RESUMO
NOTCH pathway proteins, including the transcriptional factor HES1, play crucial roles in the development of the inner ear by means of the lateral inhibition mechanism, in which supporting cells have their phenotype preserved while they are prevented from becoming hair cells. Genetic manipulation of this pathway has been demonstrated to increase hair cell number. The present study aimed to investigate gene expression effects in hair cells and supporting cells after Hes1-shRNA lentivirus transduction in organotypic cultures of the organ of Corti from postnatal-day-3 mice. Forty-eight hours after in vitro knockdown, Hes1 gene expression was reduced at both mRNA and protein levels. Myo7a (hair cell marker) and Sox2 (progenitor cell marker) mRNA levels also significantly increased. The modulation of gene expression in the organ of Corti upon Hes1 knockdown is consistent with cell phenotypes related to lateral inhibition mechanism interference in the inner ear. The lentivirus-based expression of Hes1-shRNA is a valuable strategy for genetic interference in the organ of Corti and for future evaluation of its efficacy in protocols aiming at the regeneration of hair cells in vivo.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Cóclea , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular , Células Ciliadas Auditivas , Camundongos , Órgão Espiral , Receptores Notch , Fatores de Transcrição HES-1/genéticaRESUMO
NOTCH pathway proteins, including the transcriptional factor HES1, play crucial roles in the development of the inner ear by means of the lateral inhibition mechanism, in which supporting cells have their phenotype preserved while they are prevented from becoming hair cells. Genetic manipulation of this pathway has been demonstrated to increase hair cell number. The present study aimed to investigate gene expression effects in hair cells and supporting cells after Hes1-shRNA lentivirus transduction in organotypic cultures of the organ of Corti from postnatal-day-3 mice. Forty-eight hours after in vitro knockdown, Hes1 gene expression was reduced at both mRNA and protein levels. Myo7a (hair cell marker) and Sox2 (progenitor cell marker) mRNA levels also significantly increased. The modulation of gene expression in the organ of Corti upon Hes1 knockdown is consistent with cell phenotypes related to lateral inhibition mechanism interference in the inner ear. The lentivirus-based expression of Hes1-shRNA is a valuable strategy for genetic interference in the organ of Corti and for future evaluation of its efficacy in protocols aiming at the regeneration of hair cells in vivo.
Assuntos
Animais , Ratos , Cóclea , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Órgão Espiral , Diferenciação Celular , Receptores Notch , Fatores de Transcrição HES-1/genética , Células Ciliadas AuditivasRESUMO
DNA methylation is essential in X chromosome inactivation and genomic imprinting, maintaining repression of XIST in the active X chromosome and monoallelic repression of imprinted genes. Disruption of the DNA methyltransferase genes DNMT1 and DNMT3B in the HCT116 cell line (DKO cells) leads to global DNA hypomethylation and biallelic expression of the imprinted gene IGF2 but does not lead to reactivation of XIST expression, suggesting that XIST repression is due to a more stable epigenetic mark than imprinting. To test this hypothesis, we induced acute hypomethylation in HCT116 cells by 5-aza-2′-deoxycytidine (5-aza-CdR) treatment (HCT116-5-aza-CdR) and compared that to DKO cells, evaluating DNA methylation by microarray and monitoring the expression of XIST and imprinted genes IGF2, H19, and PEG10. Whereas imprinted genes showed biallelic expression in HCT116-5-aza-CdR and DKO cells, the XIST locus was hypomethylated and weakly expressed only under acute hypomethylation conditions, indicating the importance of XIST repression in the active X to cell survival. Given that DNMT3A is the only active DNMT in DKO cells, it may be responsible for ensuring the repression of XIST in those cells. Taken together, our data suggest that XIST repression is more tightly controlled than genomic imprinting and, at least in part, is due to DNMT3A.
Assuntos
Humanos , Metilação de DNA/genética , Repressão Epigenética/genética , Genoma Humano , Genoma/genética , Impressão Genômica/genética , Fator de Crescimento Insulin-Like II/genética , RNA Longo não Codificante/genética , Azacitidina/administração & dosagem , Azacitidina/análogos & derivados , /genética , Metilação de DNA/efeitos dos fármacos , Técnicas de Inativação de Genes , Genoma Humano/efeitos dos fármacos , Hibridização in Situ Fluorescente/métodos , Análise em Microsséries , Polimorfismo de Nucleotídeo Único , Proteínas/metabolismo , RNA Longo não Codificante/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosRESUMO
DNA methylation is essential in X chromosome inactivation and genomic imprinting, maintaining repression of XIST in the active X chromosome and monoallelic repression of imprinted genes. Disruption of the DNA methyltransferase genes DNMT1 and DNMT3B in the HCT116 cell line (DKO cells) leads to global DNA hypomethylation and biallelic expression of the imprinted gene IGF2 but does not lead to reactivation of XIST expression, suggesting that XIST repression is due to a more stable epigenetic mark than imprinting. To test this hypothesis, we induced acute hypomethylation in HCT116 cells by 5-aza-2'-deoxycytidine (5-aza-CdR) treatment (HCT116-5-aza-CdR) and compared that to DKO cells, evaluating DNA methylation by microarray and monitoring the expression of XIST and imprinted genes IGF2, H19, and PEG10. Whereas imprinted genes showed biallelic expression in HCT116-5-aza-CdR and DKO cells, the XIST locus was hypomethylated and weakly expressed only under acute hypomethylation conditions, indicating the importance of XIST repression in the active X to cell survival. Given that DNMT3A is the only active DNMT in DKO cells, it may be responsible for ensuring the repression of XIST in those cells. Taken together, our data suggest that XIST repression is more tightly controlled than genomic imprinting and, at least in part, is due to DNMT3A.
Assuntos
Metilação de DNA/genética , Repressão Epigenética/genética , Genoma Humano , Genoma/genética , Impressão Genômica/genética , Fator de Crescimento Insulin-Like II/genética , RNA Longo não Codificante/genética , Proteínas Reguladoras de Apoptose , Azacitidina/administração & dosagem , Azacitidina/análogos & derivados , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/efeitos dos fármacos , Proteínas de Ligação a DNA , Decitabina , Técnicas de Inativação de Genes , Genoma Humano/efeitos dos fármacos , Células HCT116 , Humanos , Hibridização in Situ Fluorescente/métodos , Análise em Microsséries , Polimorfismo de Nucleotídeo Único , Proteínas/metabolismo , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , DNA Metiltransferase 3BRESUMO
Mammals perform dosage compensation of X-linked gene products between XY males and XX females by transcriptionally silencing all but one X chromosome per diploid cell, a process called X chromosome inactivation (XCI). XCI involves counting X chromosomes in a cell, random or imprinted choice of one X to remain active, initiation and spread of the inactivation signal in CIS throughout the other X chromosomes, and maintenance of the inactive state of those X chromosomes during cell divisions thereafter. Most of what is known of the molecular mechanisms involved in the different steps of XCI has been studied in the mouse. In this review we compare XCI in mouse and human, and discuss how much of the murine data can be extrapolated to humans.
Assuntos
Mecanismo Genético de Compensação de Dose , Cromossomo X/genética , Animais , Metilação de DNA , Humanos , Camundongos , RNA Longo não Codificante , RNA não Traduzido/genética , Fatores de Transcrição/genéticaRESUMO
In mammals, dosage compensation at X-linked loci is achieved by the process of X chromosome inactivation in the homogametic sex. While most genes on the inactive X chromosome (Xi) are subjected to transcriptional inactivation, some escape inactivation and present biallelic expression. The expression status of X-linked genes has been extensively studied in somatic cell hybrids containing only the human Xi. Although this approach has recently been used to generate a profile of X-linked gene activity, it may not reflect what happens in a normal human cell. The recent development of a database of single nucleotide polymorphisms (SNPs) throughout the human genome enables investigation of allele-specific gene expression in normal human cells. In this study, we established a panel of X-linked expressed SNPs (cSNPs). These markers were used for monitoring gene expression in primary human fibroblast cell lines with completely skewed XCI, demonstrating the potential of this system for studying X-linked gene expression in normal human cells.
Assuntos
Alelos , Polimorfismo de Nucleotídeo Único , Cromossomo X/genética , Sequência de Bases , Linhagem Celular , DNA Complementar/genética , Mecanismo Genético de Compensação de Dose , Feminino , Expressão Gênica , Ligação Genética , Marcadores Genéticos , Genótipo , Heterozigoto , Humanos , Hipoxantina Fosforribosiltransferase/genética , MutaçãoRESUMO
X-chromosome inactivation (XCI) is the process by which mammals perform dosage compensation of X-linked gene products between XY males and XX females, resulting in the transcriptional silencing of all but one X chromosome per diploid cell. XCI involves counting the X chromosomes in a cell, randomly choosing those to be inactivated, spreading the inactivation signal in cis throughout the chromosome, and maintaining the inactive state of those X chromosomes during cell divisions thereafter. How the cell performs all these tasks is a fascinating problem and, together with epigenetic inheritance, a basic cellular mechanism that remains to be fully understood. In this review, we describe recent experiments aimed at understanding the first events of XCI and propose a model for initiation of XCI.
