RESUMO
The recent development of the CRISPR/Cas9 system as an efficient and accessible programmable genome-editing tool has revolutionized basic science research. CRISPR/Cas9 system-based technologies have armed researchers with new powerful tools to unveil the impact of genetics on disease development by enabling the creation of precise cellular and animal models of human diseases. The therapeutic potential of these technologies is tremendous, particularly in gene therapy, in which a patient-specific mutation is genetically corrected in order to treat human diseases that are untreatable with conventional therapies. However, the translation of CRISPR/Cas9 into the clinics will be challenging, since we still need to improve the efficiency, specificity and delivery of this technology. In this review, we focus on several in vitro, in vivo and ex vivo applications of the CRISPR/Cas9 system in human disease-focused research, explore the potential of this technology in translational medicine and discuss some of the major challenges for its future use in patients.
Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/tendências , Terapia Genética/tendências , Pesquisa Translacional Biomédica/tendências , Animais , Humanos , Modelos Animais , Mutação/genéticaRESUMO
Neural progenitor cells (NeuPCs) possess a unique nuclear architecture that changes during differentiation. Nucleoporins are linked with cell-type-specific gene regulation, coupling physical changes in nuclear structure to transcriptional output; but, whether and how they coordinate with key fate-determining transcription factors is unclear. Here we show that the nucleoporin Nup153 interacts with Sox2 in adult NeuPCs, where it is indispensable for their maintenance and controls neuronal differentiation. Genome-wide analyses show that Nup153 and Sox2 bind and co-regulate hundreds of genes. Binding of Nup153 to gene promoters or transcriptional end sites correlates with increased or decreased gene expression, respectively, and inhibiting Nup153 expression alters open chromatin configurations at its target genes, disrupts genomic localization of Sox2, and promotes differentiation in vitro and a gliogenic fate switch in vivo. Together, these findings reveal that nuclear structural proteins may exert bimodal transcriptional effects to control cell fate.
Assuntos
Regulação da Expressão Gênica , Células-Tronco Neurais/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Animais , Cromatina/metabolismo , Genoma , Camundongos , Neurogênese/genética , Ligação Proteica , Transcrição GênicaRESUMO
Nucleoporins (Nups) are a family of proteins best known as the constituent building blocks of nuclear pore complexes (NPCs), membrane-embedded channels that mediate nuclear transport across the nuclear envelope. Recent evidence suggests that several Nups have additional roles in controlling the activation and silencing of developmental genes; however, the mechanistic details of these functions remain poorly understood. Here, we show that depletion of Nup153 in mouse embryonic stem cells (mESCs) causes the derepression of developmental genes and induction of early differentiation. This loss of stem cell identity is not associated with defects in the nuclear import of key pluripotency factors. Rather, Nup153 binds around the transcriptional start site (TSS) of developmental genes and mediates the recruitment of the polycomb-repressive complex 1 (PRC1) to a subset of its target loci. Our results demonstrate a chromatin-associated role of Nup153 in maintaining stem cell pluripotency by functioning in mammalian epigenetic gene silencing.
Assuntos
Células-Tronco Embrionárias/fisiologia , Inativação Gênica , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Células-Tronco Pluripotentes/fisiologia , Animais , Sítios de Ligação , Diferenciação Celular , Núcleo Celular/metabolismo , Cromatina/metabolismo , Mapeamento Cromossômico , Células-Tronco Embrionárias/citologia , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Camundongos , Células-Tronco Pluripotentes/citologia , Complexo Repressor Polycomb 1/metabolismo , Ligação ProteicaRESUMO
Monozygotic (MZ) twins are partially concordant for most complex diseases, including autoimmune disorders. Whereas phenotypic concordance can be used to study heritability, discordance suggests the role of non-genetic factors. In autoimmune diseases, environmentally driven epigenetic changes are thought to contribute to their etiology. Here we report the first high-throughput and candidate sequence analyses of DNA methylation to investigate discordance for autoimmune disease in twins. We used a cohort of MZ twins discordant for three diseases whose clinical signs often overlap: systemic lupus erythematosus (SLE), rheumatoid arthritis, and dermatomyositis. Only MZ twins discordant for SLE featured widespread changes in the DNA methylation status of a significant number of genes. Gene ontology analysis revealed enrichment in categories associated with immune function. Individual analysis confirmed the existence of DNA methylation and expression changes in genes relevant to SLE pathogenesis. These changes occurred in parallel with a global decrease in the 5-methylcytosine content that was concomitantly accompanied with changes in DNA methylation and expression levels of ribosomal RNA genes, although no changes in repetitive sequences were found. Our findings not only identify potentially relevant DNA methylation markers for the clinical characterization of SLE patients but also support the notion that epigenetic changes may be critical in the clinical manifestations of autoimmune disease.
Assuntos
Metilação de DNA , Doenças em Gêmeos/genética , Lúpus Eritematoso Sistêmico/genética , Gêmeos Monozigóticos/genética , 5-Metilcitosina/química , Artrite Reumatoide/genética , Doenças Autoimunes/genética , Estudos de Coortes , Ilhas de CpG/genética , Dermatomiosite/genética , Feminino , Genes de RNAr , Humanos , Masculino , Regiões Promotoras Genéticas , Análise de Sequência de DNA/métodosRESUMO
The molecular basis underlying the aberrant DNA-methylation patterns in human cancer is largely unknown. Altered DNA methyltransferase (DNMT) activity is believed to contribute, as DNMT expression levels increase during tumorigenesis. Here, we present evidence that the expression of DNMT3b is post-transcriptionally regulated by HuR, an RNA-binding protein that stabilizes and/or modulates the translation of target mRNAs. The presence of a putative HuR-recognition motif in the DNMT3b 3'UTR prompted studies to investigate if this transcript associated with HuR. The interaction between HuR and DNMT3b mRNA was studied by immunoprecipitation of endogenous HuR ribonucleoprotein complexes followed by RT-qPCR detection of DNMT3b mRNA, and by in vitro pulldown of biotinylated DNMT3b RNAs followed by western blotting detection of HuR. These studies revealed that binding of HuR stabilized the DNMT3b mRNA and increased DNMT3b expression. Unexpectedly, cisplatin treatment triggered the dissociation of the [HuR-DNMT3b mRNA] complex, in turn promoting DNMT3b mRNA decay, decreasing DNMT3b abundance, and lowering the methylation of repeated sequences and global DNA methylation. In summary, our data identify DNMT3b mRNA as a novel HuR target, present evidence that HuR affects DNMT3b expression levels post-transcriptionally, and reveal the functional consequences of the HuR-regulated DNMT3b upon DNA methylation patterns.
Assuntos
Antígenos de Superfície/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Antígenos de Superfície/análise , Antineoplásicos/farmacologia , Sequência de Bases , Linhagem Celular Tumoral , Cisplatino/farmacologia , DNA (Citosina-5-)-Metiltransferases/metabolismo , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Humanos , Dados de Sequência Molecular , Proteínas de Ligação a RNA/análise , DNA Metiltransferase 3BRESUMO
One of the most challenging projects in the field of epigenetics is the generation of detailed functional maps of DNA methylation in different cell and tissue types in normal and disease-associated conditions. This information will help us not only understand the role of DNA methylation but also identify targets for therapeutic treatment. The completion of the various epigenome projects depends on the design of novel strategies to survey and generate detailed cartograms of the DNA methylome. Methyl-DNA immunoprecipitation (MeDIP) assays, in combination with hybridization on high-resolution microarrays or high-throughput sequencing (HTS) techniques, are excellent methods for identifying methylated CpG-rich sequences. We provide a critical overview of different genome-wide techniques for DNA methylation analysis and propose that MeDIP assays may constitute a key method for elucidating the hypermethylome of cancer cells.
Assuntos
Metilação de DNA , DNA/isolamento & purificação , DNA/metabolismo , Imunoprecipitação/métodos , Animais , Anticorpos Antinucleares , DNA/genética , Metilases de Modificação do DNA/metabolismo , DNA de Neoplasias/metabolismo , HumanosRESUMO
CpG island promoter hypermethylation of tumor suppressor genes is a common hallmark of human cancer, and new large-scale epigenomic technologies might be useful in our attempts to define the complete DNA hypermethylome of tumor cells. Here, we report a functional search for hypermethylated CpG islands using the colorectal cancer cell line HCT-116, in which two major DNA methyltransferases, DNMT1 and DNMT3b, have been genetically disrupted (DKO cells). Using methylated DNA immunoprecipitation methodology in conjunction with promoter microarray analyses, we found that DKO cells experience a significant loss of hypermethylated CpG islands. Further characterization of these candidate sequences shows CpG island promoter hypermethylation and silencing of genes with potentially important roles in tumorigenesis, such as the Ras guanine nucleotide-releasing factor (RASGRF2), the apoptosis-associated basic helix-loop transcription factor (BHLHB9), and the homeobox gene (HOXD1). Hypermethylation of these genes occurs in premalignant lesions and accumulates during tumorigenesis. Thus, our results show the usefulness of DNMT genetic disruption strategies combined with methylated DNA immunoprecipitation in searching for unknown hypermethylated candidate genes in human cancer that might aid our understanding of the biology of the disease and be of potential translational use.
Assuntos
Neoplasias do Colo/genética , Metilação de DNA , Inativação Gênica , Linhagem Celular Tumoral , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/deficiência , DNA (Citosina-5-)-Metiltransferases/genética , Deleção de Genes , Humanos , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , DNA Metiltransferase 3BRESUMO
Alkylation of DNA at the O(6)-position of guanine is one of the most critical events leading to mutation, cancer, and cell death. O(6)-alkylguanine-DNA alkyltransferase (AGT), also known as O(6)-methylguanine-DNA methyltransferase (MGMT), is the DNA repair protein responsible for removing alkylation adducts from the O(6)-position of guanine in DNA. The promoter CpG island hypermethylation-associated gene silencing of MGMT is associated with a wide spectrum of human tumors. This epigenetic inactivation of MGMT has two main consequences in human cancer. First, it uncovers a new mutator pathway that causes the accumulation of G-to-A transition mutations that can affect genes required for genomic stability. Second, there is a strong and significant positive correlation between MGMT promoter hypermethylation and increased tumor sensitivity to alkylating drugs. These findings underline the importance of MGMT promoter hypermethylation in basic and translational cancer research.
Assuntos
O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Alquilantes/toxicidade , Animais , Ilhas de CpG , Metilação de DNA , Reparo do DNA , Epigênese Genética , Inativação Gênica , Humanos , Mutação , Neoplasias/genética , Neoplasias/metabolismo , O(6)-Metilguanina-DNA Metiltransferase/antagonistas & inibidores , O(6)-Metilguanina-DNA Metiltransferase/genética , Prognóstico , Regiões Promotoras GenéticasRESUMO
Human cancers exhibit genomic instability and an increased mutation rate due to underlying defects in DNA repair genes. Hypermethylation of CpG islands in gene promoter regions is an important mechanism of gene inactivation in cancer. Many cellular pathways, including DNA repair, are inactivated by this type of epigenetic lesion, resulting in mutator pathways. In this review, we discuss the adverse consequences suffered by a cell when DNA repair genes such as the DNA mismatch repair gene hMLH1, the DNA alkyl-repair gene O(6)-methylguanine-DNA methyltransferase, the familial breast cancer gene BRCA1 and the Werner syndrome gene WRN become epigenetically silenced in human cancer.