Abstract
Stable inheritance of DNA methylation is critical for maintaining differentiated phenotypes in multicellular organisms. We have recently identified dual mono-ubiquitylation of histone H3 (H3Ub2) by UHRF1 as an essential mechanism to recruit DNMT1 to chromatin. Here, we show that PCNA-associated factor 15 (PAF15) undergoes UHRF1-dependent dual mono-ubiquitylation (PAF15Ub2) on chromatin in a DNA replication-coupled manner. This event will, in turn, recruit DNMT1. During early S-phase, UHRF1 preferentially ubiquitylates PAF15, whereas H3Ub2 predominates during late S-phase. H3Ub2 is enhanced under PAF15 compromised conditions, suggesting that H3Ub2 serves as a backup for PAF15Ub2. In mouse ES cells, loss of PAF15Ub2 results in DNA hypomethylation at early replicating domains. Together, our results suggest that there are two distinct mechanisms underlying replication timing-dependent recruitment of DNMT1 through PAF15Ub2 and H3Ub2, both of which are prerequisite for high fidelity DNA methylation inheritance.
Highlights
Stable inheritance of DNA methylation is critical for maintaining differentiated phenotypes in multicellular organisms
Given that the ubiquitin ligase activity of UHRF1 and the ubiquitin binding activity of DNA methyltransferase 1 (DNMT1) are essential for the recruitment of DNMT1 to hemi-methylated DNA sites, we speculated that factors associated with the DNA replication machinery utilize ubiquitin signals to recruit DNMT1
RxDNMT1WT interacted with H3Ub2 as well as with unmodified and mono-ubiquitylated histone H3 (Supplementary Fig. 1d, −SDS), suggesting that indirect binding is preserved under this condition
Summary
Stable inheritance of DNA methylation is critical for maintaining differentiated phenotypes in multicellular organisms. We show that PCNA-associated factor 15 (PAF15) undergoes UHRF1-dependent dual monoubiquitylation (PAF15Ub2) on chromatin in a DNA replication-coupled manner This event will, in turn, recruit DNMT1. UHRF1 recognizes specific epigenetic modifications on DNA strands and histone H3 tails through its SET- and RING-associated (SRA) domain and tandem Tudor domain (TTD)–plant homeodomain (PHD), respectively[9,10,11,12,13,14] The former binds to hemi-methylated DNA, while the latter recognizes N-terminal 1ARTK4 residues and tri-methylated Lys[9] of H3 (H3K9me[3]). We and others have recently reported that UHRF1-mediated dual monoubiquitylation of histone H3 (H3Ub2) on lysine residues 14, 18, and 23 plays a role in the RFTS-dependent recruitment of DNMT1 and its enzymatic activation, ensuring the high fidelity of DNA maintenance methylation[18,23,24]. When the PAF15-dependent mechanism is perturbed, DNMT1 utilizes H3Ub2, suggesting that H3Ub2 functions as a backup system for the maintenance of DNA methylation
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