CDCA7 is a hemimethylated DNA adaptor for the nucleosome remodeler HELLS

Abstract

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.

Fig. 1. CDCA7 and HELLS accumulate on chromatin upon inhibition of maintenance DNA methylation

(A) X. laevis sperm nuclei were incubated with interphase egg extracts depleted with mock IgG (ΔMOCK), anti-DNMT1 (ΔDNMT1), anti-HELLS (ΔHELLS), or anti-CDCA7e (ΔCDCA7e) antibodies for 3 h in the presence of cycloheximide. Chromatin was isolated and analyzed by western blotting. (B) X. laevis sperm nuclei were isolated at indicated time points after incubation with interphase Xenopus egg extracts in the presence or absence of 0.5 µM mouse DPPA3 (mDPPA3), a protein that inhibits binding of UHRF1 and DNMT1 to chromatin. Chromatin-associated proteins were analyzed by western blotting.

Fig. 2. CDCA7 selectively binds hemimethylated DNA

(A) Magnetic beads coupled with pBluescript DNA with unmethylated CpGs (un-Me), hemimethylated CpGs (hemi-Me), or fully methylated CpGs (full-Me), were incubated with interphase Xenopus egg extracts. Beads were collected after 60 min and analyzed by western blotting. (B) ³⁵S-labeled X. laevis CDCA7e proteins (wildtype or with the indicated ICF3-patient associated mutation) were incubated with control beads, or beads conjugated 200 bp unmethylated or hemimethylated DNA (Table S1). ³⁵S-labeled Xkid (85), a nonspecific DNA-binding protein, was used as a loading control. Autoradiography of ³⁵S-labeled proteins in input and beads fraction is shown. (C) and (D) Native gel electrophoresis mobility shift assay (EMSA) using recombinant X. laevis CDCA7e^WT and CDCA7e^R232H. (E) Left: schematic of H. sapiens CDCA7 (isoform 2 NP_665809). Positions of the zf-4CXXC_R1 domain (purple), three ICF3-patient mutations (cyan), and conserved cysteine residues (yellow) are shown. Right: EMSA assay using the purified zf-4CXXC_R1 domain (aa 264–371) of H. sapiens CDCA7. For C-E, double-stranded DNA oligonucleotides with an unmethylated, hemimethylated or fully-methylated CpG used for protein binding were visualized.

Fig. 3:. Cryo-EM structure of hCDCA7:nucleosome complex

(A) Native gel electrophoresis mobility shift assay analyzing the interaction of hCDCA7_264–371 C339S with nucleosomes. (B) A composite cryo-EM map (left) and the model structure of hCDCA7_264–371 C339S (generated from AF2) bound to nucleosome harboring a hemimethylated CpG at the 3’-linker DNA (right). The map corresponding to CDCA7 is colored purple. (C) Overlay of AF2 model of hCDCA7_264–371 C339S on the cryo-EM map. (D) Electrostatic surface potential of hCDCA7_264–371, where red and blue indicate negative and positive charges, respectively. Linker DNA is depicted in gray, orange indicates the location of 5-methylcytosine (5mC). (E) A model structure of hCDCA7_264–371 C339S bound to 3’-linker DNA. ICF mutation residues, R274 and R304, are shown as cyan stick model superimposed on a transparent sphere model.

Fig. 4. Identification of HELLS-CDCA7 interaction interface

(A) Schematics of X. laevis HELLS and CDCA7e. Positions of the signature 11 conserved cysteine residues and 3 ICF disease-associated mutations in CDCA7e are marked in yellow and cyan, respectively. CC1 is a coiled-coil domain important for autoinhibition. (B) The best predicted structure model of X. laevis HELLS-CDCA7e complex by AF2. (C) Sequence alignment of the putative HELLS/DDM1-binding interface of CDCA7. (D) Sequence alignment of the putative CDCA7-binding interface 1 in HELLS/DDM1. (E) Sequence alignment of the putative CDCA7-binding interface 2 in HELLS. (F) Immunoprecipitation by control IgG or anti-CDCA7e antibodies from Xenopus egg extracts containing ³⁵S-labeled wild-type or deletion mutant of X. laevis HELLS and CDCA7e. (G) Immunoprecipitation by control IgG or anti-HELLS antibody from Xenopus egg extracts containing ³⁵S-labeled HELLS and wild-type or Δ74–105 deletion mutant of CDCA7e. Autoradiography is shown in F and G.

Fig. 5. CDCA7 recruits HELLS to hemimethylated DNA

(A) Beads coated with unmethylated or hemimethylated DNA (pBluescript) were incubated with interphase Xenopus egg control mock IgG-depleted extracts (ΔMOCK) or CDCA7e-depleted extracts (ΔCDCA7e) for 30 min. Beads were isolated and analyzed by western blotting. (B) ³⁵S-labeled HELLS or HELLS Δ63–96 was incubated with beads coated with 200 bp unmethylated or hemimethylated DNA. Beads were isolated and associated ³⁵S-labeled proteins were visualized by autoradiography. Nonspecific DNA-binding protein Xkid DNA-binding domain (Xkid-DBD) was used as a loading control.

Fig. 6. CDCA7e and HELLS regulate replication-uncoupled maintenance DNA methylation

(A) Xenopus sperm nuclei were incubated for 120 min in interphase Xenopus egg extract in the presence of 0.5 µM recombinant mDPPA3. Chromatin was isolated and reincubated in interphase egg extract in the presence or absence of 150 μM aphidicolin (APH). (B, C). Sperm nuclei were incubated for 120 min in Mock-depleted extracts or either CDCA7e-depleted (B) or HELLS-depleted (C) extracts supplemented with mDPPA3. Chromatin was isolated and reincubated in Mock-depleted and either CDCA7e-depleted (B) or HELLS-depleted (C) extracts in the presence of aphidicolin. Chromatin was then isolated at indicated time points and chromatin-bound proteins were analyzed by western blotting using indicated antibodies.