The termination of UHRF1-dependent PAF15 ubiquitin signaling is regulated by USP7 and ATAD5
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Evaluation Summary:
DNA methylation inheritance through the UHRF1-DNMT1 signaling axis is becoming increasingly appreciated as a ubiquitin-regulated process. This study builds on the observation that UHRF1 multi mono-ubiquitinates the PCNA-associated protein PAF15, and that, similarly to H3 substrates, these mono-ubiquitin sites are bound by DNMT1 and may contribute to its S-phase chromatin association. The authors focus on players involved in ubiquitin removal and PAF15 release from chromatin and they identify the deubiquitinase USP7 and the DNA replication regulator ATAD5 as important to this termination process. While manipulation of these factors using Xenopus egg extracts shows quite striking effects on DNMT1 chromatin association, effects on DNA methylation are minimal and this brings to question the importance and potential impact of the pathway involving PAF15. In addition, how the findings from Xenopus egg extracts translate to regulation of DNA methylation maintenance in mammalian cells is currently unclear.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
UHRF1-dependent ubiquitin signaling plays an integral role in the regulation of maintenance DNA methylation. UHRF1 catalyzes transient dual mono-ubiquitylation of PAF15 (PAF15Ub2), which regulates the localization and activation of DNMT1 at DNA methylation sites during DNA replication. Although the initiation of UHRF1-mediated PAF15 ubiquitin signaling has been relatively well characterized, the mechanisms underlying its termination and how they are coordinated with the completion of maintenance DNA methylation have not yet been clarified. This study shows that deubiquitylation by USP7 and unloading by ATAD5 (ELG1 in yeast) are pivotal processes for the removal of PAF15 from chromatin. On replicating chromatin, USP7 specifically interacts with PAF15Ub2 in a complex with DNMT1. USP7 depletion or inhibition of the interaction between USP7 and PAF15 results in abnormal accumulation of PAF15Ub2 on chromatin. Furthermore, we also find that the non-ubiquitylated form of PAF15 (PAF15Ub0) is removed from chromatin in an ATAD5-dependent manner. PAF15Ub2 was retained at high levels on chromatin when the catalytic activity of DNMT1 was inhibited, suggesting that the completion of maintenance DNA methylation is essential for the termination of UHRF1-mediated ubiquitin signaling. This finding provides a molecular understanding of how the maintenance DNA methylation machinery is disassembled at the end of the S phase.
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Evaluation Summary:
DNA methylation inheritance through the UHRF1-DNMT1 signaling axis is becoming increasingly appreciated as a ubiquitin-regulated process. This study builds on the observation that UHRF1 multi mono-ubiquitinates the PCNA-associated protein PAF15, and that, similarly to H3 substrates, these mono-ubiquitin sites are bound by DNMT1 and may contribute to its S-phase chromatin association. The authors focus on players involved in ubiquitin removal and PAF15 release from chromatin and they identify the deubiquitinase USP7 and the DNA replication regulator ATAD5 as important to this termination process. While manipulation of these factors using Xenopus egg extracts shows quite striking effects on DNMT1 chromatin association, effects on DNA methylation are minimal and this brings to question the importance and potential …
Evaluation Summary:
DNA methylation inheritance through the UHRF1-DNMT1 signaling axis is becoming increasingly appreciated as a ubiquitin-regulated process. This study builds on the observation that UHRF1 multi mono-ubiquitinates the PCNA-associated protein PAF15, and that, similarly to H3 substrates, these mono-ubiquitin sites are bound by DNMT1 and may contribute to its S-phase chromatin association. The authors focus on players involved in ubiquitin removal and PAF15 release from chromatin and they identify the deubiquitinase USP7 and the DNA replication regulator ATAD5 as important to this termination process. While manipulation of these factors using Xenopus egg extracts shows quite striking effects on DNMT1 chromatin association, effects on DNA methylation are minimal and this brings to question the importance and potential impact of the pathway involving PAF15. In addition, how the findings from Xenopus egg extracts translate to regulation of DNA methylation maintenance in mammalian cells is currently unclear.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The manuscript by Miyashita et al describes deubiquitylation of PAF15 by USP7 and its role in regulation of replication-coupled DNA methylation maintenance. Previous studies from the same group showed that UHRF1-mediated dual mono-ubiquitylation of PAF15 (PAF15Ub2) promotes PAF15 chromatin loading and acts as a unique and critical molecular mark to recruit DNMT1 to the DNA replication sites. Here they show that termination of PAF15Ub2 signaling is regulated by USP7-mediated deubiquitylation and ATAD5-mediated removal from chromatin. These findings provide a molecular understanding of how the maintenance DNA methylation machinery is disassembled post replication.
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Reviewer #2 (Public Review):
In this work, Miyashita et al investigated the mechanism for the termination of the PAF15 ubiquitin signaling in DNMT1-mediated maintenance DNA methylation. Using a cell-free system, they showed that chromatin dissociation of PAF15 and DNMT1 is coupled with completion of maintenance DNA methylation and requires the deubiquitylation activity of USP7. Through GST pulldown and mutational analyses, they further identified and mapped an interaction between USP7 and PAF15, which is important for the USP7-mediated deubiquitylation of PAF15 with dual mono-ubiquitin (PAF15Ub2). In addition, they found that ATAD5, a protein that regulates the unloading of PCNA, promotes the release of PAF15Ub1/PAF15Ub0 from chromatin. Co-depletion of USP7 and ATAD5 from egg extracts results in an elevated global DNA methylation, …
Reviewer #2 (Public Review):
In this work, Miyashita et al investigated the mechanism for the termination of the PAF15 ubiquitin signaling in DNMT1-mediated maintenance DNA methylation. Using a cell-free system, they showed that chromatin dissociation of PAF15 and DNMT1 is coupled with completion of maintenance DNA methylation and requires the deubiquitylation activity of USP7. Through GST pulldown and mutational analyses, they further identified and mapped an interaction between USP7 and PAF15, which is important for the USP7-mediated deubiquitylation of PAF15 with dual mono-ubiquitin (PAF15Ub2). In addition, they found that ATAD5, a protein that regulates the unloading of PCNA, promotes the release of PAF15Ub1/PAF15Ub0 from chromatin. Co-depletion of USP7 and ATAD5 from egg extracts results in an elevated global DNA methylation, suggesting a cooperative effect between USP7 and ATAD5. Together, these studies identified USP7 and ATAD5 as two components critical for termination of PAF15 ubiquitin signaling pathway. Overall, the study is well designed and performed, largely supporting the central conclusion.
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Reviewer #3 (Public Review):
DNA methylation inheritance through the UHRF1-DNMT1 signaling axis is becoming increasingly appreciated as a ubiquitin-regulated process. Mechanistic studies of the enzymes and ubiquitin-dependent interactions that facilitate the association of DNMT1 with replicating chromatin are of fundamental importance for the epigenetics field and have potential to reveal mechanisms of dysregulation that are associated with abnormal DNA methylation patterning in human cancers. This study builds on the observation that UHRF1 multi mono-ubiquitinates the PCNA-associated protein PAF15, and that, similarly to H3 substrates, these mono-ubiquitin sites are bound by DNMT1 and may contribute to its S-phase chromatin association. The authors focus on players involved in ubiquitin removal and PAF15 release from chromatin. They …
Reviewer #3 (Public Review):
DNA methylation inheritance through the UHRF1-DNMT1 signaling axis is becoming increasingly appreciated as a ubiquitin-regulated process. Mechanistic studies of the enzymes and ubiquitin-dependent interactions that facilitate the association of DNMT1 with replicating chromatin are of fundamental importance for the epigenetics field and have potential to reveal mechanisms of dysregulation that are associated with abnormal DNA methylation patterning in human cancers. This study builds on the observation that UHRF1 multi mono-ubiquitinates the PCNA-associated protein PAF15, and that, similarly to H3 substrates, these mono-ubiquitin sites are bound by DNMT1 and may contribute to its S-phase chromatin association. The authors focus on players involved in ubiquitin removal and PAF15 release from chromatin. They identify the deubiquitinase USP7 and the DNA replication regulator ATAD5 as important to this termination process. However, while manipulation of these factors shows quite striking effects on DNMT1 chromatin association using Xenopus egg extracts as a model system for the process of DNA methylation maintenance, effects on DNA methylation are minimal, which brings to question the importance and potential impact of this pathway involving PAF15 and its role in regulating DNA methylation inheritance through mitotic cell divisions. This and other major concerns that limit my enthusiasm for this study in its current form are bulleted below.
Major concerns:
• Intro paragraph beginning on Line 86 needs more detail to support 1) a role for PAF15 in DNA methylation maintenance; 2) the contribution of PAF15Ub2 to DNMT1 chromatin association; and 3) the association of inefficient termination of PAF15Ub signaling with abnormal DNA methylation maintenance in human cancers. The way this paragraph is written makes it unclear whether these are speculations or whether there is strong data to support these claims.
• To what extent does inhibition of PAF15Ub2 support DNA methylation maintenance? Is it an absolute requirement, or only a partial contributor? And how does PAF15 compete with H3 (which is presumably in vast excess) for these Ub ligase, DUB, and reader activities? These questions are important to consider for framing the potential impact of this study that focuses on the PAF15-Ub regulatory mechanism. Does it really matter in a physiologic context and for the faithful propagation of DNA methylation patterns through mitotic cell divisions?
• (Line 116) Again, the way this is written 'to understand how the termination of DNA methylation maintenance is regulated' implies that PAF15Ub2 is a major regulator of DNMT1 function. I'm not so sure the data strongly support this claim.
• Fig 1A - with only a chromatin fraction shown, the authors cannot claim that 'PAF15 underwent dual mono-ub on chromatin and then dissociated from chromatin.' More generally, the paper relies exclusively on this chromatin association western blotting assay for querying chromatin interaction dynamics. Orthogonal approaches should be considered to strengthen conclusions being drawn.
• The extent to which these findings in Xenopus extracts translate to mammalian regulation of DNA methylation maintenance is unclear.
• Do the mutations that disrupt PAF15-USP7 interaction also affect PAF15-PCNA interaction?
• It is difficult to interpret Fig 3C since USP7 is also elevated in the 90 and 120 min time points in the presence of the inhibitor.
• For such striking effects of these perturbations on DNMT1 chromatin association, it is surprising that the effects on DNA methylation are subtle. This comes back to my comment above regarding whether this mechanism that has now been elegantly dissected actually matters for DNA methylation maintenance. To strengthen the impact of this work, it will be important to expand DNA methylation analyses and extend these findings to mammalian cells.
• (Line 517) How can the authors claim - or even suggest - that 'PAF15 Ub signaling is the primary pathway to maintain DNA methylation during S-phase' when there is limited evidence to support significant effects on DNA methylation maintenance in the absence of these factors? -
