A Chromosomal Memory Triggered by <em>Xist</em> Regulates Histone Methylation in X Inactivation

<div><p>We have elucidated the kinetics of histone methylation during X inactivation using an inducible <em>Xist</em> expression system in mouse embryonic stem (ES) cells. Previous reports showed that the ability of <em>Xist</em> to trigger silencing is restricted to an early window in ES cell differentiation. Here we show that this window is also important for establishing methylation patterns on the potential inactive X chromosome. By immunofluorescence and chromatin immunoprecipitation experiments we show that histone H3 lysine 27 trimethylation (H3K27m3) and H4 lysine 20 monomethylation (H4K20m1) are associated with <em>Xist</em> expression in undifferentiated ES cells and mark the initiation of X inactivation. Both marks depend on <em>Xist</em> RNA localisation but are independent of silencing. Induction of <em>Xist</em> expression after the initiation window leads to a markedly reduced ability to induce H3K27m3, whereas expression before the restrictive time point allows efficient H3K27m3 establishment. Our data show that <em>Xist</em> expression early in ES cell differentiation establishes a chromosomal memory, which is maintained in the absence of silencing. One consequence of this memory is the ability to introduce H3K27m3 efficiently after the restrictive time point on the chromosome that has expressed <em>Xist</em> early. Our results suggest that this silencing-independent chromosomal memory has important implications for the maintenance of X inactivation, where previously self-perpetuating heterochromatin structures were viewed as the principal form of memory.</p>