DNMT1 is recruited by PCNA and UHRF1 to maintain DNA methylation after replication. UHRF1 recognizes hemimethylated DNA substrates via the SRA domain, but also repressive H3K9me3 histone marks with its TTD. With systematic mutagenesis and functional assays, we could show that chromatin binding further involved UHRF1 PHD binding to unmodified H3R2. These complementation assays clearly demonstrated that the ubiquitin ligase activity of the UHRF1 RING domain is required for maintenance DNA methylation. Mass spectrometry of UHRF1-deficient cells revealed H3K18 as a novel ubiquitination target of UHRF1 in mammalian cells. With bioinformatics and mutational analyses, we identified a ubiquitin interacting motif (UIM) in the N-terminal regulatory domain of DNMT1 that binds to ubiquitinated H3 tails and is essential for DNA methylation in vivo. H3 ubiquitination and subsequent DNA methylation required UHRF1 PHD binding to H3R2. These results show the manifold regulatory mechanisms controlling DNMT1 activity that require the reading and writing of epigenetic marks by UHRF1 and illustrate the multifaceted interplay between DNA and histone modifications. The identification and functional characterization of the DNMT1 UIM suggests a novel regulatory principle and we speculate that histone H2AK119 ubiquitination might also lead to UIM-dependent recruitment of DNMT1 and DNA methylation beyond classic maintenance.

Np95 is an important determinant in cell cycle progression. Its expression is tightly regulated and becomes detectable shortly before the entry of cells into S phase. Accordingly, Np95 is absolutely required for the G 1 /S transition. Its continued expression throughout the S/G 2 /M phases further suggests additional roles. Indeed, Np95 has been implicated in DNA damage response. Here, we show that Np95 is tightly bound to chromatin in vivo and that it binds to histones in vivo and in vitro. The binding to histones is direct and shows a remarkable preference for histone H3 and its N-terminal tail. A novel protein domain, the SRA-YDG domain, contained in Np95 is indispensable both for the interaction with histones and for chromatin binding in vivo. Np95 contains a RING finger. We show that this domain confers E3 ubiquitin ligase activity on Np95, which is specific for core histones, in vitro. Finally, Np95 shows specific E3 activity for histone H3 when the endogenous core octamer, coimmunoprecipitating with Np95, is used as a substrate. Histone ubiquitination is an important determinant in the regulation of chromatin structure and gene transcription. Thus, the demonstration that Np95 is a chromatin-associated ubiquitin ligase suggests possible molecular mechanisms for its action as a cell cycle regulator.Ubiquitination is a frequent posttranslational modification with a vast impact on cell physiology. Ubiquitin (Ub) is a conserved 76-amino-acid polypeptide that is covalently attached to target proteins via an isopeptide bond between its carboxyl-terminal glycine and the ε-amino group of a lysine in substrate proteins (23). A complex enzymatic cascade leads to ubiquitination. Ub is first activated through the formation of a thiol ester bond with the Ub-activating enzyme (E1) and then transferred to a Ub-conjugating enzyme (E2). Finally, Ub is transferred to the substrate through the action of a Ub ligase (E3). Two major families of E3s exist, the HECT type and the RING type. In HECT-E3-mediated catalysis, Ub is transferred from E2 to HECT-E3 and then by E3 to the substrate. In RING-E3-mediated catalysis, E3 mediates the direct transfer of Ub from E2 to the substrate. The E3 ligases, therefore, are the substrate recognition components of the system and confer specificity on the process (23, 47).The best-characterized type of ubiquitination is polyubiquitination, in which the substrate-bound Ub serves as an acceptor for further cycles of ubiquitination (23). By and large, polyubiquitin functions as a general device for targeting of the polyubiquitinated substrate to the proteasome, with ensuing proteolytic degradation (25,48). An emerging body of evidence indicates, however, that when Ub is appended as a single moiety to a target protein (monoubiquitination), the posttranslational modification has a completely different biological impact and serves primarily to modulate protein function and/or interaction(s) (10,24,57).Histones are among the major monoubiquitinated proteins in the cell, and histones H2A, H2B, and H3...

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits distribution, and reproduction in any medium, provided the original author and source are credited. This license does not permit commercial exploitation without specific permission.Recent studies have indicated that nuclear protein of 95 kDa (Np95) is essential for maintaining genomic methylation by recruiting DNA methyltransferase (Dnmt) 1 to hemi-methylated sites. Here, we show that Np95 interacts more strongly with regulatory domains of the de novo methyltransferases Dnmt3a and Dnmt3b. To investigate possible functions, we developed an epigenetic silencing assay using fluorescent reporters in embryonic stem cells (ESCs). Interestingly, silencing of the cytomegalovirus promoter in ESCs preceded DNA methylation and was strictly dependent on the presence of either Np95, histone H3 methyltransferase G9a or Dnmt3a and Dnmt3b. Our results indicate a regulatory role for Np95, Dnmt3a and Dnmt3b in mediating epigenetic silencing through histone modification followed by DNA methylation.

DNA methylation and histone modifications play a central role in the epigenetic regulation of gene expression and cell differentiation. Recently, Np95 (also known as UHRF1 or ICBP90) has been found to interact with Dnmt1 and to bind hemimethylated DNA, indicating together with genetic studies a central role in the maintenance of DNA methylation. Using in vitro binding assays we observed a weak preference of Np95 and its SRA (SET- and Ring-associated) domain for hemimethylated CpG sites. However, the binding kinetics of Np95 in living cells was not affected by the complete loss of genomic methylation. Investigating further links with heterochromatin, we could show that Np95 preferentially binds histone H3 N-terminal tails with trimethylated (H3K9me3) but not acetylated lysine 9 via a tandem Tudor domain. This domain contains three highly conserved aromatic amino acids that form an aromatic cage similar to the one binding H3K9me3 in the chromodomain of HP1ß. Mutations targeting the aromatic cage of the Np95 tandem Tudor domain (Y188A and Y191A) abolished specific H3 histone tail binding. These multiple interactions of the multi-domain protein Np95 with hemimethylated DNA and repressive histone marks as well as with DNA and histone methyltransferases integrate the two major epigenetic silencing pathways.