During X chromosome inactivation (XCI), in female placental mammals, gene silencing is initiated by the Xist long non‐coding RNA. Xist accumulation at the X leads to enrichment of specific chromatin marks, including PRC2‐dependent H3K27me3 and SETD8‐dependent H4K20me1. However, the dynamics of this process in relation to Xist RNA accumulation remains unknown as is the involvement of H4K20me1 in initiating gene silencing. To follow XCI dynamics in living cells, we developed a genetically encoded, H3K27me3‐specific intracellular antibody or H3K27me3‐mintbody. By combining live‐cell imaging of H3K27me3, H4K20me1, the X chromosome and Xist RNA, with ChIP‐seq analysis we uncover concurrent accumulation of both marks during XCI, albeit with distinct genomic distributions. Furthermore, using a Xist B and C repeat mutant, which still shows gene silencing on the X but not H3K27me3 deposition, we also find a complete lack of H4K20me1 enrichment. This demonstrates that H4K20me1 is dispensable for the initiation of gene silencing, although it may have a role in the chromatin compaction that characterises facultative heterochromatin.

During X chromosome inactivation (XCI), in female mammals, gene silencing is initiated by the Xist long-noncoding RNA. Xist accumulation at the X leads to enrichment of specific chromatin marks, including PRC2-dependent H3K27me3 and SETD8-dependent H4K20me1. However, the dynamics of this process in relation to Xist RNA accumulation remains unknown as is the molecular mechanism allowing for H4K20me1 enrichment. To follow XCI dynamics in living cells, we developed a genetically-encoded, H3K27me3-specific intracellular antibody, or H3K27me3-mintbody. By combining it with live-imaging of H4K20me1, the X chromosome and Xist RNA we uncover similarities in the initial accumulation dynamics of H3K27me3 and H4K20me1. Further ChIP-seq analysis confirmed concurrent accumulation of both marks during XCI albeit with distinct genomic distributions. Using a Xist B and C repeat mutant, which can silence the X but does not allow for H3K27me3 enrichment, we also found a lack of H4K20me1 deposition. Thus, these two marks are brought onto the X by the same region of Xist and H4K20me1 in particular may well have a role in the chromatin compaction that characterises facultative heterochromatin.