13 can extend the postnatal window of cardiomyocyte proliferation and reactivate the cardiomyocyte cell cycle in the adult heart. Moreover, adult Meis1-Hoxb13 double-knockout hearts display widespread cardiomyocyte mitosis, sarcomere disassembly and improved left ventricular systolic function following myocardial infarction, as demonstrated by echocardiography and magnetic resonance imaging. Chromatin immunoprecipitation with sequencing demonstrates that Meis1 and Hoxb13 act cooperatively to regulate cardiomyocyte maturation and cell cycle. Finally, we show that the calcium-activated protein phosphatase calcineurin dephosphorylates Hoxb13 at serine-204, resulting in its nuclear localization and cell cycle arrest. These results demonstrate that Meis1 and Hoxb13 act cooperatively to regulate cardiomyocyte maturation and proliferation and provide mechanistic insights into the link between hyperplastic and hypertrophic growth of cardiomyocytes.
Assuntos
Calcineurina/metabolismo , Proliferação de Células , Proteínas de Homeodomínio/metabolismo , Proteína Meis1/metabolismo , Miócitos Cardíacos/citologia , Animais , Animais Recém-Nascidos , Feminino , Deleção de Genes , Regulação da Expressão Gênica , Coração/fisiologia , Proteínas de Homeodomínio/genética , Masculino , Camundongos , Miocárdio/citologia , Ligação Proteica , RegeneraçãoRESUMO
Direct reprogramming of fibroblasts to cardiomyocytes represents a potential means of restoring cardiac function following myocardial injury. AKT1 in the presence of four cardiogenic transcription factors, GATA4, HAND2, MEF2C, and TBX5 (AGHMT), efficiently induces the cardiac gene program in mouse embryonic fibroblasts but not adult fibroblasts. To identify additional regulators of adult cardiac reprogramming, we performed an unbiased screen of transcription factors and cytokines for those that might enhance or suppress the cardiogenic activity of AGHMT in adult mouse fibroblasts. Among a collection of inducers and repressors of cardiac reprogramming, we discovered that the zinc finger transcription factor 281 (ZNF281) potently stimulates cardiac reprogramming by genome-wide association with GATA4 on cardiac enhancers. Concomitantly, ZNF281 suppresses expression of genes associated with inflammatory signaling, suggesting the antagonistic convergence of cardiac and inflammatory transcriptional programs. Consistent with an inhibitory influence of inflammatory pathways on cardiac reprogramming, blockade of these pathways with anti-inflammatory drugs or components of the nucleosome remodeling deacetylase (NuRD) complex, which associate with ZNF281, stimulates cardiac gene expression. We conclude that ZNF281 acts at a nexus of cardiac and inflammatory gene programs, which exert opposing influences on fibroblast to cardiac reprogramming.
Assuntos
Reprogramação Celular/genética , Regulação da Expressão Gênica/genética , Fatores de Transcrição/metabolismo , Anti-Inflamatórios/farmacologia , Reprogramação Celular/efeitos dos fármacos , Fibroblastos/fisiologia , Fator de Transcrição GATA4/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Estudo de Associação Genômica Ampla , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Proteínas Repressoras , TranscriptomaRESUMO
Aroma or fragrance in rice is a genetically controlled trait; Its high appreciation by consumers increases the rice market price. Previous studies have revealed that the rice aroma is controlled by a specific gene called BETAINE ALDEHYDE DEHYDROGENASE (OsBADH2), and mutation of this gene leads to the accumulation of an aromatic substance 2-acetyl-1-pyrroline (2-AP). The use of genetic engineering to produce aroma in commercial and cultivated hybrids is a contemporary need for molecular breeding. The current study reports the generation of aroma in the three-line hybrid restorer line Shu-Hui-313 (SH313). We created knock-out (KO) lines of OsBADH2 through the CRISPR/Cas9. The analysis of KO lines revealed a significantly increased content of 2AP in the grains compared with the control. However, other phenotypic traits (plant height, seed setting rate, and 1000-grain weight) were significantly decreased. These KO lines were crossed with a non-aromatic three-line hybrid rice male sterile line (Rong-7-A) to produce Rong-7-You-626 (R7Y626), R7Y627 and R7Y628. The measurement of 2-AP revealed significantly increased contents in these cross combinations. We compared the content of 2-AP in tissues at the booting stage. Data revealed that young spike stalk base contained the highest content of 2-AP and can be used for identification (by simple chewing) of aromatic lines under field conditions. In conclusion, our dataset offers a genetic source and illustrates the generation of aroma in non-aromatic hybrids, and outlines a straightforward identification under field conditions.
Assuntos
Betaína/análogos & derivados , Oryza , Oryza/genética , Sistemas CRISPR-Cas/genética , Odorantes , Genes de PlantasRESUMO
[Figure: see text].
Assuntos
Cardiomiopatias/terapia , Edição de Genes/métodos , Terapia Genética/métodos , Distrofia Muscular de Duchenne/terapia , Animais , Sistemas CRISPR-Cas , Cardiomiopatias/etiologia , Linhagem Celular , Células Cultivadas , Dependovirus/genética , Distrofina/genética , Distrofina/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Distrofia Muscular de Duchenne/complicações , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , TranscriptomaRESUMO
[Figure: see text].
Assuntos
Infarto do Miocárdio/metabolismo , Miofibroblastos/metabolismo , Pirofosfatases/metabolismo , Animais , Células Cultivadas , Fibrose , Células HEK293 , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/patologia , Miocárdio/metabolismo , Miocárdio/patologia , Miofibroblastos/patologia , Pirofosfatases/genéticaRESUMO
Duchenne muscular dystrophy (DMD) is a fatal muscle disorder characterized by cycles of degeneration and regeneration of multinucleated myofibers and pathological activation of a variety of other muscle-associated cell types. The extent to which different nuclei within the shared cytoplasm of a myofiber may display transcriptional diversity and whether individual nuclei within a multinucleated myofiber might respond differentially to DMD pathogenesis is unknown. Similarly, the potential transcriptional diversity among nonmuscle cell types within dystrophic muscle has not been explored. Here, we describe the creation of a mouse model of DMD caused by deletion of exon 51 of the dystrophin gene, which represents a prevalent disease-causing mutation in humans. To understand the transcriptional abnormalities and heterogeneity associated with myofiber nuclei, as well as other mononucleated cell types that contribute to the muscle pathology associated with DMD, we performed single-nucleus transcriptomics of skeletal muscle of mice with dystrophin exon 51 deletion. Our results reveal distinctive and previously unrecognized myonuclear subtypes within dystrophic myofibers and uncover degenerative and regenerative transcriptional pathways underlying DMD pathogenesis. Our findings provide insights into the molecular underpinnings of DMD, controlled by the transcriptional activity of different types of muscle and nonmuscle nuclei.
Assuntos
Degeneração Macular/genética , Distrofia Muscular Animal/genética , Distrofia Muscular de Duchenne/genética , Regeneração/genética , Transdução de Sinais/genética , Animais , Modelos Animais de Doenças , Éxons/genética , Deleção de Genes , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/patologia , Mutação/genética , Miofibrilas/genética , Análise de Sequência de RNA/métodos , Transcrição Gênica/genética , Transcriptoma/genéticaRESUMO
Polybrominated diphenyl ethers (PBDEs) are classic and emerging pollutants that are potentially harmful to the human immune system. Research on their immunotoxicity and mechanisms suggests that they play an important role in the resulting pernicious effects of PBDEs. 2,2',4,4'-Tetrabrominated biphenyl ether (BDE-47) is the most biotoxic PBDE congener, and, in this study, we evaluated its toxicity toward RAW264.7 cells of mouse macrophages. The results show that exposure to BDE-47 led to a significant decrease in cell viability and a prominent increase in apoptosis. A decrease in mitochondrial membrane potential (MMP) and an increase in cytochrome C release and caspase cascade activation thus demonstrate that cell apoptosis induced by BDE-47 occurs via the mitochondrial pathway. In addition, BDE-47 inhibits phagocytosis in RAW264.7 cells, changes the related immune factor index, and causes immune function damage. Furthermore, we discovered a significant increase in the level of cellular reactive oxygen species (ROS), and the regulation of genes linked to oxidative stress was also demonstrated using transcriptome sequencing. The degree of apoptosis and immune function impairment caused by BDE-47 could be reversed after treatment with the antioxidant NAC and, conversely, exacerbated by treatment with the ROS-inducer BSO. These findings indicate that oxidative damage caused by BDE-47 is a critical event that leads to mitochondrial apoptosis in RAW264.7 macrophages, ultimately resulting in the suppression of immune function.
Assuntos
Éteres Difenil Halogenados , Mitocôndrias , Camundongos , Animais , Humanos , Espécies Reativas de Oxigênio/metabolismo , Éteres Difenil Halogenados/farmacologia , Mitocôndrias/metabolismo , Macrófagos/metabolismoRESUMO
The adult mammalian heart has limited capacity for regeneration following injury, whereas the neonatal heart can readily regenerate within a short period after birth. To uncover the molecular mechanisms underlying neonatal heart regeneration, we compared the transcriptomes and epigenomes of regenerative and nonregenerative mouse hearts over a 7-d time period following myocardial infarction injury. By integrating gene expression profiles with histone marks associated with active or repressed chromatin, we identified transcriptional programs underlying neonatal heart regeneration, and the blockade to regeneration in later life. Our results reveal a unique immune response in regenerative hearts and a retained embryonic cardiogenic gene program that is active during neonatal heart regeneration. Among the unique immune factors and embryonic genes associated with cardiac regeneration, we identified Ccl24, which encodes a cytokine, and Igf2bp3, which encodes an RNA-binding protein, as previously unrecognized regulators of cardiomyocyte proliferation. Our data provide insights into the molecular basis of neonatal heart regeneration and identify genes that can be modulated to promote heart regeneration.
Assuntos
Animais Recém-Nascidos/fisiologia , Coração/fisiologia , Código das Histonas/fisiologia , Regeneração/fisiologia , Transcriptoma/fisiologia , Animais , Animais Recém-Nascidos/crescimento & desenvolvimento , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Traumatismos Cardíacos/genética , Traumatismos Cardíacos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Infarto do Miocárdio/genética , Regeneração/genética , Transcriptoma/genéticaRESUMO
Embryonic and postnatal life depend on the uninterrupted function of cardiac muscle cells. These cells, termed cardiomyocytes, display many fascinating behaviors, including complex morphogenic movements, interactions with other cell types of the heart, persistent contractility and quiescence after birth. Each of these behaviors depends on complex interactions between both cardiac-restricted and widely expressed transcription factors, as well as on epigenetic modifications. Here, we review recent advances in our understanding of the genetic and epigenetic control of cardiomyocyte differentiation and proliferation during heart development, regeneration and disease. We focus on those regulators that are required for both heart development and disease, and highlight the regenerative principles that might be manipulated to restore function to the injured adult heart.
Assuntos
Epigênese Genética , Cardiopatias/genética , Coração/embriologia , Miócitos Cardíacos/metabolismo , Regeneração/genética , Animais , Regulação da Expressão Gênica no Desenvolvimento , HumanosRESUMO
Twist transcription factors function as ancestral regulators of mesodermal cell fates in organisms ranging from Drosophila to mammals. Through lineage tracing of Twist2 (Tw2)-expressing cells with tamoxifen-inducible Tw2-CreERT2 and tdTomato (tdTO) reporter mice, we discovered a unique cell population that progressively contributes to cardiomyocytes (CMs), endothelial cells, and fibroblasts in the adult heart. Clonal analysis confirmed the ability of Tw2-derived tdTO+ (Tw2-tdTO+) cells to form CMs in vitro. Within the adult heart, Tw2-tdTO+ CMs accounted for â¼13% of total CMs, the majority of which resulted from fusion of Tw2-tdTO+ cells with existing CMs. Tw2-tdTO+ cells also contribute to cardiac remodeling after injury. We conclude that Tw2-tdTO+ cells participate in lifelong maintenance of cardiac function, at least in part through de novo formation of CMs and fusion with preexisting CMs, as well as in the genesis of other cellular components of the adult heart.
Assuntos
Células-Tronco Multipotentes/metabolismo , Miocárdio/metabolismo , Proteínas Repressoras/biossíntese , Proteína 1 Relacionada a Twist/biossíntese , Animais , Drosophila melanogaster , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Camundongos , Camundongos Transgênicos , Células-Tronco Multipotentes/citologia , Miocárdio/citologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Proteínas Repressoras/genética , Proteína 1 Relacionada a Twist/genéticaRESUMO
Epigenetic factors and related small molecules have emerged to be strongly involved in autophagy process. Here we report that 2-PCPA and GSK-LSD1, two inhibitors of histone H3K4 demethylase KDM1A/LSD1, induce autophagy in multiple mammalian cell lines. The two small molecules induce accumulation of LC3II, formation of autophagosome and autolysosome, and SQSTM1/p62 degradation. 2-PCPA treatment inhibits cell proliferation through cell cycle arrest but does not inducing cell death. Exogenous expression of KDM1A/LSD1 impaired the autophagic phenotypes triggered by 2-PCPA. The autophagy induced by 2-PCPA requires LC3-II processing machinery. But depletion of BECN1 and ULK1 with siRNA did not affect the LC3-II accumulation triggered by 2-PCPA. 2-PCPA treatment induces the change of global gene expression program, including a series of autophagy-related genes, such as SQSTM1/p62. Taken together, our data indicate that KDM1A/LSD1 inhibitors induce autophagy through affecting the expression of autophagy-related genes and in a BECN1-independent manner.
Assuntos
Autofagia/genética , Histona Desmetilases/genética , Proteínas Associadas aos Microtúbulos/genética , Proteína Sequestossoma-1/metabolismo , Animais , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Autofagia/efeitos dos fármacos , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Beclina-1/genética , Epigênese Genética/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Histona Desmetilases/antagonistas & inibidores , Histona Desmetilases/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Piperazinas/farmacologia , Proteína Sequestossoma-1/genéticaRESUMO
RATIONALE: Inflammation in post-myocardial infarction (MI) is necessary for myocyte repair and wound healing. Unfortunately, it is also a key component of subsequent heart failure pathology. Transcription factor forkhead box O4 (FoxO4) regulates a variety of biological processes, including inflammation. However, its role in MI remains unknown. OBJECTIVE: To test the hypothesis that FoxO4 promotes early post-MI inflammation via endothelial arginase 1 (Arg1). METHODS AND RESULTS: We induced MI in wild-type and FoxO4(-/-) mice. FoxO4(-/-) mice had a significantly higher post-MI survival, better cardiac function, and reduced infarct size. FoxO4(-/-) hearts had significantly fewer neutrophils, reduced expression of cytokines, and competitive nitric oxide synthase inhibitor Arg1. We generated conditional FoxO4 knockout mice with FoxO4 deleted in cardiac mycoytes or endothelial cells. FoxO4 endothelial cell-specific knockout mice showed significant post-MI improvement of cardiac function and reduction of neutrophil accumulation and cytokine expression, whereas FoxO4 cardiac mycoyte-specific knockout mice had no significant difference in cardiac function and post-MI inflammation from those of control littermates. FoxO4 binds the Foxo-binding site in the Arg1 promoter and activates Arg1 transcription. FoxO4 knockdown in human aortic endothelial cells upregulated nitric oxide on ischemia and suppressed monocyte adhesion that can be reversed by ectopic-expression of Arg1. Furthermore, chemical inhibition of Arg1 in wild-type mice had similar cardioprotection and reduced inflammation after MI as FoxO4 inactivation and administration of nitric oxide synthase inhibitor to FoxO4 KO mice reversed the beneficial effects of FoxO4 deletion on post-MI cardiac function. CONCLUSIONS: FoxO4 activates Arg1 transcription in endothelial cells in response to MI, leading to downregulation of nitric oxide and upregulation of neutrophil infiltration to the infarct area.
Assuntos
Arginase/biossíntese , Células Endoteliais/enzimologia , Fatores de Transcrição Forkhead/metabolismo , Inflamação/enzimologia , Infarto do Miocárdio/enzimologia , Miócitos Cardíacos/enzimologia , Animais , Anti-Inflamatórios/farmacologia , Arginase/antagonistas & inibidores , Arginase/genética , Sítios de Ligação , Adesão Celular , Proteínas de Ciclo Celular , Técnicas de Cocultura , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/imunologia , Indução Enzimática , Fatores de Transcrição Forkhead/deficiência , Fatores de Transcrição Forkhead/genética , Células HEK293 , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Inflamação/fisiopatologia , Inflamação/prevenção & controle , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infarto do Miocárdio/genética , Infarto do Miocárdio/imunologia , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Infarto do Miocárdio/prevenção & controle , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/imunologia , Miócitos Cardíacos/patologia , Infiltração de Neutrófilos , Óxido Nítrico/metabolismo , Regiões Promotoras Genéticas , Interferência de RNA , Transdução de Sinais , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Transfecção , Células U937RESUMO
Methylation of histone H3 on the lysine-4 residue (H3K4me) is found throughout the eukaryotic domain, and its initial discovery as a conserved epigenetic mark of active transcription from yeast to mammalian cells has contributed to the histone code hypothesis. However, recent studies have raised questions on whether the different forms of H3K4me play a direct role in gene regulation or are simply by-products of the transcription process. Here, we review the often-conflicting experimental evidence, focusing on the monomethylation of lysine 4 on histone H3 that has been linked to the transcriptional state of enhancers in metazoans. We suggest that this epigenetic mark acts in a context-dependent manner to directly facilitate the transcriptional output of the genome and the establishment of cellular identity.
Assuntos
Histonas , Lisina , Animais , Histonas/genética , Histonas/metabolismo , Lisina/genética , Lisina/metabolismo , Metilação , Saccharomyces cerevisiae/genética , Regulação da Expressão Gênica/genética , MamíferosRESUMO
AIMS: RNA binding proteins play essential roles in mediating RNA splicing and are key post-transcriptional regulators in the heart. Our recent study demonstrated that RBPMS (RNA binding protein with multiple splicing) is crucial for cardiac development through modulating mRNA splicing, but little is known about its functions in the adult heart. In this study, we aim to characterize the post-natal cardiac function of Rbpms and its mechanism of action. METHODS AND RESULTS: We generated a cardiac-specific knockout mouse line and found that cardiac-specific loss of Rbpms caused severe cardiomyocyte contractile defects, leading to dilated cardiomyopathy and early lethality in adult mice. We showed by proximity-dependent biotin identification assay and mass spectrometry that RBPMS associates with spliceosome factors and other RNA binding proteins, such as RBM20, that are important in cardiac function. We performed paired-end RNA sequencing and RT-PCR and found that RBPMS regulates mRNA alternative splicing of genes associated with sarcomere structure and function, such as Ttn, Pdlim5, and Nexn, generating new protein isoforms. Using a minigene splicing reporter assay, we determined that RBPMS regulates target gene splicing through recognizing tandem intronic CAC motifs. We also showed that RBPMS knockdown in human induced pluripotent stem cell-derived cardiomyocytes impaired cardiomyocyte contraction. CONCLUSION: This study identifies RBPMS as an important regulator of cardiomyocyte contraction and cardiac function by modulating sarcomeric gene alternative splicing.
Assuntos
Processamento Alternativo , Células-Tronco Pluripotentes Induzidas , Animais , Humanos , Camundongos , Conectina/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos Knockout , Miócitos Cardíacos/metabolismo , RNA/metabolismo , Splicing de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Comprehensive analysis of chromatin architecture is crucial for understanding the gene regulatory programs during development and in disease pathogenesis, yet current methods often inadequately address the unique challenges presented by analysis of heterogeneous tissue samples. Here, we introduce Droplet Hi-C, which employs a commercial microfluidic device for high-throughput, single-cell chromatin conformation profiling in droplets. Using Droplet Hi-C, we mapped the chromatin architecture at single-cell resolution from the mouse cortex and analyzed gene regulatory programs in major cortical cell types. Additionally, we used this technique to detect copy number variation (CNV), structural variations (SVs) and extrachromosomal DNA (ecDNA) in cancer cells, revealing clonal dynamics and other oncogenic events during treatment. We further refined this technique to allow for joint profiling of chromatin architecture and transcriptome in single cells, facilitating a more comprehensive exploration of the links between chromatin architecture and gene expression in both normal tissues and tumors. Thus, Droplet Hi-C not only addresses critical gaps in chromatin analysis of heterogeneous tissues but also emerges as a versatile tool enhancing our understanding of gene regulation in health and disease.
RESUMO
Smc5/6 is a member of the eukaryotic structural maintenance of chromosomes (SMC) family of complexes with important roles in genome maintenance and viral restriction. However, limited structural understanding of Smc5/6 hinders the elucidation of its diverse functions. Here, we report cryo-EM structures of the budding yeast Smc5/6 complex in eight-subunit, six-subunit and five-subunit states. Structural maps throughout the entire length of these complexes reveal modularity and key elements in complex assembly. We show that the non-SMC element (Nse)2 subunit supports the overall shape of the complex and uses a wedge motif to aid the stability and function of the complex. The Nse6 subunit features a flexible hook region for attachment to the Smc5 and Smc6 arm regions, contributing to the DNA repair roles of the complex. Our results also suggest a structural basis for the opposite effects of the Nse1-3-4 and Nse5-6 subcomplexes in regulating Smc5/6 ATPase activity. Collectively, our integrated structural and functional data provide a framework for understanding Smc5/6 assembly and function.
RESUMO
Cancer cells exhibit phenotypical plasticity and epigenetic reprogramming that allows them to evade lineage-dependent targeted treatments by adopting lineage plasticity. The underlying mechanisms by which cancer cells exploit the epigenetic regulatory machinery to acquire lineage plasticity and therapy resistance remain poorly understood. We identified zinc finger protein 397 (ZNF397) as a bona fide coactivator of the androgen receptor (AR), essential for the transcriptional program governing AR-driven luminal lineage. ZNF397 deficiency facilitates the transition of cancer cell from an AR-driven luminal lineage to a ten-eleven translocation 2 (TET2)-driven lineage plastic state, ultimately promoting resistance to therapies inhibiting AR signaling. Intriguingly, our findings indicate that a TET2 inhibitor can eliminate the resistance to AR-targeted therapies in ZNF397-deficient tumors. These insights uncover a novel mechanism through which prostate cancer acquires lineage plasticity via epigenetic rewiring and offer promising implications for clinical interventions designed to overcome therapy resistance dictated by lineage plasticity. Significance: This study reveals a bifurcated role of ZNF397, and a TET2-driven epigenetic mechanism regulating tumor lineage plasticity and therapy response in prostate cancer, enhances the understanding of drug resistance, and unveils a new therapeutic strategy for overcoming androgen receptor-targeted therapy resistance.
Assuntos
Proteínas de Ligação a DNA , Dioxigenases , Resistencia a Medicamentos Antineoplásicos , Neoplasias da Próstata , Receptores Androgênicos , Masculino , Humanos , Proteínas de Ligação a DNA/genética , Receptores Androgênicos/metabolismo , Receptores Androgênicos/genética , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Neoplasias da Próstata/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Camundongos , Animais , Linhagem Celular Tumoral , Epigênese Genética , Linhagem da CélulaRESUMO
Simultaneous detection of chromatin accessibility and transcription from the same cells promises to greatly facilitate the dissection of cell-type-specific gene regulatory programs in complex tissues. Paired-seq enables joint analysis of open chromatin and nuclear transcriptome from up to a million cells in parallel. It achieves ultra-high-throughput single-cell multiomics with the use of a combinatorial barcoding strategy involving sequential ligation of multiplexed DNA barcodes to chromatin DNA fragments and reverse transcription products, followed by high-throughput DNA sequencing of the resulting DNA libraries and deconvolution of single-cell multiomic maps based on cell-specific barcodes.
Assuntos
Cromatina , Transcriptoma , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Biblioteca Gênica , Análise de Célula Única/métodosRESUMO
We previously reported Paired-Tag, a combinatorial indexing-based method that can simultaneously map histone modifications and gene expression at single-cell resolution at scale. However, the lengthy procedure of Paired-Tag has hindered its general adoption in the community. To address this bottleneck, we developed a droplet-based Paired-Tag protocol that is faster and more accessible than the previous method. Using cultured mammalian cells and primary brain tissues, we demonstrate its superior performance at identifying candidate cis-regulatory elements and associating their dynamic chromatin state to target gene expression in each constituent cell type in a complex tissue.