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CGG-repeat dynamics and FMR1 gene silencing in fragile X syndrome stem cells and stem cell-derived neurons / Y. ZHOU in Molecular Autism, 7 (2016)
[article]
Titre : CGG-repeat dynamics and FMR1 gene silencing in fragile X syndrome stem cells and stem cell-derived neurons Type de document : Texte imprimé et/ou numérique Auteurs : Y. ZHOU, Auteur ; D. KUMARI, Auteur ; N. SCIASCIA, Auteur ; K. USDIN, Auteur Article en page(s) : 42p. Langues : Anglais (eng) Mots-clés : 5' Untranslated Regions Alleles Cell Differentiation Cell Line DNA Methylation Embryonic Stem Cells/metabolism/pathology Fragile X Mental Retardation Protein/genetics/metabolism Fragile X Syndrome/genetics/metabolism/pathology Gene Silencing Humans Induced Pluripotent Stem Cells/metabolism/pathology Male Neurons/metabolism/pathology Primary Cell Culture Time Factors Trinucleotide Repeat Expansion Fragile X syndrome Repeat contractions Repeat expansion mutation Repeat-mediated gene silencing Stem cells Index. décimale : PER Périodiques Résumé : BACKGROUND: Fragile X syndrome (FXS), a common cause of intellectual disability and autism, results from the expansion of a CGG-repeat tract in the 5' untranslated region of the FMR1 gene to >200 repeats. Such expanded alleles, known as full mutation (FM) alleles, are epigenetically silenced in differentiated cells thus resulting in the loss of FMRP, a protein important for learning and memory. The timing of repeat expansion and FMR1 gene silencing is controversial. METHODS: We monitored the repeat size and methylation status of FMR1 alleles with expanded CGG repeats in patient-derived induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) that were grown for extended period of time either as stem cells or differentiated into neurons. We used a PCR assay optimized for the amplification of large CGG repeats for sizing, and a quantitative methylation-specific PCR for the analysis of FMR1 promoter methylation. The FMR1 mRNA levels were analyzed by qRT-PCR. FMRP levels were determined by western blotting and immunofluorescence. Chromatin immunoprecipitation was used to study the association of repressive histone marks with the FMR1 gene in FXS ESCs. RESULTS: We show here that while FMR1 gene silencing can be seen in FXS embryonic stem cells (ESCs), some silenced alleles contract and when the repeat number drops below ~400, DNA methylation erodes, even when the repeat number remains >200. The resultant active alleles do not show the large step-wise expansions seen in stem cells from other repeat expansion diseases. Furthermore, there may be selection against large active alleles and these alleles do not expand further or become silenced on neuronal differentiation. CONCLUSIONS: Our data support the hypotheses that (i) large expansions occur prezygotically or in the very early embryo, (ii) large unmethylated alleles may be deleterious in stem cells, (iii) methylation can occur on alleles with >400 repeats very early in embryogenesis, and (iv) expansion and contraction may occur by different mechanisms. Our data also suggest that the threshold for stable methylation of FM alleles may be higher than previously thought. A higher threshold might explain why some carriers of FM alleles escape methylation. It may also provide a simple explanation for why silencing has not been observed in mouse models with >200 repeats. En ligne : http://dx.doi.org/10.1186/s13229-016-0105-9 Permalink : https://www.cra-rhone-alpes.org/cid/opac_css/index.php?lvl=notice_display&id=329
in Molecular Autism > 7 (2016) . - 42p.[article] CGG-repeat dynamics and FMR1 gene silencing in fragile X syndrome stem cells and stem cell-derived neurons [Texte imprimé et/ou numérique] / Y. ZHOU, Auteur ; D. KUMARI, Auteur ; N. SCIASCIA, Auteur ; K. USDIN, Auteur . - 42p.
Langues : Anglais (eng)
in Molecular Autism > 7 (2016) . - 42p.
Mots-clés : 5' Untranslated Regions Alleles Cell Differentiation Cell Line DNA Methylation Embryonic Stem Cells/metabolism/pathology Fragile X Mental Retardation Protein/genetics/metabolism Fragile X Syndrome/genetics/metabolism/pathology Gene Silencing Humans Induced Pluripotent Stem Cells/metabolism/pathology Male Neurons/metabolism/pathology Primary Cell Culture Time Factors Trinucleotide Repeat Expansion Fragile X syndrome Repeat contractions Repeat expansion mutation Repeat-mediated gene silencing Stem cells Index. décimale : PER Périodiques Résumé : BACKGROUND: Fragile X syndrome (FXS), a common cause of intellectual disability and autism, results from the expansion of a CGG-repeat tract in the 5' untranslated region of the FMR1 gene to >200 repeats. Such expanded alleles, known as full mutation (FM) alleles, are epigenetically silenced in differentiated cells thus resulting in the loss of FMRP, a protein important for learning and memory. The timing of repeat expansion and FMR1 gene silencing is controversial. METHODS: We monitored the repeat size and methylation status of FMR1 alleles with expanded CGG repeats in patient-derived induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) that were grown for extended period of time either as stem cells or differentiated into neurons. We used a PCR assay optimized for the amplification of large CGG repeats for sizing, and a quantitative methylation-specific PCR for the analysis of FMR1 promoter methylation. The FMR1 mRNA levels were analyzed by qRT-PCR. FMRP levels were determined by western blotting and immunofluorescence. Chromatin immunoprecipitation was used to study the association of repressive histone marks with the FMR1 gene in FXS ESCs. RESULTS: We show here that while FMR1 gene silencing can be seen in FXS embryonic stem cells (ESCs), some silenced alleles contract and when the repeat number drops below ~400, DNA methylation erodes, even when the repeat number remains >200. The resultant active alleles do not show the large step-wise expansions seen in stem cells from other repeat expansion diseases. Furthermore, there may be selection against large active alleles and these alleles do not expand further or become silenced on neuronal differentiation. CONCLUSIONS: Our data support the hypotheses that (i) large expansions occur prezygotically or in the very early embryo, (ii) large unmethylated alleles may be deleterious in stem cells, (iii) methylation can occur on alleles with >400 repeats very early in embryogenesis, and (iv) expansion and contraction may occur by different mechanisms. Our data also suggest that the threshold for stable methylation of FM alleles may be higher than previously thought. A higher threshold might explain why some carriers of FM alleles escape methylation. It may also provide a simple explanation for why silencing has not been observed in mouse models with >200 repeats. En ligne : http://dx.doi.org/10.1186/s13229-016-0105-9 Permalink : https://www.cra-rhone-alpes.org/cid/opac_css/index.php?lvl=notice_display&id=329 CRISPR/Cas9-mediated heterozygous knockout of the autism gene CHD8 and characterization of its transcriptional networks in cerebral organoids derived from iPS cells / P. WANG in Molecular Autism, 8 (2017)
[article]
Titre : CRISPR/Cas9-mediated heterozygous knockout of the autism gene CHD8 and characterization of its transcriptional networks in cerebral organoids derived from iPS cells Type de document : Texte imprimé et/ou numérique Auteurs : P. WANG, Auteur ; R. MOKHTARI, Auteur ; E. PEDROSA, Auteur ; M. KIRSCHENBAUM, Auteur ; C. BAYRAK, Auteur ; D. ZHENG, Auteur ; H. M. LACHMAN, Auteur Article en page(s) : 11p. Langues : Anglais (eng) Mots-clés : Autism Spectrum Disorder/genetics Bipolar Disorder/genetics CRISPR-Cas Systems Cell Differentiation Cells, Cultured DNA-Binding Proteins/*genetics Gene Expression Profiling/*methods Gene Expression Regulation Gene Knockout Techniques *Gene Regulatory Networks Humans Induced Pluripotent Stem Cells/*cytology Mental Disorders/*genetics Mutation Organoids/*cytology Schizophrenia/genetics Sequence Analysis, RNA/*methods Telencephalon/*cytology Transcription Factors/*genetics *Autism *Beta-catenin *Bipolar disorder *Cancer *Dlx6-as1 *Distal-less homeobox *Gabaergic *Hmga2 *Schizophrenia *Tcf4 *Wnt *Znf132 Index. décimale : PER Périodiques Résumé : BACKGROUND: CHD8 (chromodomain helicase DNA-binding protein 8), which codes for a member of the CHD family of ATP-dependent chromatin-remodeling factors, is one of the most commonly mutated genes in autism spectrum disorders (ASD) identified in exome-sequencing studies. Loss of function mutations in the gene have also been found in schizophrenia (SZ) and intellectual disabilities and influence cancer cell proliferation. We previously reported an RNA-seq analysis carried out on neural progenitor cells (NPCs) and monolayer neurons derived from induced pluripotent stem (iPS) cells that were heterozygous for CHD8 knockout (KO) alleles generated using CRISPR-Cas9 gene editing. A significant number of ASD and SZ candidate genes were among those that were differentially expressed in a comparison of heterozygous KO lines (CHD8(+/-)) vs isogenic controls (CHD8(+/-)), including the SZ and bipolar disorder (BD) candidate gene TCF4, which was markedly upregulated in CHD8(+/-) neuronal cells. METHODS: In the current study, RNA-seq was carried out on CHD8(+/-) and isogenic control (CHD8(+/+)) cerebral organoids, which are 3-dimensional structures derived from iPS cells that model the developing human telencephalon. RESULTS: TCF4 expression was, again, significantly upregulated. Pathway analysis carried out on differentially expressed genes (DEGs) revealed an enrichment of genes involved in neurogenesis, neuronal differentiation, forebrain development, Wnt/beta-catenin signaling, and axonal guidance, similar to our previous study on NPCs and monolayer neurons. There was also significant overlap in our CHD8(+/-) DEGs with those found in a transcriptome analysis carried out by another group using cerebral organoids derived from a family with idiopathic ASD. Remarkably, the top DEG in our respective studies was the non-coding RNA DLX6-AS1, which was markedly upregulated in both studies; DLX6-AS1 regulates the expression of members of the DLX (distal-less homeobox) gene family. DLX1 was also upregulated in both studies. DLX genes code for transcription factors that play a key role in GABAergic interneuron differentiation. Significant overlap was also found in a transcriptome study carried out by another group using iPS cell-derived neurons from patients with BD, a condition characterized by dysregulated WNT/beta-catenin signaling in a subgroup of affected individuals. CONCLUSIONS: Overall, the findings show that distinct ASD, SZ, and BD candidate genes converge on common molecular targets-an important consideration for developing novel therapeutics in genetically heterogeneous complex traits. En ligne : http://dx.doi.org/10.1186/s13229-017-0124-1 Permalink : https://www.cra-rhone-alpes.org/cid/opac_css/index.php?lvl=notice_display&id=331
in Molecular Autism > 8 (2017) . - 11p.[article] CRISPR/Cas9-mediated heterozygous knockout of the autism gene CHD8 and characterization of its transcriptional networks in cerebral organoids derived from iPS cells [Texte imprimé et/ou numérique] / P. WANG, Auteur ; R. MOKHTARI, Auteur ; E. PEDROSA, Auteur ; M. KIRSCHENBAUM, Auteur ; C. BAYRAK, Auteur ; D. ZHENG, Auteur ; H. M. LACHMAN, Auteur . - 11p.
Langues : Anglais (eng)
in Molecular Autism > 8 (2017) . - 11p.
Mots-clés : Autism Spectrum Disorder/genetics Bipolar Disorder/genetics CRISPR-Cas Systems Cell Differentiation Cells, Cultured DNA-Binding Proteins/*genetics Gene Expression Profiling/*methods Gene Expression Regulation Gene Knockout Techniques *Gene Regulatory Networks Humans Induced Pluripotent Stem Cells/*cytology Mental Disorders/*genetics Mutation Organoids/*cytology Schizophrenia/genetics Sequence Analysis, RNA/*methods Telencephalon/*cytology Transcription Factors/*genetics *Autism *Beta-catenin *Bipolar disorder *Cancer *Dlx6-as1 *Distal-less homeobox *Gabaergic *Hmga2 *Schizophrenia *Tcf4 *Wnt *Znf132 Index. décimale : PER Périodiques Résumé : BACKGROUND: CHD8 (chromodomain helicase DNA-binding protein 8), which codes for a member of the CHD family of ATP-dependent chromatin-remodeling factors, is one of the most commonly mutated genes in autism spectrum disorders (ASD) identified in exome-sequencing studies. Loss of function mutations in the gene have also been found in schizophrenia (SZ) and intellectual disabilities and influence cancer cell proliferation. We previously reported an RNA-seq analysis carried out on neural progenitor cells (NPCs) and monolayer neurons derived from induced pluripotent stem (iPS) cells that were heterozygous for CHD8 knockout (KO) alleles generated using CRISPR-Cas9 gene editing. A significant number of ASD and SZ candidate genes were among those that were differentially expressed in a comparison of heterozygous KO lines (CHD8(+/-)) vs isogenic controls (CHD8(+/-)), including the SZ and bipolar disorder (BD) candidate gene TCF4, which was markedly upregulated in CHD8(+/-) neuronal cells. METHODS: In the current study, RNA-seq was carried out on CHD8(+/-) and isogenic control (CHD8(+/+)) cerebral organoids, which are 3-dimensional structures derived from iPS cells that model the developing human telencephalon. RESULTS: TCF4 expression was, again, significantly upregulated. Pathway analysis carried out on differentially expressed genes (DEGs) revealed an enrichment of genes involved in neurogenesis, neuronal differentiation, forebrain development, Wnt/beta-catenin signaling, and axonal guidance, similar to our previous study on NPCs and monolayer neurons. There was also significant overlap in our CHD8(+/-) DEGs with those found in a transcriptome analysis carried out by another group using cerebral organoids derived from a family with idiopathic ASD. Remarkably, the top DEG in our respective studies was the non-coding RNA DLX6-AS1, which was markedly upregulated in both studies; DLX6-AS1 regulates the expression of members of the DLX (distal-less homeobox) gene family. DLX1 was also upregulated in both studies. DLX genes code for transcription factors that play a key role in GABAergic interneuron differentiation. Significant overlap was also found in a transcriptome study carried out by another group using iPS cell-derived neurons from patients with BD, a condition characterized by dysregulated WNT/beta-catenin signaling in a subgroup of affected individuals. CONCLUSIONS: Overall, the findings show that distinct ASD, SZ, and BD candidate genes converge on common molecular targets-an important consideration for developing novel therapeutics in genetically heterogeneous complex traits. En ligne : http://dx.doi.org/10.1186/s13229-017-0124-1 Permalink : https://www.cra-rhone-alpes.org/cid/opac_css/index.php?lvl=notice_display&id=331 Autism-associated protein POGZ controls ESCs and ESC neural induction by association with esBAF / Xiaoyun SUN in Molecular Autism, 13 (2022)
[article]
Titre : Autism-associated protein POGZ controls ESCs and ESC neural induction by association with esBAF Type de document : Texte imprimé et/ou numérique Auteurs : Xiaoyun SUN, Auteur ; Linxi CHENG, Auteur ; Yuhua SUN, Auteur Article en page(s) : 24 p. Langues : Anglais (eng) Mots-clés : Animals Autism Spectrum Disorder/genetics Autistic Disorder Cell Differentiation Embryonic Development Embryonic Stem Cells/metabolism Transcription Factors/genetics/metabolism Index. décimale : PER Périodiques Résumé : BACKGROUND: The POGZ gene has been found frequently mutated in neurodevelopmental disorders (NDDs), particularly autism spectrum disorder (ASD) and intellectual disability (ID). However, little is known about its roles in embryonic stem cells (ESCs), neural development and diseases. METHODS: We generated Pogz-/- ESCs and directed ESC differentiation toward a neural fate. We performed biochemistry, ChIP-seq, ATAC-seq, and bioinformatics analyses to understand the role of POGZ. RESULTS: We show that POGZ is required for the maintenance of ESC identity and the up-regulation of neural genes during ESC differentiation toward a neural fate. Genome-wide binding analysis shows that POGZ is primarily localized to gene promoter and enhancer regions. POGZ functions as both a transcriptional activator and repressor, and its loss leads to deregulation of differentiation genes, including neural genes. POGZ physically associates with the SWI-SNF (esBAF) chromatin remodeler complex, and together they modulate enhancer activities via epigenetic modifications such as chromatin remodeling and histone modification. During ESC neural induction, POGZ-mediated recruitment of esBAF/BRG1 and H3K27ac are important for proper expression of neural progenitor genes. LIMITATIONS: The genotype and allele relevant to human neurodevelopmental disorders is heterozygous loss of function. This work is designed to study the effects of loss of POGZ function on ESCs and during ESC neural induction. Also, this work lacks of in vivo validation using animal models. CONCLUSIONS: The data suggest that POGZ is both a transcription factor and a genome regulator, and its loss leads to defects in neural induction and neurogenesis. En ligne : http://dx.doi.org/10.1186/s13229-022-00502-9 Permalink : https://www.cra-rhone-alpes.org/cid/opac_css/index.php?lvl=notice_display&id=491
in Molecular Autism > 13 (2022) . - 24 p.[article] Autism-associated protein POGZ controls ESCs and ESC neural induction by association with esBAF [Texte imprimé et/ou numérique] / Xiaoyun SUN, Auteur ; Linxi CHENG, Auteur ; Yuhua SUN, Auteur . - 24 p.
Langues : Anglais (eng)
in Molecular Autism > 13 (2022) . - 24 p.
Mots-clés : Animals Autism Spectrum Disorder/genetics Autistic Disorder Cell Differentiation Embryonic Development Embryonic Stem Cells/metabolism Transcription Factors/genetics/metabolism Index. décimale : PER Périodiques Résumé : BACKGROUND: The POGZ gene has been found frequently mutated in neurodevelopmental disorders (NDDs), particularly autism spectrum disorder (ASD) and intellectual disability (ID). However, little is known about its roles in embryonic stem cells (ESCs), neural development and diseases. METHODS: We generated Pogz-/- ESCs and directed ESC differentiation toward a neural fate. We performed biochemistry, ChIP-seq, ATAC-seq, and bioinformatics analyses to understand the role of POGZ. RESULTS: We show that POGZ is required for the maintenance of ESC identity and the up-regulation of neural genes during ESC differentiation toward a neural fate. Genome-wide binding analysis shows that POGZ is primarily localized to gene promoter and enhancer regions. POGZ functions as both a transcriptional activator and repressor, and its loss leads to deregulation of differentiation genes, including neural genes. POGZ physically associates with the SWI-SNF (esBAF) chromatin remodeler complex, and together they modulate enhancer activities via epigenetic modifications such as chromatin remodeling and histone modification. During ESC neural induction, POGZ-mediated recruitment of esBAF/BRG1 and H3K27ac are important for proper expression of neural progenitor genes. LIMITATIONS: The genotype and allele relevant to human neurodevelopmental disorders is heterozygous loss of function. This work is designed to study the effects of loss of POGZ function on ESCs and during ESC neural induction. Also, this work lacks of in vivo validation using animal models. CONCLUSIONS: The data suggest that POGZ is both a transcription factor and a genome regulator, and its loss leads to defects in neural induction and neurogenesis. En ligne : http://dx.doi.org/10.1186/s13229-022-00502-9 Permalink : https://www.cra-rhone-alpes.org/cid/opac_css/index.php?lvl=notice_display&id=491