Structure of human DPPA3 bound to the UHRF1 PHD finger reveals its functional and structural differences from mouse DPPA3.

DNA methylation maintenance is essential for cell fate inheritance. In differentiated cells, this involves orchestrated actions of DNMT1 and UHRF1. In mice, the high-affinity binding of DPPA3 to the UHRF1 PHD finger regulates UHRF1 chromatin dissociation and cytosolic localization, which is required for oocyte maturation and early embryo development. However, the human DPPA3 ortholog functions during these stages remain unclear. Here, we report the structural basis for human DPPA3 binding to the UHRF1 PHD finger. The conserved human DPPA3 85VRT87 motif binds to the acidic surface of UHRF1 PHD finger, whereas mouse DPPA3 binding additionally utilizes two unique α-helices. The binding affinity of human DPPA3 for the UHRF1 PHD finger was weaker than that of mouse DPPA3. Consequently, human DPPA3, unlike mouse DPPA3, failed to inhibit UHRF1 chromatin binding and DNA remethylation in Xenopus egg extracts effectively. Our data provide novel insights into the distinct function and structure of human DPPA3.

Cryo-electron microscopy reveals the impact of the nucleosome dynamics on transcription activity.

The structural biology of nucleosomes and their complexes with chromatin-associated factors contributes to our understanding of fundamental biological processes in the genome. With the advent of cryo-electron microscopy (cryo-EM), several structures are emerging with histone variants, various species and chromatin-associated proteins that bind to nucleosomes. Cryo-EM enables visualization of the dynamic states of nucleosomes, leading to the accumulation of knowledge on chromatin-templated biology. The cryo-EM structure of nucleosome in Komagataella pastoris, as studied by Fukushima et al., provided the insights into transcription ability of RNAPII with nucleosome dynamics. In this commentary, we review the recent advances in the structural biology of nucleosomes and their related biomolecules.

Cooperative DNA-binding activities of Chp2 are critical for its function in heterochromatin assembly.

Heterochromatin protein 1 (HP1) is an evolutionarily conserved protein that plays a critical role in heterochromatin assembly. HP1 proteins share a basic structure consisting of an N-terminal chromodomain (CD) and a C-terminal chromoshadow domain (CSD) linked by a disordered hinge region. The CD recognizes histone H3 lysine 9 methylation, a hallmark of heterochromatin, while the CSD forms a dimer to recruit other chromosomal proteins. HP1 proteins have been shown to bind DNA or RNA primarily through the hinge region. However, how DNA or RNA binding contributes to their function remains elusive. Here, we focus on Chp2, one of the two HP1 proteins in fission yeast, and investigate how Chp2's DNA-binding ability contributes to its function. Similar to other HP1 proteins, the Chp2 hinge exhibits clear DNA-binding activity. Interestingly, the Chp2 CSD also shows robust DNA-binding activity. Mutational analysis revealed that basic residues in the Chp2 hinge and at the N-terminus of the CSD are essential for DNA binding, and the combined amino acid substitutions of these residues alter Chp2 stability, impair Chp2 heterochromatin localization and lead to a silencing defect. These results demonstrate that the cooperative DNA-binding activities of Chp2 play an important role in heterochromatin assembly in fission yeast.

The termination of UHRF1-dependent PAF15 ubiquitin signaling is regulated by USP7 and ATAD5.

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.

Novel compound heterozygous mutations in UHRF1 are associated with atypical immunodeficiency, centromeric instability and facial anomalies syndrome with distinctive genome-wide DNA hypomethylation.

Immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome is in most cases caused by mutations in either DNA methyltransferase (DNMT)3B, zinc finger and BTB domain containing 24, cell division cycle associated 7 or helicase lymphoid-specific. However, the causative genes of a few ICF patients remain unknown. We, herein, identified ubiquitin-like with plant homeodomain and really interesting new gene finger domains 1 (UHRF1) as a novel causative gene of one such patient with atypical symptoms. This patient is a compound heterozygote for two previously unreported mutations in UHRF1: c.886C > T (p.R296W) and c.1852C > T (p.R618X). The R618X mutation plausibly caused nonsense-mediated decay, while the R296W mutation changed the higher order structure of UHRF1, which is indispensable for the maintenance of CG methylation along with DNMT1. Genome-wide methylation analysis revealed that the patient had a centromeric/pericentromeric hypomethylation, which is the main ICF signature, but also had a distinctive hypomethylation pattern compared to patients with the other ICF syndrome subtypes. Structural and biochemical analyses revealed that the R296W mutation disrupted the protein conformation and strengthened the binding affinity of UHRF1 with its partner LIG1 and reduced ubiquitylation activity of UHRF1 towards its ubiquitylation substrates, histone H3 and proliferating cell nuclear antigen -associated factor 15 (PAF15). We confirmed that the R296W mutation causes hypomethylation at pericentromeric repeats by generating the HEK293 cell lines that mimic the patient's UHRF1 molecular context. Since proper interactions of the UHRF1 with LIG1, PAF15 and histone H3 are essential for the maintenance of CG methylation, the mutation could disturb the maintenance process. Evidence for the importance of the UHRF1 conformation for CG methylation in humans is, herein, provided for the first time and deepens our understanding of its role in regulation of CG methylation.

CDCA7 is a hemimethylated DNA adaptor for the nucleosome remodeler HELLS.

Mutations of the SNF2 family ATPase HELLS and its activator CDCA7 cause immunodeficiency-centromeric instability-facial anomalies (ICF) syndrome, characterized by hypomethylation at heterochromatin. The unique zinc-finger domain, zf-4CXXC_R1, of CDCA7 is widely conserved across eukaryotes but is absent from species that lack HELLS and DNA methyltransferases, implying its specialized relation with methylated DNA. Here we demonstrate that zf-4CXXC_R1 acts as a hemimethylated DNA sensor. The zf-4CXXC_R1 domain of CDCA7 selectively binds to DNA with a hemimethylated CpG, but not unmethylated or fully methylated CpG, and ICF disease mutations eliminated this binding. CDCA7 and HELLS interact via their N-terminal alpha helices, through which HELLS is recruited to hemimethylated DNA. While placement of a hemimethylated CpG within the nucleosome core particle can hinder its recognition by CDCA7, cryo-EM structure analysis of the CDCA7-nucleosome complex suggests that zf-4CXXC_R1 recognizes a hemimethylated CpG in the major groove at linker DNA. Our study provides insights into how the CDCA7-HELLS nucleosome remodeling complex uniquely assists maintenance DNA methylation.

Structural basis for activation of DNMT1.

DNMT1 is an essential enzyme that maintains genomic DNA methylation, and its function is regulated by mechanisms that are not yet fully understood. Here, we report the cryo-EM structure of human DNMT1 bound to its two natural activators: hemimethylated DNA and ubiquitinated histone H3. We find that a hitherto unstudied linker, between the RFTS and CXXC domains, plays a key role for activation. It contains a conserved α-helix which engages a crucial "Toggle" pocket, displacing a previously described inhibitory linker, and allowing the DNA Recognition Helix to spring into the active conformation. This is accompanied by large-scale reorganization of the inhibitory RFTS and CXXC domains, allowing the enzyme to gain full activity. Our results therefore provide a mechanistic basis for the activation of DNMT1, with consequences for basic research and drug design.

Structural basis for the unique multifaceted interaction of DPPA3 with the UHRF1 PHD finger.

Ubiquitin-like with PHD and RING finger domain-containing protein 1 (UHRF1)-dependent DNA methylation is essential for maintaining cell fate during cell proliferation. Developmental pluripotency-associated 3 (DPPA3) is an intrinsically disordered protein that specifically interacts with UHRF1 and promotes passive DNA demethylation by inhibiting UHRF1 chromatin localization. However, the molecular basis of how DPPA3 interacts with and inhibits UHRF1 remains unclear. We aimed to determine the structure of the mouse UHRF1 plant homeodomain (PHD) complexed with DPPA3 using nuclear magnetic resonance. Induced α-helices in DPPA3 upon binding of UHRF1 PHD contribute to stable complex formation with multifaceted interactions, unlike canonical ligand proteins of the PHD domain. Mutations in the binding interface and unfolding of the DPPA3 helical structure inhibited binding to UHRF1 and its chromatin localization. Our results provide structural insights into the mechanism and specificity underlying the inhibition of UHRF1 by DPPA3.

Structure-based screening combined with computational and biochemical analyses identified the inhibitor targeting the binding of DNA Ligase 1 to UHRF1.

The accumulation of epigenetic alterations is one of the major causes of tumorigenesis. Aberrant DNA methylation patterns cause genome instability and silencing of tumor suppressor genes in various types of tumors. Therefore, drugs that target DNA methylation-regulating factors have great potential for cancer therapy. Ubiquitin-like containing PHD and RING finger domain 1 (UHRF1) is an essential factor for DNA methylation maintenance. UHRF1 is overexpressed in various cancer cells and down-regulation of UHRF1 in these cells reactivates the expression of tumor suppressor genes, thus UHRF1 is a promising target for cancer therapy. We have previously shown that interaction between the tandem Tudor domain (TTD) of UHRF1 and DNA ligase 1 (LIG1) di/trimethylated on Lys126 plays a key role in the recruitment of UHRF1 to replication sites and replication-coupled DNA methylation maintenance. An arginine binding cavity (Arg-binding cavity) of the TTD is essential for LIG1 interaction, thus the development of inhibitors that target the Arg-binding cavity could potentially repress UHRF1 function in cancer cells. To develop such an inhibitor, we performed in silico screening using not only static but also dynamic metrics based on all-atom molecular dynamics simulations, resulting in efficient identification of 5-amino-2,4-dimethylpyridine (5A-DMP) as a novel TTD-binding compound. Crystal structure of the TTD in complex with 5A-DMP revealed that the compound stably bound to the Arg-binding cavity of the TTD. Furthermore, 5A-DMP inhibits the full-length UHRF1:LIG1 interaction in Xenopus egg extracts. Our study uncovers a UHRF1 inhibitor which can be the basis of future experiments for cancer therapy.

Preparation of the ubiquitination-triggered active form of SETDB1 in Escherichia coli for biochemical and structural analyses.

Trimethylation of histone H3 at K9 by the lysine methyltransferase, SET domain bifurcated histone lysine methyltransferase 1 (SETDB1) plays a pivotal role in silencing tissue-specific genes and retrotransposable elements. In mammalian cells, SETDB1 undergoes monoubiquitination in the insertion region of the SET domain in an E3 ubiquitin ligase-independent manner. This ubiquitination has been shown to enhance the histone H3-K9 methyltransferase activity of SETDB1; however, the molecular mechanism underlying SETDB1 activation by ubiquitination is unknown. In this study, we developed an Escherichia coli ubiquitination plasmid for the preparation of ubiquitinated SETDB1. Western blotting and mutational analyses showed that co-expression of the SET domain of SETDB1 with the proteins encoded by the ubiquitination plasmid led to site-specific monoubiquitination of the SET domain at K867. An in vitro histone H3 methylation assay demonstrated that the ubiquitinated SET domain of SETDB1 acquired enzymatic activity. Taken together, these findings demonstrate successful preparation of the active form of SETDB1 with the E.coli ubiquitination system, which will aid biochemical and structural studies of ubiquitinated SETDB1. Graphical Abstract.

Crystal structure of inhibitor-bound human MSPL that can activate high pathogenic avian influenza.

Infection of certain influenza viruses is triggered when its HA is cleaved by host cell proteases such as proprotein convertases and type II transmembrane serine proteases (TTSP). HA with a monobasic motif is cleaved by trypsin-like proteases, including TMPRSS2 and HAT, whereas the multibasic motif found in high pathogenicity avian influenza HA is cleaved by furin, PC5/6, or MSPL. MSPL belongs to the TMPRSS family and preferentially cleaves [R/K]-K-K-R↓ sequences. Here, we solved the crystal structure of the extracellular region of human MSPL in complex with an irreversible substrate-analog inhibitor. The structure revealed three domains clustered around the C-terminal α-helix of the SPD. The inhibitor structure and its putative model show that the P1-Arg inserts into the S1 pocket, whereas the P2-Lys and P4-Arg interacts with the Asp/Glu-rich 99-loop that is unique to MSPL. Based on the structure of MSPL, we also constructed a homology model of TMPRSS2, which is essential for the activation of the SARS-CoV-2 spike protein and infection. The model may provide the structural insight for the drug development for COVID-19.

Structural dynamics of double-stranded DNA with epigenome modification.

Modification of cytosine plays an important role in epigenetic regulation of gene expression and genome stability. Cytosine is converted to 5-methylcytosine (5mC) by DNA methyltransferase; in turn, 5mC may be oxidized to 5-hydroxymethylcytosine (5hmC) by ten-eleven translocation enzyme. The structural flexibility of DNA is known to affect the binding of proteins to methylated DNA. Here, we have carried out a semi-quantitative analysis of the dynamics of double-stranded DNA (dsDNA) containing various epigenetic modifications by combining data from imino 1H exchange and imino 1H R1ρ relaxation dispersion NMR experiments in a complementary way. Using this approach, we characterized the base-opening (kopen) and base-closing (kclose) rates, facilitating a comparison of the base-opening and -closing process of dsDNA containing cytosine in different states of epigenetic modification. A particularly striking result is the increase in the kopen rate of hemi-methylated dsDNA 5mC/C relative to unmodified or fully methylated dsDNA, indicating that the Watson-Crick base pairs undergo selective destabilization in 5mC/C. Collectively, our findings imply that the epigenetic modulation of cytosine dynamics in dsDNA mediates destabilization of the GC Watson-Crick base pair to allow base-flipping in living cells.

Serine 298 Phosphorylation in Linker 2 of UHRF1 Regulates Ligand-Binding Property of Its Tandem Tudor Domain.

Ubiquitin-like with PHD and RING finger domains 1 (UHRF1) is an essential factor for the maintenance of mammalian DNA methylation and harbors several reader modules for recognizing epigenetic marks. The tandem Tudor domain (TTD) of UHRF1 has a peptide-binding groove that functions as a binding platform for intra- or intermolecular interactions. Besides the groove interacting with unphosphorylated linker 2 and spacer of UHRF1, it also interacts with di/tri-methylated histone H3 at Lys9 and DNA ligase 1 (LIG1) at Lys126. Here we focus on the phosphorylation of Ser298 in linker 2, which was implied to regulate the ligand-binding property of the TTD. Although the protein expression level of UHRF1 is unchanged throughout the cell cycle, Ser298 phosphorylated form of UHRF1 is notably increased in the G2/M phase, which is revealed by immunoprecipitation followed by Western blotting. Molecularly, while unphosphorylated linker 2 covers the peptide-binding groove to prevent access of other interactors, small-angle X-ray scattering, thermal stability assay and molecular dynamics simulation revealed that the phosphate group of Ser298 dissociates linker 2 from the peptide-binding groove of the TTD to permit the other interactors to access to the groove. Our data reveal a mechanism in which Ser298 phosphorylation in linker 2 triggers a change of the TTD's structure and may affect multiple functions of UHRF1 by facilitating associations with LIG1 at DNA replication sites and histone H3K9me2/me3 at heterochromatic regions.

Two distinct modes of DNMT1 recruitment ensure stable maintenance DNA methylation.

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.

Enhanced processivity of Dnmt1 by monoubiquitinated histone H3.

DNA methylation controls gene expression, and once established, DNA methylation patterns are faithfully copied during DNA replication by the maintenance DNA methyltransferase Dnmt1. In vivo, Dnmt1 interacts with Uhrf1, which recognizes hemimethylated CpGs. Recently, we reported that Uhrf1-catalyzed K18- and K23-ubiquitinated histone H3 binds to the N-terminal region (the replication focus targeting sequence, RFTS) of Dnmt1 to stimulate its methyltransferase activity. However, it is not yet fully understood how ubiquitinated histone H3 stimulates Dnmt1 activity. Here, we show that monoubiquitinated histone H3 stimulates Dnmt1 activity toward DNA with multiple hemimethylated CpGs but not toward DNA with only a single hemimethylated CpG, suggesting an influence of ubiquitination on the processivity of Dnmt1. The Dnmt1 activity stimulated by monoubiquitinated histone H3 was additively enhanced by the Uhrf1 SRA domain, which also binds to RFTS. Thus, Dnmt1 activity is regulated by catalysis (ubiquitination)-dependent and -independent functions of Uhrf1.

Structure of PCNA in complex with DNMT1 PIP box reveals the basis for the molecular mechanism of the interaction.

DNMT1 is a C5-DNA methyltransferase that plays a pivotal role in DNA methylation maintenance. During early and mid S-phase, DNMT1 accumulates at DNA replication sites by binding to proliferating cell nuclear antigen (PCNA), an essential factor for DNA replication, through a PIP box motif. However, the molecular mechanism by which the DNMT1 PIP box motif binds to PCNA remains unclear. Here, we report the crystal structure of PCNA bound to DNMT1 PIP box peptide. The structure reveals the detailed interaction between PCNA and DNMT1 PIP box; conserved glutamine and hydrophobic/aromatic residues in the PIP box are recognized by the Q- and hydrophobic pockets of PCNA, respectively. The structure also shows novel intramolecular interactions within the PIP box motif, which stabilize the helix conformation in the PIP box. Our data provide structural insight into the recruitment of DNMT1 to replication sites by PCNA.

Structure of the UHRF1 Tandem Tudor Domain Bound to a Methylated Non-histone Protein, LIG1, Reveals Rules for Binding and Regulation.

The protein UHRF1 is crucial for DNA methylation maintenance. The tandem Tudor domain (TTD) of UHRF1 binds histone H3K9me2/3 with micromolar affinity, as well as unmethylated linker regions within UHRF1 itself, causing auto-inhibition. Recently, we showed that a methylated histone-like region of DNA ligase 1 (LIG1K126me2/me3) binds the UHRF1 TTD with nanomolar affinity, permitting UHRF1 recruitment to chromatin. Here we report the crystal structure of the UHRF1 TTD bound to a LIG1K126me3 peptide. The data explain the basis for the high TTD-binding affinity of LIG1K126me3 and reveal that the interaction may be regulated by phosphorylation. Binding of LIG1K126me3 switches the overall structure of UHRF1 from a closed to a flexible conformation, suggesting that auto-inhibition is relieved. Our results provide structural insight into how UHRF1 performs its key function in epigenetic maintenance.

Structure of the Dnmt1 Reader Module Complexed with a Unique Two-Mono-Ubiquitin Mark on Histone H3 Reveals the Basis for DNA Methylation Maintenance.

The proper location and timing of Dnmt1 activation are essential for DNA methylation maintenance. We demonstrate here that Dnmt1 utilizes two-mono-ubiquitylated histone H3 as a unique ubiquitin mark for its recruitment to and activation at DNA methylation sites. The crystal structure of the replication foci targeting sequence (RFTS) of Dnmt1 in complex with H3-K18Ub/23Ub reveals striking differences to the known ubiquitin-recognition structures. The two ubiquitins are simultaneously bound to the RFTS with a combination of canonical hydrophobic and atypical hydrophilic interactions. The C-lobe of RFTS, together with the K23Ub surface, also recognizes the N-terminal tail of H3. The binding of H3-K18Ub/23Ub results in spatial rearrangement of two lobes in the RFTS, suggesting the opening of its active site. Actually, incubation of Dnmt1 with H3-K18Ub/23Ub increases its catalytic activity in vitro. Our results therefore shed light on the essential role of a unique ubiquitin-binding module in DNA methylation maintenance.

RFTS-dependent negative regulation of Dnmt1 by nucleosome structure and histone tails.

DNA methylation in promoter regions represses gene expression and is copied over mitotic divisions by Dnmt1. Dnmt1 activity is regulated by its replication foci targeting sequence (RFTS) domain which masks the catalytic pocket. It has been shown that Dnmt1 activity on unmethylated DNA is inhibited in nucleosome cores. In the present study, we aimed to assess the effect of nuclesome formation on maintenance methylation at single CpG resolution. We show that Dnmt1 fully methylates naked linker DNA in dinucleosomes, whereas maintenance methylation was repressed at all CpG sites in nucleosome core particles. Deletion of RFTS partly released obstruction of Dnmt1 activity in core particles. Histone H3 tail peptides inhibited Dnmt1 in an RFTS-dependent manner and repression was modulated by acetylation or methylation. We propose a novel function of RFTS to regulate Dnmt1 activity in nucleosomes.

Methylation of DNA Ligase 1 by G9a/GLP Recruits UHRF1 to Replicating DNA and Regulates DNA Methylation.

DNA methylation is an essential epigenetic mark in mammals that has to be re-established after each round of DNA replication. The protein UHRF1 is essential for this process; it has been proposed that the protein targets newly replicated DNA by cooperatively binding hemi-methylated DNA and H3K9me2/3, but this model leaves a number of questions unanswered. Here, we present evidence for a direct recruitment of UHRF1 by the replication machinery via DNA ligase 1 (LIG1). A histone H3K9-like mimic within LIG1 is methylated by G9a and GLP and, compared with H3K9me2/3, more avidly binds UHRF1. Interaction with methylated LIG1 promotes the recruitment of UHRF1 to DNA replication sites and is required for DNA methylation maintenance. These results further elucidate the function of UHRF1, identify a non-histone target of G9a and GLP, and provide an example of a histone mimic that coordinates DNA replication and DNA methylation maintenance.