Epigenetic Regulation of X-Chromosome Inactivation Maintenance in Somatic Cells
DOI:
https://doi.org/10.5281/zenodo.22760312Keywords:
X-chromosome inactivation, Xist, H3K27me3, DNA methylation, escape genes, reactivation, somatic cellsAbstract
X-chromosome inactivation is established during early female mammalian development and subsequently
maintained across the somatic lifespan of the organism through a combination of the long non-coding RNA Xist,
repressive histone modifications and DNA methylation, yet the relative contribution of each layer to long-term
maintenance, as opposed to initial establishment, of the inactive state in differentiated somatic cells has been
difficult to separate experimentally. Here we used doxycycline-inducible degradation systems targeting Xist RNA,
the Polycomb repressive complex 2 (PRC2) subunit EZH2, and the maintenance DNA methyltransferase DNMT1,
individually and in combination, in a clonal human female fibroblast line with a skewed, stably inactivated X
chromosome, to quantify the contribution of each maintenance layer to reactivation of silenced X-linked genes.
Acute Xist depletion for 72 hours produced detectable but modest reactivation of a minority of X-linked genes,
concentrated among genes classified as facultative escapees in other contexts, without measurable loss of
H3K27me3 or DNA methylation at their promoters within the same window. Combined Xist depletion with EZH2
degradation produced substantially greater reactivation than either perturbation alone, and addition of DNMT1
inhibition to this combination produced a further, though proportionally smaller, increment in reactivation, with a core
set of 62 genes remaining silenced even under triple perturbation. Genes that resisted reactivation under triple
perturbation were disproportionately located in gene-dense domains with the highest baseline H3K27me3 and DNA
methylation density, and time-course analysis showed that reactivation of the most resistant genes required more
than seven days of sustained triple perturbation to reach half-maximal induction. These results indicate that
maintenance of X-inactivation in somatic cells depends on layered, partially redundant epigenetic mechanisms
whose combined and prolonged disruption is required to substantially reactivate the inactive X chromosome, with a
residual core of genes exhibiting unusually stable, multi-layer-reinforced silencing.
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