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Female Naïve human pluripotent stem cells carry X chromosomes with Xa-like and Xi-like folding conformations

ABSTRACT: In naïve human pluripotent stem cells (hPSCs) that represent the pre-implantation embryonic states, epigenetic regulators for cell identity remain poorly defined. One of the major remaining questions in naïve stem cell biology is how female cells mediate and regulate X chromosome inactivation (XCI). Recent studies incorporating single-cell RNA sequencing (scRNA-seq) have demonstrated that transcript-based technologies are insufficient to unequivocally resolve XCI regulation in a human system. Instead, 3D genomics shows immense promise for studying XCI by interrogating changes to the X chromosomes’ 3D states. Here, we sought to characterize the 3D state of the X chromosome in naïve and primed hPSCs. Using chromatin tracing, we analyzed the folding conformations of whole X chromosomes with megabase genomic resolution in multiple naïve and primed hPSC lines. Our data found that X chromosomes in female naive hPSCs exhibit a range of spatial folding conformations similar to the active X chromosome (Xa) and the inactive X chromosome (Xi) in somatic cells; these data suggest that naïve hPSCs have initiated XCI. As XCI process ultimately generates detectable differences in volume between the Xa and the Xi, we measured the radius of gyration of individual X chromosome copies across various hPSC cell lines and culture conditions. In naïve hPSCs, our results show that the X chromosomes with Xi-like conformations are not more compact than those with Xa-like conformation. This finding is directly counter to the Xi compaction typically observed in post-XCI female somatic cells. These contradictory 3D signatures of XCI suggest that female naïve hPSCs have initiated the XCI process, but are poised before XCI-driven silencing in a metastable state. As observed in H7 naïve hPSCs, this metastable state can be abolished through classically noted means, such as the loss of Xp chromatin, leading to the deleted p X chromosome (dXp). The Xp loss observed here seems to drive XIST expression and accumulation around the dXp chromosome. Overall, our findings provide insight into the previously undefined X chromosome status in naïve hPSCs in single chromosome resolution, and are critical in understanding the unique epigenetic regulation in early embryonic cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE213979 | GEO | 2023/08/09

REPOSITORIES: GEO

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Project description:X chromosome inactivation (XCI) is a dosage compensation mechanism that silences the majority of genes on one X chromosome in each female cell. In order to characterize epigenetic changes that accompany this process, we measured DNA methylation levels in both 45,X Turner syndrome patients, who carry a single active X chromosome (Xa) and normal 46,XX females, who carry one Xa and one inactive X (Xi). Methylated DNA was immunoprecipitated and hybridized to tiling oligonucleotide arrays, generating epigenetic profiles of active and inactive X chromosomes. We observed that XCI is accompanied by changes in DNA methylation specifically at CpG islands. While the majority of CpG islands show increased methylation levels on the Xi, XCI results in reduced methylation at ~20% of CpG islands. Both intra- and inter-genic CpG islands are epigenetically modified, with the biggest increase in methylation occuring at the promoters of genes silenced by XCI. In contrast, genes escaping XCI have low levels of promoter methylation, while genes that undergo polymorphic silencing show intermediate increases in methylation proportionate to their frequency of inactivation. Thus promoter methylation and susceptibility to XCI are correlated. We observed a global correlation between CpG island methylation and the evolutionary age of different X chromosome strata, and that genes escaping XCI show increased methylation within gene bodies. We utilized our epigenetic map to predict both novel genes escaping XCI, and to identify sequence features that may contribute to the XCI process. Finally, as our study included Turner syndrome patients with single X chromosomes of both maternal and paternal origin we searched for parent-of-origin specific methylation differences, but found no evidence to support imprinting on the human X chromosome. Our study provides the first epigenetic profile of active and inactive X chromosomes, giving novel insights into the phenomenon of dosage compensation. Methylated DNA was enriched by immunoprecipitation using antibodies against 5-methylcytosine. meDIP and input DNA was labeled with cy5 and cy3 respectively and hybridized to Nimblegen arrays comprising 2.1 million 50-85mers covering human chromosomes 20, 21, 22, X and Y at a mean density of ~1 probe per 100bp. Resulting log2 fluorescence ratios correspond to methylation levels. Seven patients with Turner syndrome (45,X karyotype), and three normal females (46,XX karyotype) were analyzed. Of the Turner syndrome cases, four had a maternally-derived X, and three had a paternally-derived X chromosome.

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Female Naïve human pluripotent stem cells carry X chromosomes with Xa-like and Xi-like folding conformations

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