RESUMO
A first-in-human trial demonstrated that a vaccine targeting the histone mutation H3K27M can induce an immune response, in a mutation-specific manner, in patients with diffuse midline glioma. In a recent study by Boschert et al., the same group now dissects the functional immune response triggered after effective vaccination of one of the patients, who has been in remission for over 3 years. The H3K27M peptide vaccine, named H3-vac, induces a CD4+ T-cell-specific immune response in this patient and expands the repertoire of polyclonal H3K27M-specific T-cell receptors. A clonal H3K27M-reactive B-cell population was also detected in the patient's cerebrospinal fluid. Importantly, the immune response is induced across various human leukocyte antigen alleleotypes, indicating the potential efficacy of the vaccine in diverse populations. By exploring in detail the immune response linked to this patient's long-term survival, the authors prove peptide vaccinations as a viable therapeutic approach. This paves the way for personalised therapies harnessing immunogenic T- and B-cell responses against different tumour types.
Assuntos
Vacinas Anticâncer , Glioma , Humanos , Glioma/imunologia , Glioma/terapia , Glioma/genética , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/uso terapêutico , Vacinação , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/terapiaRESUMO
In muscular dystrophies, muscle fibers loose integrity and die, causing significant suffering and premature death. Strikingly, the extraocular muscles (EOMs) are spared, functioning well despite the disease progression. Although EOMs have been shown to differ from body musculature, the mechanisms underlying this inherent resistance to muscle dystrophies remain unknown. Here, we demonstrate important differences in gene expression as a response to muscle dystrophies between the EOMs and trunk muscles in zebrafish via transcriptomic profiling. We show that the LIM-protein Fhl2 is increased in response to the knockout of desmin, plectin and obscurin, cytoskeletal proteins whose knockout causes different muscle dystrophies, and contributes to disease protection of the EOMs. Moreover, we show that ectopic expression of fhl2b can partially rescue the muscle phenotype in the zebrafish Duchenne muscular dystrophy model sapje, significantly improving their survival. Therefore, Fhl2 is a protective agent and a candidate target gene for therapy of muscular dystrophies.
Assuntos
Proteínas com Domínio LIM , Proteínas Musculares , Distrofia Muscular de Duchenne , Músculos Oculomotores , Animais , Proteínas do Citoesqueleto/metabolismo , Distrofina/genética , Expressão Ectópica do Gene , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Músculos Oculomotores/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas Musculares/metabolismo , Proteínas com Domínio LIM/metabolismoRESUMO
Several preclinical models have been recently developed for metabolic associated fatty liver disease (MAFLD) and associated hepatocellular carcinoma (HCC) but comprehensive analysis of the regulatory and transcriptional landscapes underlying disease in these models are still missing. We investigated the regulatory and transcriptional landscape in fatty livers and liver tumours from DIAMOND mice that faithfully mimic human HCC development in the context of MAFLD. RNA-sequencing and ChIP-sequencing revealed rewiring of the Wnt/ß-catenin regulatory network in DIAMOND tumours, as manifested by chromatin remodelling and associated switching in the expression of the canonical TCF/LEF downstream effectors. We identified splicing as a major mechanism leading to constitutive oncogenic activation of ß-catenin in a large subset of DIAMOND tumours, a mechanism that is independent on somatic mutations in the locus and that has not been previously shown. Similar splicing events were found in a fraction of human HCC and hepatoblastoma samples.
Assuntos
Carcinoma Hepatocelular , Fígado Gorduroso , Neoplasias Hepáticas , Camundongos , Humanos , Animais , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , beta Catenina/genética , beta Catenina/metabolismo , Redes Reguladoras de Genes , Fígado Gorduroso/genética , Dieta , Via de Sinalização Wnt/genéticaRESUMO
BACKGROUND: Glioblastoma (GB) is the most aggressive of all primary brain tumours and due to its highly invasive nature, surgical resection is nearly impossible. Patients typically rely on radiotherapy with concurrent temozolomide (TMZ) treatment and face a median survival of ~ 14 months. Alterations in the Epidermal Growth Factor Receptor gene (EGFR) are common in GB tumours, but therapies targeting EGFR have not shown significant clinical efficacy. METHODS: Here, we investigated the influence of the EGFR regulatory genome on GB cells and identified novel EGFR enhancers located near the GB-associated SNP rs723527. We used CRISPR/Cas9-based approaches to target the EGFR enhancer regions, generating multiple modified GB cell lines, which enabled us to study the functional response to enhancer perturbation. RESULTS: Epigenomic perturbation of the EGFR regulatory region decreases EGFR expression and reduces the proliferative and invasive capacity of glioblastoma cells, which also undergo a metabolic reprogramming in favour of mitochondrial respiration and present increased apoptosis. Moreover, EGFR enhancer-perturbation increases the sensitivity of GB cells to TMZ, the first-choice chemotherapeutic agent to treat glioblastoma. CONCLUSIONS: Our findings demonstrate how epigenomic perturbation of EGFR enhancers can ameliorate the aggressiveness of glioblastoma cells and enhance the efficacy of TMZ treatment. This study demonstrates how CRISPR/Cas9-based perturbation of enhancers can be used to modulate the expression of key cancer genes, which can help improve the effectiveness of existing cancer treatments and potentially the prognosis of difficult-to-treat cancers such as glioblastoma.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Humanos , Temozolomida/farmacologia , Temozolomida/uso terapêutico , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Genes erbB-1 , Receptores ErbB/metabolismo , Epigenômica , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Sequências Reguladoras de Ácido NucleicoRESUMO
Chromatin organization controls transcription by modulating 3D-interactions between enhancers and promoters in the nucleus. Alterations in epigenetic states and 3D-chromatin organization result in gene expression changes contributing to cancer. Here, we map the promoter-enhancer interactome and regulatory landscape of glioblastoma, the most aggressive primary brain tumour. Our data reveals profound rewiring of promoter-enhancer interactions, chromatin accessibility and redistribution of histone marks in glioblastoma. This leads to loss of long-range regulatory interactions and overall activation of promoters, which orchestrate changes in the expression of genes associated to glutamatergic synapses, axon guidance, axonogenesis and chromatin remodelling. SMAD3 and PITX1 emerge as major transcription factors controlling genes related to synapse organization and axon guidance. Inhibition of SMAD3 and neuronal activity stimulation cooperate to promote proliferation of glioblastoma cells in co-culture with glutamatergic neurons, and in mice bearing patient-derived xenografts. Our findings provide mechanistic insight into the regulatory networks that mediate neurogliomal synaptic communication.
Assuntos
Glioblastoma , Humanos , Animais , Camundongos , Glioblastoma/genética , Elementos Facilitadores Genéticos/genética , Cromatina , Fatores de Transcrição/genética , Expressão GênicaRESUMO
Genome architecture, epigenetics and enhancer function control the fate and identity of cells. Reprogramming to induced pluripotent stem cells (iPSCs) changes the transcriptional profile and chromatin landscape of the starting somatic cell to that of the pluripotent cell in a stepwise manner. Changes in the regulatory networks are tightly regulated during normal embryonic development to determine cell fate, and similarly need to function in cell fate control during reprogramming. Switching off the somatic program and turning on the pluripotent program involves a dynamic reorganization of the epigenetic landscape, enhancer function, chromatin accessibility and 3D chromatin topology. Within this context, we will review here the current knowledge on the processes that control the establishment and maintenance of pluripotency during somatic cell reprogramming.
Assuntos
Reprogramação Celular , Células-Tronco Pluripotentes Induzidas , Reprogramação Celular/genética , Cromatina/genética , Epigênese Genética , EpigenômicaRESUMO
AMP-activated protein kinase (AMPK) has an important role in cellular energy homeostasis and has emerged as a promising target for treatment of Type 2 Diabetes (T2D) due to its beneficial effects on insulin sensitivity and glucose homeostasis. O304 is a pan-AMPK activator that has been shown to improve glucose homeostasis in both mouse models of diabetes and in human T2D subjects. Here, we describe the genome-wide transcriptional profile and chromatin landscape of pancreatic islets following O304 treatment of mice fed high-fat diet (HFD). O304 largely prevented genome-wide gene expression changes associated with HFD feeding in CBA mice and these changes were associated with remodelling of active and repressive chromatin marks. In particular, the increased expression of the ß-cell stress marker Aldh1a3 in islets from HFD-mice is completely abrogated following O304 treatment, which is accompanied by loss of active chromatin marks in the promoter as well as distant non-coding regions upstream of the Aldh1a3 gene. Moreover, O304 treatment restored dysfunctional glucose homeostasis as well as expression of key markers associated with ß-cell function in mice with already established obesity. Our findings provide preclinical evidence that O304 is a promising therapeutic compound not only for T2D remission but also for restoration of ß-cell function following remission of T2D diabetes.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Dieta Hiperlipídica/efeitos adversos , Ativadores de Enzimas/farmacologia , Expressão Gênica/efeitos dos fármacos , Código das Histonas/efeitos dos fármacos , Código das Histonas/genética , Histonas/metabolismo , Ilhotas Pancreáticas/metabolismo , Obesidade/metabolismo , Tiadiazóis/farmacologia , Proteínas Quinases Ativadas por AMP/fisiologia , Família Aldeído Desidrogenase 1/genética , Família Aldeído Desidrogenase 1/metabolismo , Animais , Diabetes Mellitus Tipo 2/fisiopatologia , Modelos Animais de Doenças , Glucose/metabolismo , Homeostase/efeitos dos fármacos , Células Secretoras de Insulina/fisiologia , Camundongos , Camundongos Endogâmicos CBA , Obesidade/etiologia , Retinal Desidrogenase/genética , Retinal Desidrogenase/metabolismoRESUMO
BACKGROUND: Organ culture models have been used over the past few decades to study development and disease. The in vitro three-dimensional (3D) culture system of organoids is well known, however, these 3D systems are both costly and difficult to culture and maintain. As such, less expensive, faster and less complex methods to maintain 3D cell culture models would complement the use of organoids. Chick embryos have been used as a model to study human biology for centuries, with many fundamental discoveries as a result. These include cell type induction, cell competence, plasticity and contact inhibition, which indicates the relevance of using chick embryos when studying developmental biology and disease mechanisms. RESULTS: Here, we present an updated protocol that enables time efficient, cost effective and long-term expansion of fetal organ spheroids (FOSs) from chick embryos. Utilizing this protocol, we generated FOSs in an anchorage-independent growth pattern from seven different organs, including brain, lung, heart, liver, stomach, intestine and epidermis. These three-dimensional (3D) structures recapitulate many cellular and structural aspects of their in vivo counterpart organs and serve as a useful developmental model. In addition, we show a functional application of FOSs to analyze cell-cell interaction and cell invasion patterns as observed in cancer. CONCLUSION: The establishment of a broad ranging and highly effective method to generate FOSs from different organs was successful in terms of the formation of healthy, proliferating 3D organ spheroids that exhibited organ-like characteristics. Potential applications of chick FOSs are their use in studies of cell-to-cell contact, cell fusion and tumor invasion under defined conditions. Future studies will reveal whether chick FOSs also can be applicable in scientific areas such as viral infections, drug screening, cancer diagnostics and/or tissue engineering.
Assuntos
Técnicas de Cultura de Células em Três Dimensões , Modelos Biológicos , Invasividade Neoplásica/patologia , Organoides/citologia , Esferoides Celulares/citologia , Animais , Comunicação Celular , Linhagem Celular Tumoral , Embrião de Galinha , Galinhas , Humanos , Organoides/ultraestrutura , Esferoides Celulares/ultraestrutura , Técnicas de Cultura de TecidosRESUMO
In the originally published version of this Letter, ref. 43 was erroneously provided twice. In the 'Estimation of relative cell-type-specific composition of AML samples' section in the Methods, the citation to ref. 43 after the GEO dataset GSE24759 is correct. However, in the 'Mice' section of the Methods, the citation to ref. 43 after 'TAMERE' should have been associated with a new reference1. The original Letter has been corrected online (with the new reference included as ref. 49).
RESUMO
The transcription factor Myc is essential for the regulation of haematopoietic stem cells and progenitors and has a critical function in haematopoietic malignancies. Here we show that an evolutionarily conserved region located 1.7 megabases downstream of the Myc gene that has previously been labelled as a 'super-enhancer' is essential for the regulation of Myc expression levels in both normal haematopoietic and leukaemic stem cell hierarchies in mice and humans. Deletion of this region in mice leads to a complete loss of Myc expression in haematopoietic stem cells and progenitors. This caused an accumulation of differentiation-arrested multipotent progenitors and loss of myeloid and B cells, mimicking the phenotype caused by Mx1-Cre-mediated conditional deletion of the Myc gene in haematopoietic stem cells. This super-enhancer comprises multiple enhancer modules with selective activity that recruits a compendium of transcription factors, including GFI1b, RUNX1 and MYB. Analysis of mice carrying deletions of individual enhancer modules suggests that specific Myc expression levels throughout most of the haematopoietic hierarchy are controlled by the combinatorial and additive activity of individual enhancer modules, which collectively function as a 'blood enhancer cluster' (BENC). We show that BENC is also essential for the maintenance of MLL-AF9-driven leukaemia in mice. Furthermore, a BENC module, which controls Myc expression in mouse haematopoietic stem cells and progenitors, shows increased chromatin accessibility in human acute myeloid leukaemia stem cells compared to blasts. This difference correlates with MYC expression and patient outcome. We propose that clusters of enhancers, such as BENC, form highly combinatorial systems that allow precise control of gene expression across normal cellular hierarchies and which also can be hijacked in malignancies.
Assuntos
Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica , Genes myc/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Leucemia/genética , Leucemia/patologia , Família Multigênica/genética , Animais , Linfócitos B/citologia , Diferenciação Celular , Linhagem da Célula/genética , Cromatina/genética , Cromatina/metabolismo , Regulação para Baixo , Feminino , Deleção de Genes , Células-Tronco Hematopoéticas/patologia , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Multipotentes/citologia , Células Mieloides/citologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Prognóstico , Deleção de Sequência , Análise de Sobrevida , Fatores de Transcrição/metabolismoRESUMO
Gene expression often requires interaction between promoters and distant enhancers, which occur within the context of highly organized topologically associating domains (TADs). Using a series of engineered chromosomal rearrangements at the Shh locus, we carried out an extensive fine-scale characterization of the factors that govern the long-range regulatory interactions controlling Shh expression. We show that Shh enhancers act pervasively, yet not uniformly, throughout the TAD. Importantly, changing intra-TAD distances had no impact on Shh expression. In contrast, inversions disrupting the TAD altered global folding of the region and prevented regulatory contacts in a distance-dependent manner. Our data indicate that the Shh TAD promotes distance-independent contacts between distant regions that would otherwise interact only sporadically, enabling functional communication between them. In large genomes where genomic distances per se can limit regulatory interactions, this function of TADs could be as essential for gene expression as the formation of insulated neighborhoods.
Assuntos
Elementos Facilitadores Genéticos , Proteínas Hedgehog/genética , Animais , Anormalidades Congênitas/embriologia , Anormalidades Congênitas/genética , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Regiões Promotoras GenéticasRESUMO
During embryonic development, complex transcriptional programs govern the precision of gene expression. Many key developmental genes are regulated via cis-regulatory elements that are located far away in the linear genome. How sequences located hundreds of kilobases away from a promoter can influence its activity has been the subject of numerous speculations, which all underline the importance of the 3D-organization of the genome. The recent advent of chromosome conformation capture techniques has put into focus the subdivision of the genome into topologically associating domains (TADs). TADs may influence regulatory activities on multiple levels. The relative invariance of TAD limits across cell types suggests that they may form fixed structural domains that could facilitate and/or confine long-range regulatory interactions. However, most recent studies suggest that interactions within TADs are more variable and dynamic than initially described. Hence, different models are emerging regarding how TADs shape the complex 3D conformations, and thereafter influence the networks of cis-interactions that govern gene expression during development. For further resources related to this article, please visit the WIREs website.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Ativação Transcricional , Animais , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos , Regiões Promotoras Genéticas , Estrutura Terciária de ProteínaRESUMO
Cohesin, which in somatic vertebrate cells consists of SMC1, SMC3, RAD21 and either SA1 or SA2, mediates higher-order chromatin organization. To determine how cohesin contributes to the establishment of tissue-specific transcriptional programs, we compared genome-wide cohesin distribution, gene expression and chromatin architecture in cerebral cortex and pancreas from adult mice. More than one third of cohesin binding sites differ between the two tissues and these show reduced overlap with CCCTC-binding factor (CTCF) and are enriched at the regulatory regions of tissue-specific genes. Cohesin/CTCF sites at active enhancers and promoters contain, at least, cohesin-SA1. Analyses of chromatin contacts at the Protocadherin (Pcdh) and Regenerating islet-derived (Reg) gene clusters, mostly expressed in brain and pancreas, respectively, revealed remarkable differences that correlate with the presence of cohesin. We could not detect significant changes in the chromatin contacts at the Pcdh locus when comparing brains from wild-type and SA1 null embryos. In contrast, reduced dosage of SA1 altered the architecture of the Reg locus and decreased the expression of Reg genes in the pancreas of SA1 heterozygous mice. Given the role of Reg proteins in inflammation, such reduction may contribute to the increased incidence of pancreatic cancer observed in these animals.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Animais , Sítios de Ligação , Fator de Ligação a CCCTC , Caderinas/genética , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/genética , Córtex Cerebral/metabolismo , Cromatina/química , Cromatina/genética , Proteínas Cromossômicas não Histona/deficiência , Proteínas Cromossômicas não Histona/genética , Expressão Gênica , Heterozigoto , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Família Multigênica , Pâncreas/metabolismo , Subunidades Proteicas/deficiência , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA/genética , RNA/metabolismo , Proteínas Repressoras/metabolismo , Distribuição Tecidual , CoesinasRESUMO
Cornelia de Lange Syndrome (CdLS) is a genetic disorder linked to mutations in cohesin and its regulators. To date, it is unclear which function of cohesin is more relevant to the pathology of the syndrome. A mouse heterozygous for the gene encoding the cohesin loader Nipbl recapitulates many features of CdLS. We have carefully examined Nipbl deficient cells and here report that they have robust cohesion all along the chromosome. DNA replication, DNA repair and chromosome segregation are carried out efficiently in these cells. While bulk cohesin loading is unperturbed, binding to certain promoters such as the Protocadherin genes in brain is notably affected and alters gene expression. These results provide further support for the idea that developmental defects in CdLS are caused by deregulated transcription and not by malfunction of cohesion-related processes.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Replicação do DNA , Síndrome de Cornélia de Lange/patologia , Modelos Animais de Doenças , Fatores de Transcrição/fisiologia , Transcrição Gênica , Animais , Western Blotting , Encéfalo/metabolismo , Encéfalo/patologia , Proteínas de Ciclo Celular/genética , Sobrevivência Celular , Células Cultivadas , Proteínas Cromossômicas não Histona/genética , Segregação de Cromossomos , Reparo do DNA , Síndrome de Cornélia de Lange/genética , Síndrome de Cornélia de Lange/metabolismo , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Imunofluorescência , Heterozigoto , Hibridização in Situ Fluorescente , Camundongos , Camundongos Knockout , Fenótipo , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , CoesinasRESUMO
Cohesin is a ring-shaped complex, conserved from yeast to human, that was named for its ability to mediate sister chromatid cohesion. This function is essential for chromosome segregation in both mitosis and meiosis, and also for DNA repair. In addition, more recent studies have shown that cohesin influences gene expression during development through mechanisms that likely involve DNA looping and interactions with several transcriptional regulators. Here, we provide an overview of how cohesin functions, highlighting its role both in development and in disease.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Doença , Desenvolvimento Embrionário , Animais , Ciclo Celular , Humanos , Complexos Multiproteicos/metabolismo , Transcrição Gênica , CoesinasRESUMO
DNA replication is facilitated by multiple factors that concentrate in the vicinity of replication forks. Here, we developed an approach that combines the isolation of proteins on nascent DNA chains with mass spectrometry (iPOND-MS), allowing a comprehensive proteomic characterization of the human replisome and replisome-associated factors. In addition to known replisome components, we provide a broad list of proteins that reside in the vicinity of the replisome, some of which were not previously associated with replication. For instance, our data support a link between DNA replication and the Williams-Beuren syndrome and identify ZNF24 as a replication factor. In addition, we reveal that SUMOylation is widespread for factors that concentrate near replisomes, which contrasts with lower UQylation levels at these sites. This resource provides a panoramic view of the proteins that concentrate in the surroundings of the replisome, which should facilitate future investigations on DNA replication and genome maintenance.
Assuntos
DNA/metabolismo , Proteômica , Linhagem Celular , Replicação do DNA , Redes Reguladoras de Genes , Células HEK293 , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Espectrometria de Massas , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Ubiquitinação , Síndrome de Williams/genética , Síndrome de Williams/metabolismoRESUMO
Cohesin is a four subunit complex, conserved from yeast to man, with the ability to hold together two DNA segments within its ring-shaped structure. When the two segments belong to sister chromatids, cohesin is mediating cohesion, which is essential for chromosome segregation in mitosis and meiosis and for homologous DNA repair. When the two DNA segments are in the same chromatid, a loop is formed. These chromatin loops are emerging as a mechanism for controlling the communication between enhancers and promoters and thereby regulate gene expression. They also facilitate DNA replication and recombination. Given all its essential functions, it is not surprising that mutations in cohesin and its interacting factors have been associated to cancer and developmental syndromes known as cohesinopathies.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Animais , Cromátides/metabolismo , Cromatina/genética , Segregação de Cromossomos , Reparo do DNA/genética , Replicação do DNA , Doença , Recombinação Homóloga , Humanos , Interfase , Meiose , Mitose , Subunidades Proteicas/metabolismo , CoesinasRESUMO
Besides its well-established role in sister chromatid cohesion, cohesin has recently emerged as major player in the organization of interphase chromatin. Such important function is related to its ability to entrap two DNA segments also in cis, thereby facilitating long-range DNA looping which is crucial for transcriptional regulation, organization of replication factories and V(D)J recombination. Vertebrate somatic cells have two different versions of cohesin, containing Smc1, Smc3, Rad21/Scc1 and either SA1 or SA2, but their functional specificity has been largely ignored. We recently generated a knockout mouse model for the gene encoding SA1, and found that this protein is essential to complete embryonic development. Cohesin-SA1 mediates cohesion at telomeres, which is required for their replication. Telomere defects in SA1- deficient cells provoke chromosome segregation errors resulting in aneuploidy despite robust centromere cohesion. This aneuploidy could explain why heterozygous animals have an earlier onset of tumorigenesis. In addition, the genome-wide distribution of cohesin changes dramatically in the absence of SA1, and the complex shows reduced accumulation at promoters and CTCF sites. As a consequence, gene expression is altered, leading to downregulation of biological processes related to a developmental disorder linked to cohesin function, the Cornelia de Lange Syndrome (CdLS). These results point out a prominent role of cohesin-SA1 in transcriptional regulation, with clear implications in the etiology of CdLS.
Assuntos
Sistema A de Transporte de Aminoácidos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Sistema A de Transporte de Aminoácidos/genética , Aneuploidia , Animais , Sítios de Ligação , Fator de Ligação a CCCTC , Transformação Celular Neoplásica , Segregação de Cromossomos , Síndrome de Cornélia de Lange/etiologia , Desenvolvimento Embrionário , Regulação da Expressão Gênica , Células HeLa , Humanos , Camundongos , Camundongos Knockout , Neoplasias/metabolismo , Neoplasias/patologia , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Telômero/metabolismo , CoesinasRESUMO
Cohesin is a protein complex originally identified for its role in sister chromatid cohesion, although increasing evidence portrays it also as a major organizer of interphase chromatin. Vertebrate cohesin consists of Smc1, Smc3, Rad21/Scc1 and either stromal antigen 1 (SA1) or SA2. To explore the functional specificity of these two versions of cohesin and their relevance for embryonic development and cancer, we generated a mouse model deficient for SA1. Complete ablation of SA1 results in embryonic lethality, while heterozygous animals have shorter lifespan and earlier onset of tumourigenesis. SA1-null mouse embryonic fibroblasts show decreased proliferation and increased aneuploidy as a result of chromosome segregation defects. These defects are not caused by impaired centromeric cohesion, which depends on cohesin-SA2. Instead, they arise from defective telomere replication, which requires cohesion mediated specifically by cohesin-SA1. We propose a novel mechanism for aneuploidy generation that involves impaired telomere replication upon loss of cohesin-SA1, with clear implications in tumourigenesis.
Assuntos
Aneuploidia , Proteínas de Ciclo Celular/deficiência , Proteínas Cromossômicas não Histona/deficiência , Subunidades Proteicas/deficiência , Telômero/metabolismo , Animais , Carcinógenos , Proteínas de Ciclo Celular/genética , Linhagem Celular , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/genética , Segregação de Cromossomos , Dietilnitrosamina , Fibrossarcoma/induzido quimicamente , Fibrossarcoma/genética , Fibrossarcoma/patologia , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Metilcolantreno , Camundongos , Camundongos Knockout , Neoplasias Experimentais/induzido quimicamente , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Subunidades Proteicas/genética , Troca de Cromátide Irmã , CoesinasRESUMO
Vertebrates have two cohesin complexes that consist of Smc1, Smc3, Rad21/Scc1 and either SA1 or SA2, but their functional specificity is unclear. Mouse embryos lacking SA1 show developmental delay and die before birth. Comparison of the genome-wide distribution of cohesin in wild-type and SA1-null cells reveals that SA1 is largely responsible for cohesin accumulation at promoters and at sites bound by the insulator protein CTCF. As a consequence, ablation of SA1 alters transcription of genes involved in biological processes related to Cornelia de Lange syndrome (CdLS), a genetic disorder linked to dysfunction of cohesin. We show that the presence of cohesin-SA1 at the promoter of myc and of protocadherin genes positively regulates their expression, a task that cannot be assumed by cohesin-SA2. Lack of SA1 also alters cohesin-binding pattern along some gene clusters and leads to dysregulation of genes within. We hypothesize that impaired cohesin-SA1 function in gene expression underlies the molecular aetiology of CdLS.