RESUMEN
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.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT , Ubiquitina-Proteína Ligasas , Animales , Humanos , Ratones , Secuencia de Aminoácidos , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/química , Cromatina/metabolismo , Metilación de ADN , Dedos de Zinc PHD/genética , Unión Proteica , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/química , Xenopus laevis/metabolismoRESUMEN
Decitabine (DAC) is clinically used to treat myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Our genome-wide CRISPR-dCas9 activation screen using MDS-derived AML cells indicates that mitotic regulation is critical for DAC resistance. DAC strongly induces abnormal mitosis (abscission failure or tripolar mitosis) in human myeloid tumors at clinical concentrations, especially in those with TP53 mutations or antecedent hematological disorders. This DAC-induced mitotic disruption and apoptosis are significantly attenuated in DNMT1-depleted cells. In contrast, overexpression of Dnmt1, but not the catalytically inactive mutant, enhances DAC-induced mitotic defects in myeloid tumors. We also demonstrate that DAC-induced mitotic disruption is enhanced by pharmacological inhibition of the ATR-CLSPN-CHK1 pathway. These data challenge the current assumption that DAC inhibits leukemogenesis through DNMT1 inhibition and subsequent DNA hypomethylation and highlight the potent activity of DAC to disrupt mitosis through aberrant DNMT1-DNA covalent bonds.
Asunto(s)
Azacitidina , Leucemia Mieloide Aguda , Humanos , Decitabina/farmacología , Decitabina/uso terapéutico , Azacitidina/farmacología , Azacitidina/uso terapéutico , Antimetabolitos Antineoplásicos/farmacología , Leucemia Mieloide Aguda/patología , Metilación de ADN/genética , ADN , Proteínas Adaptadoras Transductoras de Señales/genéticaRESUMEN
Protein misfolding is a major factor of neurodegenerative diseases. Post-mitotic neurons are highly susceptible to protein aggregates that are not diluted by mitosis. Therefore, post-mitotic cells may have a specific protein quality control system. Here, we show that LONRF2 is a bona fide protein quality control ubiquitin ligase induced in post-mitotic senescent cells. Under unperturbed conditions, LONRF2 is predominantly expressed in neurons. LONRF2 binds and ubiquitylates abnormally structured TDP-43 and hnRNP M1 and artificially misfolded proteins. Lonrf2-/- mice exhibit age-dependent TDP-43-mediated motor neuron (MN) degeneration and cerebellar ataxia. Mouse induced pluripotent stem cell-derived MNs lacking LONRF2 showed reduced survival, shortening of neurites and accumulation of pTDP-43 and G3BP1 after long-term culture. The shortening of neurites in MNs from patients with amyotrophic lateral sclerosis is rescued by ectopic expression of LONRF2. Our findings reveal that LONRF2 is a protein quality control ligase whose loss may contribute to MN degeneration and motor deficits.
Asunto(s)
Neuronas Motoras , Ubiquitina , Ratones , Animales , Neuronas Motoras/metabolismo , Ubiquitina/metabolismo , Ligasas/metabolismo , ADN Helicasas/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Proteínas de Unión al ADN/genéticaRESUMEN
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.
Asunto(s)
Ubiquitina-Proteína Ligasas , Ubiquitina , Ubiquitina/metabolismo , Peptidasa Específica de Ubiquitina 7/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/genética , Unión Proteica , Cromatina , ADN (Citosina-5-)-Metiltransferasa 1/genética , Metilación de ADNRESUMEN
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.
RESUMEN
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.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT , Dedos de Zinc PHD , Ratones , Animales , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Cromatina , Metilación de ADN , Proteínas Cromosómicas no Histona/metabolismoRESUMEN
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.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasas , Histonas , Humanos , ADN/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , Histonas/metabolismo , Ubiquitina/metabolismoRESUMEN
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.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/antagonistas & inhibidores , ADN Ligasa (ATP)/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Simulación de Dinámica Molecular , Piridinas/farmacología , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Animales , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Línea Celular Tumoral , ADN Ligasa (ATP)/metabolismo , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Humanos , Estructura Molecular , Piridinas/química , Relación Estructura-Actividad , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , XenopusRESUMEN
DNA methylation is a chemical modification that defines cell type and lineage through the control of gene expression and genome stability. Disruption of DNA methylation control mechanisms causes a variety of diseases, including cancer. Cancer cells are characterized by aberrant DNA methylation (i.e., genome-wide hypomethylation and site-specific hypermethylation), mainly targeting CpG islands in gene expression regulatory elements. In particular, the early findings that a variety of tumor suppressor genes (TSGs) are targets of DNA hypermethylation in cancer led to the proposal of a model in which aberrant DNA methylation promotes cellular oncogenesis through TSGs silencing. However, recent genome-wide analyses have revealed that this classical model needs to be reconsidered. In this review, we will discuss the molecular mechanisms of DNA methylation abnormalities in cancer as well as their therapeutic potential.
Asunto(s)
Metilación de ADN , Neoplasias , Islas de CpG/genética , Metilación de ADN/genética , Estudio de Asociación del Genoma Completo , Humanos , Neoplasias/genética , Neoplasias/metabolismoRESUMEN
DNA ligase 1 (LIG1) is known as the major DNA ligase responsible for Okazaki fragment joining. Recent studies have implicated LIG3 complexed with XRCC1 as an alternative player in Okazaki fragment joining in cases where LIG1 is not functional, although the underlying mechanisms are largely unknown. Here, using a cell-free system derived from Xenopus egg extracts, we demonstrated the essential role of PARP1-HPF1 in LIG3-dependent Okazaki fragment joining. We found that Okazaki fragments were eventually ligated even in the absence of LIG1, employing in its place LIG3-XRCC1, which was recruited onto chromatin. Concomitantly, LIG1 deficiency induces ADP-ribosylation of histone H3 in a PARP1-HPF1-dependent manner. The depletion of PARP1 or HPF1 resulted in a failure to recruit LIG3 onto chromatin and a subsequent failure in Okazaki fragment joining in LIG1-depleted extracts. Importantly, Okazaki fragments were not ligated at all when LIG1 and XRCC1 were co-depleted. Our results suggest that a unique form of ADP-ribosylation signaling promotes the recruitment of LIG3 on chromatin and its mediation of Okazaki fragment joining as a backup system for LIG1 perturbation.
Asunto(s)
ADN Ligasa (ATP)/metabolismo , ADN/metabolismo , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Sistema Libre de Células , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Xenopus laevisRESUMEN
Genome-wide DNA demethylation is a unique feature of mammalian development and naïve pluripotent stem cells. Here, we describe a recently evolved pathway in which global hypomethylation is achieved by the coupling of active and passive demethylation. TET activity is required, albeit indirectly, for global demethylation, which mostly occurs at sites devoid of TET binding. Instead, TET-mediated active demethylation is locus-specific and necessary for activating a subset of genes, including the naïve pluripotency and germline marker Dppa3 (Stella, Pgc7). DPPA3 in turn drives large-scale passive demethylation by directly binding and displacing UHRF1 from chromatin, thereby inhibiting maintenance DNA methylation. Although unique to mammals, we show that DPPA3 alone is capable of inducing global DNA demethylation in non-mammalian species (Xenopus and medaka) despite their evolutionary divergence from mammals more than 300 million years ago. Our findings suggest that the evolution of Dppa3 facilitated the emergence of global DNA demethylation in mammals.
Asunto(s)
Cromatina/metabolismo , Proteínas Cromosómicas no Histona , Desmetilación del ADN , Mamíferos/genética , Células Madre Pluripotentes/metabolismo , Animales , Evolución Biológica , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Metilación de ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Epigenómica , Evolución Molecular , Regulación de la Expresión Génica , Genes Reguladores , Células Germinativas/metabolismo , Ratones , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Cell senescence plays a key role in age-associated organ dysfunction, but the in vivo pathogenesis is largely unclear. Here, we generated a p16-CreERT2-tdTomato mouse model to analyze the in vivo characteristics of p16high cells at a single-cell level. We found tdTomato-positive p16high cells detectable in all organs, which were enriched with age. We also found that these cells failed to proliferate and had half-lives ranging from 2.6 to 4.2 months, depending on the tissue examined. Single-cell transcriptomics in the liver and kidneys revealed that p16high cells were present in various cell types, though most dominant in hepatic endothelium and in renal proximal and distal tubule epithelia, and that these cells exhibited heterogeneous senescence-associated phenotypes. Further, elimination of p16high cells ameliorated nonalcoholic steatohepatitis-related hepatic lipidosis and immune cell infiltration. Our new mouse model and single-cell analysis provide a powerful resource to enable the discovery of previously unidentified senescence functions in vivo.
Asunto(s)
Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Animales , Línea Celular , Senescencia Celular , Humanos , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Análisis de la Célula IndividualRESUMEN
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.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Metilación de ADN/genética , Ubiquitinación , Animales , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Cromatina/metabolismo , Humanos , Masculino , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Unión Proteica , Espermatozoides/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Xenopus laevisRESUMEN
We report combinations of a DMAP-based catalyst and phenyl acetate with optimal electron density as a new chemical system for high-yield, selective synthetic acetylation of histone lysine residues. The utility of this chemical system as a unique biologic tool is demonstrated by applying it to Xenopus laevis sperm chromatin.
RESUMEN
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.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasa 1/genética , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Histonas/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/genética , ADN/metabolismo , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , Replicación del ADN , Histonas/fisiología , Humanos , Unión Proteica , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
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.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/química , Metilación de ADN , Histonas/química , Ubiquitina/química , Animales , Cristalografía por Rayos X , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Unión Proteica , Estructura Cuaternaria de Proteína , Ubiquitina/genética , Ubiquitina/metabolismo , Xenopus laevisRESUMEN
Uhrf1-dependent histone H3 ubiquitylation plays a crucial role in the maintenance of DNA methylation via the recruitment of the DNA methyltransferase Dnmt1 to DNA methylation sites. However, the involvement of deubiquitylating enzymes (DUBs) targeting ubiquitylated histone H3 in the maintenance of DNA methylation is largely unknown. With the use of Xenopus egg extracts, we demonstrate here that Usp7, a ubiquitin carboxyl-terminal hydrolase, forms a stable complex with Dnmt1 and is recruited to DNA methylation sites during DNA replication. Usp7 deubiquitylates ubiquitylated histone H3 in vitro. Inhibition of Usp7 activity or its depletion in egg extracts results in enhanced and extended binding of Dnmt1 to chromatin, suppressing DNA methylation. Depletion of Usp7 in HeLa cells causes enhanced histone H3 ubiquitylation and enlargement of Dnmt1 nuclear foci during DNA replication. Our results thus suggest that Usp7 is a key factor that regulates maintenance of DNA methylation.
Asunto(s)
Metilación de ADN , Histonas/metabolismo , Peptidasa Específica de Ubiquitina 7/metabolismo , Ubiquitinación , Animales , Cromatina/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Replicación del ADN/fisiología , Células HeLa , Humanos , Óvulo , XenopusRESUMEN
The mammalian maintenance methyltransferase DNMT1 [DNA (cytosine-5-)-methyltransferase 1] mediates the inheritance of the DNA methylation pattern during replication. Previous studies have shown that depletion of DNMT1 causes a severe growth defect and apoptosis in differentiated cells. However, the detailed mechanisms behind this phenomenon remain poorly understood. Here we show that conditional ablation of Dnmt1 in murine embryonic fibroblasts (MEFs) resulted in an aberrant DNA replication program showing an accumulation of late-S phase replication and causing severely defective growth. Furthermore, we found that the catalytic activity and replication focus targeting sequence of DNMT1 are required for a proper DNA replication program. Taken together, our findings suggest that the maintenance of DNA methylation by DNMT1 plays a critical role in proper regulation of DNA replication in mammalian cells.
Asunto(s)
Daño del ADN/genética , Metilación de ADN/genética , Replicación del ADN/genética , Proteínas de Mantenimiento de Minicromosoma/genética , Origen de Réplica/genética , Proteínas Represoras/genética , Animales , Proliferación Celular/genética , Células Cultivadas , Fibroblastos/fisiología , Genes cdc/genética , RatonesRESUMEN
DNA methyltransferase 1 (DNMT1) plays an essential role in propagation of the DNA methylation pattern to daughter cells. The replication foci targeting sequence (RFTS) of DNMT1 is required for the recruitment of DNMT1 to DNA methylation sites through direct binding to ubiquitylated histone H3 mediated by UHRF1 (Ubiquitin-like containing PHD and RING finger domains 1). Recently, it has been reported that the RFTS plugs the catalytic pocket of DNMT1 in an intermediated manner and inhibits its DNA methyltransferase activity. However, it is unclear whether this binding affects RFTS function in terms of recruitment to DNA methylation sites. Using Xenopus egg extracts, we demonstrate here that abrogation of the interaction between the RFTS and the catalytic center of DNMT1, by deletion of the C-terminal portion or disruption of the hydrogen bond, results in non-ubiquitylated histone H3 binding and abnormal accumulation of DNMT1 on the chromatin. Interestingly, DNMT1 mutants identified in patients with a neurodegenerative disease, ADCA-DN, bound to non-ubiquitylated histone H3 and accumulated on chromatin during S phase in Xenopus egg extracts. These results suggest that the interaction between the RFTS and the catalytic center of DNMT1 serves as an autoinhibitory mechanism for suppressing the histone H3 binding of DNMT1 and ensuring the accurate recruitment of DNMT1 to sites of DNA methylation. The autoinhibitory mechanism may play an important role in the regulation of gene expression in neurogenesis.