RESUMO
X chromosome inactivation (XCI) is an essential mechanism for dosage compensation of X-linked genes in female cells. We report that subcultures from lines of female human embryonic stem cells (hESCs) exhibit variation (0-100%) for XCI markers, including XIST RNA expression and enrichment of histone H3 lysine 27 trimethylation (H3K27me3) on the inactive X chromosome (Xi). Surprisingly, regardless of the presence or absence of XCI markers in different cultures, all female hESCs we examined (H7, H9, and HSF6 cells) exhibit a monoallelic expression pattern for a majority of X-linked genes. Our results suggest that these established female hESCs have already completed XCI during the process of derivation and/or propagation, and the XCI pattern of lines we investigated is already not random. Moreover, XIST gene expression in subsets of cultured female hESCs is unstable and subject to stable epigenetic silencing by DNA methylation. In the absence of XIST expression, approximately 12% of X-linked promoter CpG islands become hypomethylated and a portion of X-linked alleles on the Xi are reactivated. Because alterations in dosage compensation of X-linked genes could impair somatic cell function, we propose that XCI status should be routinely checked in subcultures of female hESCs, with cultures displaying XCI markers better suited for use in regenerative medicine.
Assuntos
Células-Tronco Embrionárias/metabolismo , Epigênese Genética , Inativação do Cromossomo X/genética , Células Cultivadas , Ilhas de CpG/genética , Metilação de DNA , DNA Complementar/genética , Mecanismo Genético de Compensação de Dose , Células-Tronco Embrionárias/citologia , Feminino , Genes Ligados ao Cromossomo X , Marcadores Genéticos , Genótipo , Humanos , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas/genética , RNA Longo não Codificante , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
ATM and ATR are well documented for their roles in maintaining the integrity of genomic DNA by responding to DNA damage and preparing the cell for repair. Since ATM and ATR have been reported to exist in complexes with histone deacetylases, we asked whether Atm and Atr might also uphold gene silencing by heterochromatin. We show that the Atm/Atr inhibitor 2-aminopurine causes the inactive X chromosome to accumulate abnormal chromatin and undergo unwanted gene reactivation. We provide evidence that this gene expression from the inactive X chromosome is not a byproduct of the accumulation of DNA breaks. Individually inhibiting Atm and Atr by either small interfering RNA or the expression of dominant-negative ATM and ATR constructs also compromised X-inactivation. Atm and Atr, therefore, not only function in responding to DNA damage but perhaps also are involved in gene silencing via the maintenance of heterochromatin.