Dynamic X-chromosomal reactivation during neuronal differentiation as a mechanism of female resilience
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Abstract
In humans, females possess two X-chromosomes, while males have one X and one Y chromosome, necessitating a mechanism to balance the expression of X-linked genes. This is achieved through X- chromosome inactivation (XCI), a process involving chromatin and epigenetic modifications, as well as nuclear re-localization, which leads to the transcriptional silencing of one randomly selected X chromosome. XCI is established early in human epiblast cells after the implantation stage of the embryo and is maintained throughout the lifespan of female individuals, with the inactive X-chromosome inherited by daughter cells.
However, XCI is incomplete, with approximately 20% of X-linked genes being either partially or fully expressed from the inactive X chromosome. These genes include constitutive escape genes, which are consistently expressed, and facultative escape genes, which are variably inactivated in a tissue-specific manner. Neurodevelopmental disorders (NDDs) exhibit a notable sex bias, with males more frequently and severely affected than females, although the underlying mechanisms remain unclear. The presence of an additional X chromosome in females and the enrichment of NDD-associated genes on the X chromosome may partly explain these sex differences, with tissue-specific escape genes likely playing a key role.
In this study, we leveraged an induced pluripotent stem cell (iPSC) in vitro system to model post- implantation epiblast cells, characterized by stable XCI. We confirmed the clonal status of the iPSCs, with the same X chromosome either consistently active or inactive across the population, and established an in vitro differentiation protocol to mimic human neurodevelopment. The iPSCs were differentiated into neural stem and progenitor cells (NPCs) and neurons, and the transcriptome was analyzed at allele- specific resolution by tracking the expression of heterozygous genomic variants.
We identified three categories of escape genes based on their allele-specific expression in iPSCs, NPCs, and neurons. Constitutive escape genes, referred to as full-escape genes, were biallelically expressed across all three cell types. We also discovered two novel categories of facultative escape genes: (1) reactivated genes, which escape XCI specifically in neuronal cells, and (2) late-silenced genes that escape XCI in iPSCs but become inactivated during neuronal differentiation. Follow-up analyses of neuronal-specific reactivated genes support the idea that these genes may play a crucial role in explaining the female protection against NDDs. Additionally, we used RNA-FISH technology to investigate the reactivation mechanism at the single-cell level, confirming the neuronal-specific reactivation of two candidate genes, MID1 and GPM6B.
In summary, the data collected during my PhD demonstrate dynamic expression patterns from the inactive X chromosome, with certain genes being reactivated or silenced during neuronal differentiation. These findings suggest that differentiation-induced use of the inactive X chromosome may contribute to the observed sex-biased phenotypes in NDDs between males and females.