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1.
Cell ; 172(5): 979-992.e6, 2018 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-29456084

RESUMO

Fragile X syndrome (FXS), the most common genetic form of intellectual disability in males, is caused by silencing of the FMR1 gene associated with hypermethylation of the CGG expansion mutation in the 5' UTR of FMR1 in FXS patients. Here, we applied recently developed DNA methylation editing tools to reverse this hypermethylation event. Targeted demethylation of the CGG expansion by dCas9-Tet1/single guide RNA (sgRNA) switched the heterochromatin status of the upstream FMR1 promoter to an active chromatin state, restoring a persistent expression of FMR1 in FXS iPSCs. Neurons derived from methylation-edited FXS iPSCs rescued the electrophysiological abnormalities and restored a wild-type phenotype upon the mutant neurons. FMR1 expression in edited neurons was maintained in vivo after engrafting into the mouse brain. Finally, demethylation of the CGG repeats in post-mitotic FXS neurons also reactivated FMR1. Our data establish that demethylation of the CGG expansion is sufficient for FMR1 reactivation, suggesting potential therapeutic strategies for FXS.


Assuntos
Metilação de DNA/genética , Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/genética , Edição de Genes , Neurônios/patologia , Animais , Proteína 9 Associada à CRISPR/metabolismo , Epigênese Genética , Células HEK293 , Heterocromatina/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Cinética , Masculino , Camundongos , Neurônios/metabolismo , Fenótipo , Regiões Promotoras Genéticas , RNA Guia de Cinetoplastídeos/metabolismo , Expansão das Repetições de Trinucleotídeos/genética
2.
Hum Mol Genet ; 31(17): 2899-2917, 2022 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-35394024

RESUMO

Cellular proliferation depends on the accurate and timely replication of the genome. Several genetic diseases are caused by mutations in key DNA replication genes; however, it remains unclear whether these genes influence the normal program of DNA replication timing. Similarly, the factors that regulate DNA replication dynamics are poorly understood. To systematically identify trans-acting modulators of replication timing, we profiled replication in 184 cell lines from three cell types, encompassing 60 different gene knockouts or genetic diseases. Through a rigorous approach that considers the background variability of replication timing, we concluded that most samples displayed normal replication timing. However, mutations in two genes showed consistently abnormal replication timing. The first gene was RIF1, a known modulator of replication timing. The second was MCM10, a highly conserved member of the pre-replication complex. Cells from a single patient carrying MCM10 mutations demonstrated replication timing variability comprising 46% of the genome and at different locations than RIF1 knockouts. Replication timing alterations in the mutated MCM10 cells were predominantly comprised of replication delays and initiation site gains and losses. Taken together, this study demonstrates the remarkable robustness of the human replication timing program and reveals MCM10 as a novel candidate modulator of DNA replication timing.


Assuntos
Período de Replicação do DNA , Proteínas de Manutenção de Minicromossomo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Replicação do DNA/genética , Período de Replicação do DNA/genética , Humanos , Proteínas de Manutenção de Minicromossomo/genética , Origem de Replicação
3.
Genome Res ; 31(12): 2155-2169, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34810218

RESUMO

Haploid human embryonic stem cells (ESCs) provide a powerful genetic system but diploidize at high rates. We hypothesized that diploidization results from aberrant DNA replication. To test this, we profiled DNA replication timing in isogenic haploid and diploid ESCs. The greatest difference was the earlier replication of the X Chromosome in haploids, consistent with the lack of X-Chromosome inactivation. We also identified 21 autosomal regions that had delayed replication in haploids, extending beyond the normal S phase and into G2/M. Haploid-delays comprised a unique set of quiescent genomic regions that are also underreplicated in polyploid placental cells. The same delays were observed in female ESCs with two active X Chromosomes, suggesting that increased X-Chromosome dosage may cause delayed autosomal replication. We propose that incomplete replication at the onset of mitosis could prevent cell division and result in re-entry into the cell cycle and whole genome duplication.

4.
Brain Behav Immun ; 96: 40-53, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33989746

RESUMO

Brain stimulation by electroconvulsive therapy is effective in neuropsychiatric disorders by unknown mechanisms. Microglial toxicity plays key role in neuropsychiatric, neuroinflammatory and degenerative diseases. We examined the mechanism by which electroconvulsive seizures (ECS) regulates microglial phenotype and response to stimuli. Microglial responses were examined by morphological analysis, Iba1 and cytokine expression. ECS did not affect resting microglial phenotype or morphology but regulated their activation by Lipopolysaccharide stimulation. Microglia were isolated after ECS or sham sessions in naïve mice for transcriptome analysis. RNA sequencing identified 141 differentially expressed genes. ECS modulated multiple immune-associated gene families and attenuated neurotoxicity-associated gene expression. Blood brain barrier was examined by injecting Biocytin-TMR tracer. There was no breakdown of the BBB, nor increase in gene-signature of peripheral monocytes, suggesting that ECS effect is mainly on resident microglia. Unbiased analysis of regulatory sequences identified the induction of microglial retinoic acid receptor α (RARα) gene expression and a putative common RARα-binding motif in multiple ECS-upregulated genes. The effects of AM580, a selective RARα agonist on microglial response to LPS was examined in vitro. AM580 prevented LPS-induced cytokine expression and reactive oxygen species production. Chronic murine experimental autoimmune encephalomyelitis (EAE) was utilized to confirm the role RARα signaling as mediator of ECS-induced transcriptional pathway in regulating microglial toxicity. Continuous intracerebroventricular delivery of AM580 attenuated effectively EAE severity. In conclusion, ECS regulates CNS innate immune system responses by activating microglial retinoic acid receptor α pathway, signifying a novel therapeutic approach for chronic neuroinflammatory, neuropsychiatric and neurodegenerative diseases.


Assuntos
Encefalomielite Autoimune Experimental , Microglia , Receptor alfa de Ácido Retinoico , Animais , Eletroconvulsoterapia , Lipopolissacarídeos , Camundongos , Transdução de Sinais
5.
PLoS Genet ; 13(8): e1006979, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28837588

RESUMO

Human pluripotent stem cells (hPSCs) are an important player in disease modeling and regenerative medicine. Nonetheless, multiple studies uncovered their inherent genetic instability upon prolonged culturing, where specific chromosomal aberrations provide cells with a growth advantage. These positively selected modifications have dramatic effects on multiple cellular characteristics. Epigenetic aberrations also possess the potential of changing gene expression and altering cellular functions. In the current study we assessed the landscape of DNA methylation aberrations during prolonged culturing of hPSCs, and defined a set of genes which are recurrently hypermethylated and silenced. We further focused on one of these genes, testis-specific Y-encoded like protein 5 (TSPYL5), and demonstrated that when silenced, differentiation-related genes and tumor-suppressor genes are downregulated, while pluripotency- and growth promoting genes are upregulated. This process is similar to the hypermethylation-mediated inactivation of certain genes during tumor development. Our analysis highlights the existence and importance of recurrent epigenetic aberrations in hPSCs during prolonged culturing.


Assuntos
Aberrações Cromossômicas , Epigênese Genética/genética , Instabilidade Genômica/genética , Células-Tronco Pluripotentes/citologia , Técnicas de Cultura de Células , Diferenciação Celular/genética , Metilação de DNA/genética , Regulação da Expressão Gênica/genética , Humanos , Medicina Regenerativa
6.
Stem Cell Reports ; 17(5): 1048-1058, 2022 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-35427485

RESUMO

Fragile X syndrome (FXS), the most prevalent heritable form of intellectual disability, is caused by the transcriptional silencing of the FMR1 gene. The epigenetic factors responsible for FMR1 inactivation are largely unknown. Here, we initially demonstrated the feasibility of FMR1 reactivation by targeting a single epigenetic factor, DNMT1. Next, we established a model system for FMR1 silencing using a construct containing the FXS-related mutation upstream to a reporter gene. This construct was methylated in vitro and introduced into a genome-wide loss-of-function (LOF) library established in haploid human pluripotent stem cells (PSCs), allowing the identification of genes whose functional loss reversed the methylation-induced silencing of the FMR1 reporter. Selected candidate genes were further analyzed in haploid- and FXS-patient-derived PSCs, highlighting the epigenetic and metabolic pathways involved in FMR1 regulation. Our work sheds light on the mechanisms responsible for CGG-expansion-mediated FMR1 inactivation and offers novel targets for therapeutic FMR1 reactivation.


Assuntos
Síndrome do Cromossomo X Frágil , Metilação de DNA/genética , Epigênese Genética , Epigenômica , Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Síndrome do Cromossomo X Frágil/genética , Humanos
7.
Nat Commun ; 12(1): 6718, 2021 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-34795250

RESUMO

In mammals, imprinted genes are regulated by differentially methylated regions (DMRs) that are inherited from germ cells, leading to monoallelic expression in accordance with parent-of-origin. Yet, it is largely unknown how imprinted DMRs are maintained in human embryos despite global DNA demethylation following fertilization. Here, we explored the mechanisms involved in imprinting regulation by employing human parthenogenetic embryonic stem cells (hpESCs), which lack paternal alleles. We show that although global loss of DNA methylation in hpESCs affects most imprinted DMRs, many paternally-expressed genes (PEGs) remain repressed. To search for factors regulating PEGs, we performed a genome-wide CRISPR/Cas9 screen in haploid hpESCs. This revealed ATF7IP as an essential repressor of a set of PEGs, which we further show is also required for silencing sperm-specific genes. Our study reinforces an important role for histone modifications in regulating imprinted genes and suggests a link between parental imprinting and germ cell identity.


Assuntos
Sistemas CRISPR-Cas , Regulação da Expressão Gênica , Impressão Genômica , Haploidia , Células-Tronco Embrionárias Humanas/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Células Cultivadas , Sequenciamento de Cromatina por Imunoprecipitação/métodos , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Células-Tronco Embrionárias Humanas/citologia , Humanos , Sistema de Sinalização das MAP Quinases/genética , Masculino , Partenogênese/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Espermatogênese/genética
8.
Cell Rep ; 26(10): 2531-2539.e4, 2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30840878

RESUMO

Fragile X syndrome (FXS) is caused primarily by a CGG repeat expansion in the FMR1 gene that triggers its transcriptional silencing. In order to investigate the regulatory layers involved in FMR1 inactivation, we tested a collection of chromatin modulators for the ability to reactivate the FMR1 locus. Although inhibitors of DNA methyltransferase (DNMT) induced the highest levels of FMR1 expression, a combination of a DNMT inhibitor and another compound potentiated the effect of reactivating treatment. To better assess the rescue effect following direct demethylation, we characterized the long-term and genome-wide effects of FMR1 reactivation and established an in vivo system to analyze FMR1-reactivating therapies. Systemic treatment with a DNMT inhibitor in mice carrying FXS induced pluripotent stem cell (iPSC)-derived transplants robustly induced FMR1 expression in the affected tissue, which was maintained for a prolonged period of time. Finally, we show a proof of principle for FMR1-reactivating therapy in the context of the CNS.


Assuntos
Inibidores Enzimáticos/farmacologia , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Síndrome do Cromossomo X Frágil/tratamento farmacológico , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Animais , Metilases de Modificação do DNA/antagonistas & inibidores , Metilases de Modificação do DNA/metabolismo , Feminino , Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/metabolismo , Síndrome do Cromossomo X Frágil/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neurais
9.
Regen Med ; 12(1): 53-68, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27900874

RESUMO

Human pluripotent stem cells (PSCs) generated from affected blastocysts or from patient-derived somatic cells are an emerging platform for disease modeling and drug discovery. Fragile X syndrome (FXS), the leading cause of inherited intellectual disability, was one of the first disorders modeled in both embryonic stem cells and induced PCSs and can serve as an exemplary case for the utilization of human PSCs in the study of human diseases. Over the past decade, FXS-PSCs have been used to address the fundamental questions regarding the pathophysiology of FXS. In this review we summarize the methodologies for generation of FXS-PSCs, discuss their advantages and disadvantages compared with existing modeling systems and describe their utilization in the study of FXS pathogenesis and in the development of targeted treatment.


Assuntos
Síndrome do Cromossomo X Frágil/patologia , Células-Tronco Pluripotentes/patologia , Humanos
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