RESUMO
In this report we demonstrate primary non-random X chromosome inactivation following targeted mutagenesis of a region immediately upstream of XIST promoter P(1). In heterozygous animals there is a preferential inactivation of the targeted X chromosome in 80--90% of cells. The phenotype correlates with inappropriate activation of XIST in a proportion of the mutant XY embryonic stem cells. Strand-specific analysis revealed increased sense transcription initiating upstream of XIST promoter P(1). There was, however, no discernible effect on transcription from the antisense Tsix gene. We demonstrate that the in vitro and in vivo phenotypes are specifically attributable to the presence of a PGKneo cassette at the targeted locus. These findings are discussed in the context of understanding mechanisms of XIST gene regulation in X inactivation.
Assuntos
Mecanismo Genético de Compensação de Dose , Regiões Promotoras Genéticas/genética , RNA não Traduzido/genética , Fatores de Transcrição/genética , Alelos , Animais , Células Cultivadas , Deleção de Genes , Camundongos , Mutagênese , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacologia , Fenótipo , RNA Longo não Codificante , RNA não Traduzido/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidoresRESUMO
Developmental regulation of the mouse Xist gene at the onset of X chromosome inactivation is mediated by RNA stabilization. Here, we show that alternate promoter usage gives rise to distinct stable and unstable RNA isoforms. Unstable Xist transcript initiates at a novel upstream promoter, whereas stable Xist RNA is transcribed from the previously identified promoter and from a novel downstream promoter. Analysis of cells undergoing X inactivation indicates that a developmentally regulated promoter switch mediates stabilization and accumulation of Xist RNA on the inactive X chromosome.
Assuntos
Mecanismo Genético de Compensação de Dose , Regulação da Expressão Gênica no Desenvolvimento , Regiões Promotoras Genéticas/fisiologia , RNA não Traduzido , Fatores de Transcrição/genética , Cromossomo X , Animais , Linhagem Celular , Desenvolvimento Embrionário e Fetal/genética , Hibridização in Situ Fluorescente , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , RNA Longo não Codificante , RNA Mensageiro/metabolismo , Transcrição Gênica/genéticaRESUMO
The onset of X inactivation is preceded by a marked increase in the level of Xist RNA. Here we demonstrate that increased stability of Xist RNA is the primary determinant of developmental up-regulation. Unstable transcript is produced by both alleles in XX ES cells and in XX embryos prior to the onset of random X inactivation. Following differentiation, transcription of unstable RNA from the active X chromosome allele continues for a period following stabilization and accumulation of transcript on the inactive X allele. We discuss the implications of these findings in terms of models for the initiation of random and imprinted X inactivation.
Assuntos
Mecanismo Genético de Compensação de Dose , RNA Mensageiro/metabolismo , RNA não Traduzido , Fatores de Transcrição/genética , Alelos , Animais , Blastocisto , Diferenciação Celular , Células Cultivadas , Dactinomicina/farmacologia , Feminino , Regulação da Expressão Gênica/fisiologia , Masculino , Camundongos , Modelos Genéticos , Inibidores da Síntese de Ácido Nucleico/farmacologia , RNA Longo não Codificante , RNA Mensageiro/biossíntese , Células-Tronco , Transcrição Gênica/fisiologiaRESUMO
The Xist gene plays a central role in regulating X chromosome inactivation and Xist transcription has recently been shown to be necessary for X inactivation in mouse. We are currently analysing regulation of the Xist gene in order to determine the mechanisms underlying initiation of Xist expression and X inactivation. Sequence comparisons indicate that a region of approximately 0.4 kb upstream of the the major transcriptional start site comprises the Xist minimal promoter. Analysis of reporter constructs demonstrates that the minimal promoter region is active both in embryonic stem (ES) cells and in differentiated derivatives, indicating that sequences either further upstream or downstream are required for appropriate developmental control of Xist transcription. We have examined the minimal promoter region in detail, and in addition to common promoter elements have identified two previously uncharacterised transcription-factor binding sites. Mutation of these sites in reporter constructs indicates that they are functionally important.
