Investigating the maintenance of X- chromosome inactivation in extra-embryonic endoderm and epiblast stem cells

Abstract

In eutherian female mammals, one of the X-chromosomes gets inactivated to compensate for the expression dosage of X-linked genes between the sexes. X-chromosome inactivation (XCI) serves as a paradigm of epigenetic memory propagation. In mice, there are two forms of XCI: imprinted and random. Imprinted XCI initiates at the two to four-cell stages of embryogenesis where the paternal X-chromosome gets inactivated. Subsequently, inactive X gets reactivated in the inner cell mass (ICM) of the late blastocyst, and random XCI is initiated in the epiblast stem cells (EpiSCs) of post-implantation embryos. In recent years, the mechanism of XCI initiation and establishment has been studied extensively. Xist, a 17 Kb long non-coding RNA acts as a master regulator of the XCI. Xist solely transcribes from the inactive-X chromosome and coats the entire inactive-X followed by recruits a series of chromatin modifiers to facilitate the heterochromatinisation. Subsequently, DNA methylation is thought to stabilise the inactivation. However, the role of Xist and DNA methylation in the maintenance of the inactive-X remains underexplored. In the present study, we have explored the contribution of Xist and DNA methylation in the maintenance of the inactive state of X-linked genes in imprinted and random XCI.

We used extra-embryonic endoderm stem cells (XEN) and EpiSC, which undergo imprinted and random XCI, respectively as our model system. XEN cells are stem cells derived from the primitive endoderm of the blastocyst, whereas EpiSC represents the epiblast of post-implantation embryos. Both lineages represent a developmental window, where they just transitioned from the initiation to the maintenance phase of XCI. Importantly, these cells are hybrid and therefore have polymorphic X-chromosomes (XMus: Mus musculus origin and XMol : Mus molossinus origin), which enabled us to disentangle the expression of X-linked genes between active-X vs. inactive-X through allele-specific expression analysis. We ablated Xist in XEN and EpiSC using CRISPR-Cas9 and performed allele-specific RNA-sequencing analysis to explore the effect of Xist loss on X-linked gene silencing. We find that only a subset of genes were reactivated upon Xist loss, while the majority of X-linked genes maintained the silent state in both XEN and EpiSC. Interestingly, many genes reactivated upon Xist loss were common between XEN and EpiSC. Taken together, we conclude that the majority of X-linked genes do not rely on Xist to maintain their inactive state.

Next, we explored the role of DNA methylation in the maintenance of imprinted XCI in XEN cells. To explore this, we treated XEN cells with 5-AzaDC, an inhibitor of DNA methyl transferase DNMT1, followed by performed allele-specific RNA-seq analysis. Surprisingly, we find that most of the X-linked genes are able to maintain their silent state despite the loss of DNA methylation. However, we show that the removal of DNA methylation in Xist-ablated XEN cells leads to the reactivation of a subset of X-linked genes, indicating these genes are dependent on both Xist and DNA methylation to maintain their inactive state. Collectively, we conclude that many X-linked genes can maintain their inactive state despite the loss of Xist and DNA methylation, indicating that other factors are involved in the maintenance of XCI.

Next, we investigated whether epigenomic states contribute to the reactivation potential of X-linked genes upon loss of Xist. We show that X-linked genes with high H3K27me3 but low H3K9me3 around the transcriptional start site (TSS) on the inactive-X are more prone to reactivation upon Xist loss. Interestingly, TSS of homologous copies of reactivated genes in the active-X chromosomes were highly enriched with active marks such as H3K4me3 and H3K27ac. Altogether, we conclude that the epigenomic states of X-linked genes are linked to the gene-specific reactivation potential upon the loss of Xist.

Separately, we investigated the role of CELF1 in the maintenance of the imprinted XCI in XEN cells. CELF1 is an Xist RNA binding protein, which forms a condensate on the inactive-X chromosome along with other proteins; therefore, it is believed that CELF1 is required for X-inactivation. We show that loss of CELF1 in XEN cells leads to defect in H3K27me3 enrichment on the inactive-X, however, it does not affect the X-linked gene silencing. Taken together, we conclude that CELF1 is dispensable for the maintenance of XCI.

In summary, our study provides significant insights into the maintenance of XCI. We demonstrate that many genes are able to maintain their inactive state despite the loss of Xist, DNA methylation or CELF1, suggesting that other factors govern the maintenance of XCI.