Preventing erosion of X-chromosome inactivation in human embryonic stem cells.

X-chromosome inactivation is a paradigm of epigenetic transcriptional regulation. Female human embryonic stem cells (hESCs) often undergo erosion of X-inactivation upon prolonged culture. Here, we investigate the sources of X-inactivation instability by deriving new primed pluripotent hESC lines. We find that culture media composition dramatically influenced the expression of XIST lncRNA, a key regulator of X-inactivation. hESCs cultured in a defined xenofree medium stably maintained XIST RNA expression and coating, whereas hESCs cultured in the widely used mTeSR1 medium lost XIST RNA expression. We pinpointed lithium chloride in mTeSR1 as a cause of XIST RNA loss. The addition of lithium chloride or inhibitors of GSK-3 proteins that are targeted by lithium to the defined hESC culture medium impeded XIST RNA expression. GSK-3 inhibition in differentiating female mouse embryonic stem cells and epiblast stem cells also resulted in a loss of XIST RNA expression. Together, these data may reconcile observed variations in X-inactivation in hESCs and inform the faithful culture of pluripotent stem cells.

Highly Resolved Detection of Long Non-coding RNAs In Situ.

Long non-coding RNAs (lncRNAs) have been postulated to function in a number of DNA-based processes, most notably transcription. The detection of lncRNAs in situ can offer insights into their function. Fluorescence in situ hybridization (FISH) enables the detection of specific nucleic acid sequences, including lncRNAs, within individual cells. Current RNA FISH techniques can inform both the localization and expression level of RNA transcripts. Together with advances in microscopy, these in situ techniques now allow for visualization and quantification of even lowly expressed or unstable lncRNAs. When combined with detection of associated proteins and chromatin modifications by immunofluorescence, RNA FISH can lend essential insights into lncRNA function. Here, we describe an integrated set of protocols to detect, individually or in combination, specific RNAs, DNAs, proteins, and histone modifications in single cells at high sensitivity using conventional fluorescence microscopy.

Conversion of random X-inactivation to imprinted X-inactivation by maternal PRC2.

Imprinted X-inactivation silences genes exclusively on the paternally-inherited X-chromosome and is a paradigm of transgenerational epigenetic inheritance in mammals. Here, we test the role of maternal vs. zygotic Polycomb repressive complex 2 (PRC2) protein EED in orchestrating imprinted X-inactivation in mouse embryos. In maternal-null (Eedm-/-) but not zygotic-null (Eed-/-) early embryos, the maternal X-chromosome ectopically induced Xist and underwent inactivation. Eedm-/- females subsequently stochastically silenced Xist from one of the two X-chromosomes and displayed random X-inactivation. This effect was exacerbated in embryos lacking both maternal and zygotic EED (Eedmz-/-), suggesting that zygotic EED can also contribute to the onset of imprinted X-inactivation. Xist expression dynamics in Eedm-/- embryos resemble that of early human embryos, which lack oocyte-derived maternal PRC2 and only undergo random X-inactivation. Thus, expression of PRC2 in the oocyte and transmission of the gene products to the embryo may dictate the occurrence of imprinted X-inactivation in mammals.

Experimental Analysis of Imprinted Mouse X-Chromosome Inactivation.

X-chromosome inactivation is a dosage compensation mechanism that equalizes X-linked gene expression between male and female mammals through the transcriptional silencing of most genes on one of the two X-chromosomes in females. With a few key exceptions, once the X-chromosome is inactivated replicated copies of that X-chromosome are maintained as inactive in all descendant cells. X-inactivation is therefore a paradigm of epigenetic inheritance. Imprinted X-inactivation is a specialized form of X-inactivation that results in the silencing of the paternally derived X-chromosome. Due to its parent-of-origin-specific pattern of inactivation, imprinted X-inactivation is a model of mitotic as well as meiotic, i.e., transgenerational, epigenetic inheritance. All cells of the early mouse embryo undergo imprinted X-inactivation, a pattern that is subsequently maintained in extraembryonic cell types in vivo and in vitro. Here, we describe both high- and low-throughput approaches to interrogate imprinted X-inactivation in the mouse embryo as well in cultured extraembryonic stem cells.