DNA was isolated using AMPure XP beads as described for the input sample. For each nChIP\seq, 0.5?l of each sample was used for qPCR validation of enrichment at control regions. of both marks during XCI, albeit with distinct genomic distributions. Furthermore, using a B Acemetacin (Emflex) and C repeat mutant, which still shows gene silencing around the X but not H3K27me3 deposition, we also find a complete lack of H4K20me1 enrichment. This demonstrates that H4K20me1 is usually dispensable for the initiation of gene silencing, although it may have a role in the chromatin compaction that characterises facultative heterochromatin. by differentiating female mouse embryonic stem cells (ESC) (Rastan & Robertson, 1985). Both and (X\inactive\specific transcript) (Penny RNA accumulation along IL10 the Xi induces not only the silencing of over a 1,000 genes but also a cascade of chromatin alterations (reviewed in Zylicz & Heard, 2020). is usually a modular non\coding RNA with specific regions playing distinct roles. Its 5 A\repeat region is vital for the induction of gene silencing (Wutz RNA coating Xi becomes rapidly enriched with the PRC1\dependent H2AK119Ub and only subsequently with PRC2\dependent H3K27me3 (Plath RNA directly recruits hnRNPK, which in turn binds non\canonical PRC1 allowing for rapid H2AK119Ub deposition (Almeida deposition of H3K27me3 (Blackledge mutants lacking the B and C repeat region can still induce XCI albeit with slightly lower efficiency (Bousard RNA coating of the Xi. Indeed, the time that lapses between RNA upregulation and H3K27me3 deposition is not known. Nor how this relates to other repressive histone marks. Another modification that rapidly accumulates around the Xi is Acemetacin (Emflex) usually H4K20me1 (Kohlmaier (Brejc RNA and H3K27me3 deposition remains unclear. Furthermore, the precise distribution of first H4K20me1 enrichment along the X and the molecular mechanisms underlying its dynamic accumulation around the Xi have not previously been investigated. To address these questions, it is important to follow the spatio\temporal dynamics of histone modifications as they accumulate around the X chromosome in living cells. To this end, we have previously developed genetically encoded, modification\specific intracellular antibodies or mintbodies, by fusing a single\chain variable fragment (scFv) of the specific antibody with a fluorescent protein (Sato RNA in live cells (Masui RNA, we demonstrate that H4K20me1 accumulation, just like that of PcG\dependent H3K27me3, relies on the RNA B and C repeats. This also reveals that both marks are dispensable for the initiation of gene silencing. Taken together, our analysis uncovers that this H4K20me1 and H3K27me3 histone marks accumulate at the Xi with comparable dynamics but with rather different distributions. Reliance of both marks on the region for their enrichment suggests a mechanistic link between Polycomb and H4K20me1 accumulation during facultative heterochromatin formation around the X chromosome. These observations also imply that the general function of H4K20me1 may be in chromatin compaction Acemetacin (Emflex) that characterises facultative heterochromatin rather than in the initiation of gene silencing. Results H3K27me3 mintbody (2E12LI) specifically tracks H3K27me3 in living cells Methods for tracking polycomb\dependent histone marks in living cells have Acemetacin (Emflex) been lacking up until now. Such tools would be hugely beneficial for the study of dynamic epigenetic processes such as XCI. To address this, we decided to generate an H3K27me3\specific mintbody. Upon screening of mouse hybridomas, we selected a 2E12 clone, which expresses an H3K27me3\specific antibody. We decided the cDNA sequence of the IgG heavy and light chains in 2E12 by deep sequencing (Kuniyoshi using a modified histone peptide array (Fig?EV2A). H3K27me3\made up of peptides were highlighted over other peptides, regardless of the neighbouring R26 modifications (Fig?EV2B). The 2E12LI\sfGFP binding was, however, occluded by S28 phosphorylation (S28ph), as commonly.