Abstract
In mammals, DNA methylation plays important roles in embryogenesis and terminal differentiation via regulation of the transcription-competent chromatin state. The methylation patterns are propagated to the next generation during replication by maintenance DNA methyltransferase, Dnmt1, in co-operation with Uhrf1. In the N-terminal regulatory region, Dnmt1 contains proliferating cell nuclear antigen (PCNA)-binding and replication foci targeting sequence (RFTS) domains, which are thought to contribute to maintenance methylation during replication. To determine the contributions of the N-terminal regulatory domains to the DNA methylation during replication, Dnmt1 lacking the RFTS and/or PCNA-binding domains was ectopically expressed in embryonic stem cells, and then the effects were analyzed. Deletion of both the PCNA-binding and RFTS domains did not significantly affect the global DNA methylation level. However, replication-dependent DNA methylation of the differentially methylated regions of three imprinted genes, Kcnq1ot1/Lit1, Peg3, and Rasgrf1, was impaired in cells expressing the Dnmt1 with not the PCNA-binding domain alone but both the PCNA-binding and RFTS domains deleted. Even in the absence of Uhrf1, which is a prerequisite factor for maintenance DNA methylation, Dnmt1 with both the domains deleted apparently maintained the global DNA methylation level, whilst the wild type and the forms containing the RFTS domain could not perform global DNA methylation under the conditions used. This apparent maintenance of the global DNA methylation level by the Dnmt1 lacking the RFTS domain was dependent on its own DNA methylation activity as well as the presence of de novo-type DNA methyltransferases. We concluded that the RFTS domain, not the PCNA-binding domain, is solely responsible for the replication-coupled DNA methylation. Furthermore, the RFTS domain acts as a safety lock by protecting the genome from replication-independent DNA methylation.
Highlights
In vertebrates, the 5th position of cytosine bases in CpG sequences is often methylated
The replication foci targeting sequence (RFTS) domain is reported to be a prerequisite for the tethering of Dnmt1 to the replication region [16]
To examine the roles of the PCNA-binding domain (PBD) and RFTS domain, stable clones expressing the full-length Dnmt1, oocyte-type Dnmt1, Dnmt1(291–1620), and Dnmt1(602–1620) with a myc-tag at their N-termini in mouse embryonic stem cells (ESC), in which the endogenous Dnmt1 gene in the genome is sandwiched between loxP sequences (Dnmt1-F/F), were established
Summary
The 5th position of cytosine bases in CpG sequences is often methylated. It is assumed that the NTD acts as a platform for the factors regulating Dnmt through tethering to specific regions. Among these factors, PCNA, which binds DNA polymerases and the factors necessary for replication, and is a prerequisite factor for replication, is thought to contribute to maintenance methylation during replication [9]. Dnmt processively methylates hemi-methylated DNA at replication foci in vivo For this processive methylation activity, the PCNA-binding domain (PBD) is dispensable under in vitro conditions [4]
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