X-inactivation in female human embryonic stem cells is in a nonrandom pattern and prone to epigenetic alterations

Abstract

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, Ϸ12% 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.culture variation ͉ DNA methylation ͉ gene regulation H uman embryonic stem cells (hESCs) are regarded as one of the most promising stem cells for regenerative medicine because of their unusual capacity of self-renewal and pluripotency (1). However, given the variations in the derivation and propagation of hESCs in different laboratories, it is imperative to establish a common set of criteria for the quality control of hESCs. Efforts have been devoted to characterizing whether established lines of hESCs carry inherent differences in gene expression and epigenetic modifications such as DNA methylation (2). Although different lines of hESCs can exhibit a common set of stem cell markers, differences in gene expression are observed including allelic expression of several imprinted genes and XIST, a crucial gene for X-inactivation (2). Several studies also demonstrated that in vitro cultures or differentiation of hESCs can contribute to changes in CpG methylation patterns and genome stability in different lines of hESCs (2-4). Thus, routine and thorough characterization of genetic and epigenetic stability in hESCs is a necessary step to ensure the quality of hESCs for regenerative medicine.X chromosome inactivation (XCI) is required for dosage compensation of X-linked genes in female cells (5). So far, only a few studies have examined XCI in female hESCs and conflicting data exist regarding the nature of XCI. It has been reported that Ϸ50% of all established female hESC lines exhibit XCI markers such as XIST expression and/or punctate histone H3 lysine 27 trimethylation (H3K27me3) staining on the inactive X chromosome (Xi), whereas other lines do not (2, 6-9). Moreover, discrepan...

Systematic Mapping of RNA-Chromatin Interactions In Vivo

“…If this was the case, we would anticipate to see differences in diRNA activities on XIST associated lincRNAs TSIX [18,19] and FTX [20] between HEK and H9 cells. The two lincRNAs exhibited similar degrees of pxRNA activities between HEK and H9 ( Figure S4A-B), consistent with the observed XIST pxRNA activities and reported XCI in H9 cells [13]. However, TSIX and FTX exhibited much reduced diRNA activities in H9 as compared to HEK (Figure S4C-D), consistent with the minimal diRNA activity of XIST in H9.…”

Xist Deficiency and Disorders of X-Inactivation in Rabbit Embryonic Stem Cells Can Be Rescued by Transcription-Factor-Mediated Conversion

“…Xi erosion in hiPSCs and hESCs is believed to be closely associated with the loss of XIST and may broadly affect the characteristics of these cells [16,27]. These epigenetic modifications not only affect X-linked genes but also impact genome-wide gene expression, resulting in the upregulation of oncogenes and the downregulation of several tumor suppressors, thereby broadly affecting the characteristics of these cells [15][16][17][27][28][29][30]. Previous studies have suggested that female hiPSCs that have undergone XCI erosion should not be used clinically [16].…”

Rapid and efficient reprogramming of somatic cells to induced pluripotent stem cells by retinoic acid receptor gamma and liver receptor homolog 1

“…The ground-state mouse female ES cells or iPSCs have two active X chromosomes, whereas human female ES cells show signs of X chromosome inactivation detected by XIST expression or punctuate immunostaining pattern for H3K27me3 (31). qRT-PCR detected little XIST in our female iPSCs compared with the parental female dermal fibroblast cells (Fig.…”