RESUMO
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.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT , Dedos de Zinco PHD , Camundongos , Animais , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Cromatina , Metilação de DNA , Proteínas Cromossômicas não Histona/metabolismoRESUMO
Mutations of the SNF2 family ATPase HELLS and its activator CDCA7 cause immunodeficiency, centromeric instability, and facial anomalies syndrome, characterized by DNA hypomethylation at heterochromatin. It remains unclear why CDCA7-HELLS is the sole nucleosome remodeling complex whose deficiency abrogates the maintenance of DNA methylation. We here identify the unique zinc-finger domain of CDCA7 as an evolutionarily conserved hemimethylation-sensing zinc finger (HMZF) domain. Cryo-electron microscopy structural analysis of the CDCA7-nucleosome complex reveals that the HMZF domain can recognize hemimethylated CpG in the outward-facing DNA major groove within the nucleosome core particle, whereas UHRF1, the critical activator of the maintenance methyltransferase DNMT1, cannot. CDCA7 recruits HELLS to hemimethylated chromatin and facilitates UHRF1-mediated H3 ubiquitylation associated with replication-uncoupled maintenance DNA methylation. We propose that the CDCA7-HELLS nucleosome remodeling complex assists the maintenance of DNA methylation on chromatin by sensing hemimethylated CpG that is otherwise inaccessible to UHRF1 and DNMT1.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT , Metilação de DNA , Nucleossomos , Ubiquitina-Proteína Ligases , Humanos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Nucleossomos/metabolismo , Nucleossomos/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Microscopia Crioeletrônica , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/química , Ilhas de CpG , Ubiquitinação , Evolução Molecular , DNA/metabolismo , DNA/química , DNA/genética , Dedos de Zinco , Cromatina/metabolismo , Cromatina/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA Helicases/metabolismo , DNA Helicases/genética , DNA Helicases/química , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/química , Eucariotos/genética , Eucariotos/metabolismo , Ligação Proteica , Histonas/metabolismo , Histonas/genética , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/químicaRESUMO
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.