Integrated multi-omics reveal polycomb repressive complex 2 restricts human trophoblast induction.

Human naive pluripotent stem cells have unrestricted lineage potential. Underpinning this property, naive cells are thought to lack chromatin-based lineage barriers. However, this assumption has not been tested. Here we define the chromatin-associated proteome, histone post-translational modifications and transcriptome of human naive and primed pluripotent stem cells. Our integrated analysis reveals differences in the relative abundance and activities of distinct chromatin modules. We identify a strong enrichment of polycomb repressive complex 2 (PRC2)-associated H3K27me3 in the chromatin of naive pluripotent stem cells and H3K27me3 enrichment at promoters of lineage-determining genes, including trophoblast regulators. PRC2 activity acts as a chromatin barrier restricting the differentiation of naive cells towards the trophoblast lineage, whereas inhibition of PRC2 promotes trophoblast-fate induction and cavity formation in human blastoids. Together, our results establish that human naive pluripotent stem cells are not epigenetically unrestricted, but instead possess chromatin mechanisms that oppose the induction of alternative cell fates.

Human 8-cell-like cells discovered.

Human naive pluripotent stem cells have the remarkable ability to generate blastoids comprising trophectoderm, epiblast, and hypoblast-like cells. In this issue, Taubenschmid-Stowers et al. (2022) show that human naive pluripotent stem cell cultures contain cells that resemble the 8-cell human embryo, providing a model to study zygotic genome activation.

Contribution of genetic and epigenetic changes to escape from X-chromosome inactivation.

BACKGROUND: X-chromosome inactivation (XCI) is the epigenetic inactivation of one of two X chromosomes in XX eutherian mammals. The inactive X chromosome is the result of multiple silencing pathways that act in concert to deposit chromatin changes, including DNA methylation and histone modifications. Yet over 15% of genes escape or variably escape from inactivation and continue to be expressed from the otherwise inactive X chromosome. To the extent that they have been studied, epigenetic marks correlate with this expression. RESULTS: Using publicly available data, we compared XCI status calls with DNA methylation, H3K4me1, H3K4me3, H3K9me3, H3K27ac, H3K27me3 and H3K36me3. At genes subject to XCI we found heterochromatic marks enriched, and euchromatic marks depleted on the inactive X when compared to the active X. Genes escaping XCI were more similar between the active and inactive X. Using sample-specific XCI status calls, we found some marks differed significantly with variable XCI status, but which marks were significant was not consistent between genes. A model trained to predict XCI status from these epigenetic marks obtained over 75% accuracy for genes escaping and over 90% for genes subject to XCI. This model made novel XCI status calls for genes without allelic differences or CpG islands required for other methods. Examining these calls across a domain of variably escaping genes, we saw XCI status vary across individual genes rather than at the domain level. Lastly, we compared XCI status calls to genetic polymorphisms, finding multiple loci associated with XCI status changes at variably escaping genes, but none individually sufficient to induce an XCI status change. CONCLUSION: The control of expression from the inactive X chromosome is multifaceted, but ultimately regulated at the individual gene level with detectable but limited impact of distant polymorphisms. On the inactive X, at silenced genes euchromatic marks are depleted while heterochromatic marks are enriched. Genes escaping inactivation show a less significant enrichment of heterochromatic marks and depletion of H3K27ac. Combining all examined marks improved XCI status prediction, particularly for genes without CpG islands or polymorphisms, as no single feature is a consistent feature of silenced or expressed genes.

Cross-species examination of X-chromosome inactivation highlights domains of escape from silencing.

BACKGROUND: X-chromosome inactivation (XCI) in eutherian mammals is the epigenetic inactivation of one of the two X chromosomes in XX females in order to compensate for dosage differences with XY males. Not all genes are inactivated, and the proportion escaping from inactivation varies between human and mouse (the two species that have been extensively studied). RESULTS: We used DNA methylation to predict the XCI status of X-linked genes with CpG islands across 12 different species: human, chimp, bonobo, gorilla, orangutan, mouse, cow, sheep, goat, pig, horse and dog. We determined the XCI status of 342 CpG islands on average per species, with most species having 80-90% of genes subject to XCI. Mouse was an outlier, with a higher proportion of genes subject to XCI than found in other species. Sixteen genes were found to have discordant X-chromosome inactivation statuses across multiple species, with five of these showing primate-specific escape from XCI. These discordant genes tended to cluster together within the X chromosome, along with genes with similar patterns of escape from XCI. CTCF-binding, ATAC-seq signal and LTR repeats were enriched at genes escaping XCI when compared to genes subject to XCI; however, enrichment was only observed in three or four of the species tested. LINE and DNA repeats showed enrichment around subject genes, but again not in a consistent subset of species. CONCLUSIONS: In this study, we determined XCI status across 12 species, showing mouse to be an outlier with few genes that escape inactivation. Inactivation status is largely conserved across species. The clustering of genes that change XCI status across species implicates a domain-level control. In contrast, the relatively consistent, but not universal correlation of inactivation status with enrichment of repetitive elements or CTCF binding at promoters demonstrates gene-based influences on inactivation state. This study broadens enrichment analysis of regulatory elements to species beyond human and mouse.

Genes that escape from X-chromosome inactivation: Potential contributors to Klinefelter syndrome.

One of the two X chromosomes in females is epigenetically inactivated, thereby compensating for the dosage difference in X-linked genes between XX females and XY males. Not all X-linked genes are completely inactivated, however, with 12% of genes escaping X chromosome inactivation and another 15% of genes varying in their X chromosome inactivation status across individuals, tissues or cells. Expression of these genes from the second and otherwise inactive X chromosome may underlie sex differences between males and females, and feature in many of the symptoms of XXY Klinefelter males, who have both an inactive X and a Y chromosome. We review the approaches used to identify genes that escape from X-chromosome inactivation and discuss the nature of their sex-biased expression. These genes are enriched on the short arm of the X chromosome, and, in addition to genes in the pseudoautosomal regions, include genes with and without Y-chromosomal counterparts. We highlight candidate escape genes for some of the features of Klinefelter syndrome and discuss our current understanding of the mechanisms underlying silencing and escape on the X chromosome as well as additional differences between the X in males and females that may contribute to Klinefelter syndrome.

How do genes that escape from X-chromosome inactivation contribute to Turner syndrome?

X-chromosome inactivation generally results in dosage equivalence for expression of X-linked genes between 46,XY males and 46,XX females. The 20-30% of genes that escape silencing are thus candidates for having a role in the phenotype of Turner syndrome. Understanding which genes escape from silencing, and how they avoid this chromosome-wide inactivation is therefore an important step toward understanding Turner Syndrome. We have examined the mechanism of escape using a previously reported knock-in of a BAC containing the human escape gene RPS4X in mouse. We now demonstrate that escape from inactivation for RPS4X is already established by embryonic Day 9.5, and that both silencing and escape are faithfully maintained across the lifespan. No overt abnormalities were observed for transgenic mice up to 1 year of age despite robust transcription of the human RPS4X gene with no detectable downregulation of the mouse homolog. However, there was no significant increase in protein levels, suggesting translational compensation in the mouse. Finally, while many of the protein-coding genes have been assessed for their inactivation status, less is known about the X-linked RNA genes, and we propose that for many microRNA genes their inactivation status can be predicted as they are intronic to genes for which the inactivation status is known.

The eXceptional nature of the X chromosome.

The X chromosome is unique in the genome. In this review we discuss recent advances in our understanding of the genetics and epigenetics of the X chromosome. The X chromosome shares limited conservation with its ancestral homologue the Y chromosome and the resulting difference in X-chromosome dosage between males and females is largely compensated for by X-chromosome inactivation. The process of inactivation is initiated by the long non-coding RNA X-inactive specific transcript (XIST) and achieved through interaction with multiple synergistic silencing pathways. Identification of Xist-interacting proteins has given insight into these processes yet the cascade of events from initiation to maintenance have still to be resolved. In particular, the initiation of inactivation in humans has been challenging to study as: it occurs very early in development; most human embryonic stem cell lines already have an inactive X; and the process seems to differ from mouse. Another difference between human and mouse X inactivation is the larger number of human genes that escape silencing. In humans over 20% of X-linked genes continue to be expressed from the otherwise inactive X chromosome. We are only beginning to understand how such escape occurs but there is growing recognition that escapees contribute to sexually dimorphic traits. The unique biology and epigenetics of the X chromosome have often led to its exclusion from disease studies, yet the X constitutes 5% of the genome and is an important contributor to disease, often in a sex-specific manner.

YY1 binding association with sex-biased transcription revealed through X-linked transcript levels and allelic binding analyses.

Sex differences in susceptibility and progression have been reported in numerous diseases. Female cells have two copies of the X chromosome with X-chromosome inactivation imparting mono-allelic gene silencing for dosage compensation. However, a subset of genes, named escapees, escape silencing and are transcribed bi-allelically resulting in sexual dimorphism. Here we conducted in silico analyses of the sexes using human datasets to gain perspectives into such regulation. We identified transcription start sites of escapees (escTSSs) based on higher transcription levels in female cells using FANTOM5 CAGE data. Significant over-representations of YY1 transcription factor binding motif and ChIP-seq peaks around escTSSs highlighted its positive association with escapees. Furthermore, YY1 occupancy is significantly biased towards the inactive X (Xi) at long non-coding RNA loci that are frequent contacts of Xi-specific superloops. Our study suggests a role for YY1 in transcriptional activity on Xi in general through sequence-specific binding, and its involvement at superloop anchors.

Escape Artists of the X Chromosome.

Inactivation of one X chromosome in mammalian females achieves dosage compensation between XX females and XY males; however, over 15% of human X-linked genes continue to be expressed from the inactive X chromosome. New genomic methodologies have improved our identification and characterization of these escape genes, revealing the importance of DNA sequence, chromatin structure, and chromosome ultrastructure in regulating expression from an otherwise inactive chromosome. Study of these exceptions to the rule of silencing highlights the interconnectedness of chromatin and chromosome structure in X-chromosome inactivation (XCI). Recent advances also demonstrate the importance of these genes in sexually dimorphic disease risk, particularly cancer.

Landscape of DNA methylation on the X chromosome reflects CpG density, functional chromatin state and X-chromosome inactivation.

X-chromosome inactivation (XCI) achieves dosage compensation between males and females through the silencing of the majority of genes on one of the female X chromosomes. Thus, the female X chromosomes provide a unique opportunity to study euchromatin and heterochromatin of allelic regions within the same nuclear environment. We examined the interplay of DNA methylation (DNAm) with CpG density, transcriptional activity and chromatin state at genes on the X chromosome using over 1800 female samples analysed with the Illumina Infinium Human Methylation450 BeadChip. DNAm was used to predict an inactivation status for 63 novel transcription start sites (TSSs) across 27 tissues. There was high concordance of inactivation status across tissues, with 62% of TSSs subject to XCI in all 27 tissues examined, whereas 9% escaped from XCI in all tissues, and the remainder showed variable escape from XCI between females in subsets of tissues. Inter-female and twin data supported a model of predominately cis-acting influences on inactivation status. The level of expression from the inactive X relative to the active X correlated with the amount of female promoter DNAm to a threshold of ∼30%, beyond which genes were consistently subject to inactivation. The inactive X showed lower DNAm than the active X at intragenic and intergenic regions for genes subject to XCI, but not at genes that escape from inactivation. Our categorization of genes that escape from X inactivation provides candidates for sex-specific differences in disease.

Derivation of consensus inactivation status for X-linked genes from genome-wide studies.

BACKGROUND: X chromosome inactivation is the epigenetic silencing of the majority of the genes on one of the X chromosomes in XX therian mammals. In humans, approximately 15 % of genes consistently escape from this inactivation and another 15 % of genes vary between individuals or tissues in whether they are subject to, or escape from, inactivation. Multiple studies have provided inactivation status calls for a large subset of the genes on the X chromosome; however, these studies vary in which genes they were able to make calls for and in some cases which call they give a specific gene. METHODS: This analysis aggregated three published studies that have examined X chromosome inactivation status of genes across the X chromosome, generating consensus calls and identifying discordancies. The impact of expression level and chromosomal location on X chromosome inactivation status was also assessed. RESULTS: Overall, we assigned a consensus XCI status 639 genes, including 78 % of protein-coding genes expressed outside of the testes, with a lower frequency for non-coding RNA and testis-specific genes. Study-specific discordancies suggest that there may be instability of XCI during cell culture and also highlight study-specific variations in call type. We observe an enrichment of discordant genes at boundaries between genes subject to and escaping from inactivation. CONCLUSIONS: This study has compiled a comprehensive list of X-chromosome inactivation statuses for genes and also discovered some biases which will help guide future studies examining X-chromosome inactivation.