Genetic Ablation of lncRNA <i>XACT</i> Does Not Change Expression Dosage of X-Linked 1 Genes But Impacts Differentiation Potential in Female Human Pluripotent Stem Cells

Abstract

Female human pluripotent stem cells (hPSCs) regularly show erosion of X-chromosome inactivation (XCI) featured by loss of the long non-coding (lnc) RNA XIST and accumulation of XACT. Although both lncRNAs are expressed from X-chromosome, XACT is mainly expressed in pluripotent cells, and the expression pattern is reciprocal to XIST, suggesting an important role in dosage compensation and differentiation. In this study, we aimed to dissect the role of XACT in the erosion of dosage compensation and differentiation potential in female hPSCs. We found that XACT RNA accumulation on inactive X-chromosomes is hPSC line dependent. XIST RNA overlapped with H3K27me3 but not H3K9me3, and XIST/H3K27me3 loss occurred during erosion in all female hPSC lines analyzed. These results indicate that a common mechanism for initiation of erosion depends on XIST loss but not XACT accumulation on inactive X-chromosomes. We further demonstrated that XACT deletion does not affect X-linked gene dosage in eroded hPSCs. Additionally, aberrant XIST RNA diffusion induced by the CRISPR activation system was independent of the presence of XACT RNA. In contrast, genetic ablation of XACT in female hPSCs resulted in the up-regulation of neuron-related genes to facilitate neural differentiation. Finally, we confirmed that XACT repression by the CRISPR inhibition system leads to dysregulation of the genes and affects neural differentiation, indicating that XACT RNA is involved in gene regulation affecting neural differentiation. Our study demonstrates that XACT is dispensable for the maintenance of dosage of X-linked genes, but impacts neural differentiation in female hPSCs, implying that XACT might play important roles in the regulation of spatiotemporal development rather than X-chromosome dosage compensation in human pluripotent cells.