
==== Front
Nucleic Acids Res
Nucleic Acids Res
nar
Nucleic Acids Research
0305-1048
1362-4962
Oxford University Press

39142653
10.1093/nar/gkae677
gkae677
AcademicSubjects/SCI00010
Gene regulation, Chromatin and Epigenetics
The C-terminal 4CXXC-type zinc finger domain of CDCA7 recognizes hemimethylated DNA and modulates activities of chromatin remodeling enzyme HELLS
Shinkai Akeo Cellular Memory Laboratory, RIKEN Cluster for Pioneering Research, Wako City, Saitama 351-0198, Japan

Hashimoto Hideharu Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, 19107, USA

Shimura Chikako Cellular Memory Laboratory, RIKEN Cluster for Pioneering Research, Wako City, Saitama 351-0198, Japan

Fujimoto Hiroaki Cellular Memory Laboratory, RIKEN Cluster for Pioneering Research, Wako City, Saitama 351-0198, Japan
Division of Life Science, Graduate School of Science & Engineering, Saitama University, Shimo-Ohkubo 255, Sakura Ward, Saitama City, Saitama 338-8570, Japan

Fukuda Kei Faculty of Life and Environmental Sciences, University of Yamanashi, Yamanashi 400-8510, Japan

https://orcid.org/0000-0003-1842-6934
Horikoshi Naoki Laboratory of Chromatin Structure and Function, Institute for Quantitative Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan

Okano Masaki Department of Pluripotent Stem Cell Biology, IMEG, Kumamoto university, Honjo 2-2-1, Chuo-ku, Kumamoto, Kumamoto 860-0811, Japan

Niwa Hitoshi Department of Pluripotent Stem Cell Biology, IMEG, Kumamoto university, Honjo 2-2-1, Chuo-ku, Kumamoto, Kumamoto 860-0811, Japan

https://orcid.org/0000-0002-2587-2150
Debler Erik W Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, 19107, USA

https://orcid.org/0000-0001-7412-3722
Kurumizaka Hitoshi Laboratory of Chromatin Structure and Function, Institute for Quantitative Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan

https://orcid.org/0000-0002-6051-2484
Shinkai Yoichi Cellular Memory Laboratory, RIKEN Cluster for Pioneering Research, Wako City, Saitama 351-0198, Japan
Division of Life Science, Graduate School of Science & Engineering, Saitama University, Shimo-Ohkubo 255, Sakura Ward, Saitama City, Saitama 338-8570, Japan

To whom correspondence should be addressed. Tel: +81 50 3502 7462; Email: yshinkai@riken.jp
Correspondence may also be addressed to Akeo Shinkai. Tel: +81 50 3502 7462; Email: akeo.shinkai@riken.jp
23 9 2024
15 8 2024
15 8 2024
52 17 1019410219
25 7 2024
25 6 2024
11 1 2024
© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Abstract

The chromatin-remodeling enzyme helicase lymphoid-specific (HELLS) interacts with cell division cycle-associated 7 (CDCA7) on nucleosomes and is involved in the regulation of DNA methylation in higher organisms. Mutations in these genes cause immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome, which also results in DNA hypomethylation of satellite repeat regions. We investigated the functional domains of human CDCA7 in HELLS using several mutant CDCA7 proteins. The central region is critical for binding to HELLS, activation of ATPase, and nucleosome sliding activities of HELLS-CDCA7. The N-terminal region tends to inhibit ATPase activity. The C-terminal 4CXXC-type zinc finger domain contributes to CpG and hemimethylated CpG DNA preference for DNA-dependent HELLS-CDCA7 ATPase activity. Furthermore, CDCA7 showed a binding preference to DNA containing hemimethylated CpG, and replication-dependent pericentromeric heterochromatin foci formation of CDCA7 with HELLS was observed in mouse embryonic stem cells; however, all these phenotypes were lost in the case of an ICF syndrome mutant of CDCA7 mutated in the zinc finger domain. Thus, CDCA7 most likely plays a role in the recruitment of HELLS, activates its chromatin remodeling function, and efficiently induces DNA methylation, especially at hemimethylated replication sites.

Graphical Abstract

Graphical Abstract

Japan Ministry of Education, Culture, Sports, Science and Technology Grant-in-Aid for Scientific Research 18H05530 18H03991 18H05534 23H05475 Japan Science and Technology Agency 10.13039/501100002241 JPMJER1901 RIKEN 10.13039/501100006264 National Institute of Allergy and Infectious Diseases 10.13039/100000060 National Institutes of Health 10.13039/100000002 R01 AI165840 Research Support Project for Life Science and Drug Discovery AMED 10.13039/100009619 JP23ama121009 Japan Society for the Promotion of Science 10.13039/501100001691
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pmcIntroduction

It has been indicated that the chromatin-remodeling enzyme helicase lymphoid-specific (HELLS) [also called lymphoid-specific (LSH), proliferation-associated SNF2-like (PASG), or SWI/SNF-related matrix-associated, actin-dependent regulator of chromatin group A6 (SMARCA6)] or decrease in DNA methylation 1 (DDM1), a plant homolog of HELLS, is involved in the regulation of genome-wide DNA methylation (1–3). DNA methylation is involved in various biological processes, such as the regulation of gene expression, inactivation of retrotransposons, X chromosome inactivation, and genomic imprinting (4–6). In mammals, DNA is preferentially methylated at the C5 position of cytosine at CpG sites. DNA methylation is categorized into two types. One is de novo DNA methylation, which methylates unmethylated DNA and is mainly catalyzed by DNA methyltransferases (DNMT)3A and DNMT3B (7). The other is the maintenance DNA methylation, which methylates hemimethylated DNA after DNA replication, mainly catalyzed by DNMT1 (8), assisted by ubiquitin-like with PHD and RING finger domains 1 (UHRF1) to target hemimethylated DNA (9,10). It has been indicated that HELLS is involved in both de novo (11,12) and maintenance (13,14) DNA methylation.

Cell division cycle-associated 7 (CDCA7; also called JPO1) is a nuclear protein that is transcriptionally regulated by c-Myc and E2F1 (15,16) and is highly expressed in human cancers (17). Human CDCA7 consists of 371 amino acid residues with a c-Myc and 14–3–3 protein-binding region and a C-terminal 4CXXC-type zinc finger (ZF) domain (18,19). In addition to DNMT3b, ZBTB24, CDCA7, HELLS and UHRF1 are causative genes of immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome, a rare autosomal recessive immunological/neurological disorder characterized by the loss of DNA methylation and chromosomal instability (18,20–22). Thus, CDCA7 and HELLS have been suggested to be involved in regulating DNA methylation. Jenness et al. (23) showed that CDCA7 from Xenopus eggs recruits HELLS onto nucleosomes to form the HELLS-CDCA7 complex, which activates the ATPase and nucleosome sliding activities of HELLS. Based on these observations, they proposed a model in which CpG sites exposed from the nucleosome by HELLS-CDCA7 are methylated by DNMTs. However, the details of how the chromatin-remodeling function of HELLS is regulated by CDCA7 are not well understood. It has also recently been suggested that HELLS contributes to the regulation of maintenance methylation via UHRF1/DNMT1 (14); but, the involvement of CDCA7 in this function is not known.

To clarify the role of CDCA7 in HELLS function and HELLS-mediated regulation of DNA methylation, we generated recombinant HELLS and various mutant CDCA7 proteins and examined their effects on HELLS function in vitro. We also examined the nuclear localization of CDCA7 and HELLS in wild-type and Cdca7 knockout (Cdca7 KO) mouse embryonic stem cells (mESCs). We found that the central domain of CDCA7 was critical for binding to HELLS, ATPase activity, and nucleosome sliding activity of HELLS. We also found that the C-terminal 4CXXC-type ZF domain of CDCA7 was involved in the recognition of CpG sites to guide HELLS to activate ATPase activity and nucleosome sliding activity. Furthermore, the C-terminal 4CXXC-type ZF domain preferentially recognizes hemimethylated CpG sites rather than unmethylated CpG sites to activate HELLS and is involved in replication-dependent pericentromeric heterochromatin (PHC) foci formation with HELLS. In response to its ability to recognize hemimethylated DNA, CDCA7 accumulates at the PHC (PHC foci formation) in a replication-dependent manner, and HELLS also accumulates at the PHC in the same mode, which is CDCA7-dependent; the ICF mutant CDCA7, which loses the hemimethylated DNA recognition preference and replication-dependent PHC foci formation, abolishes the PHC localization of HELLS. These results suggest that CDCA7 forms a complex with HELLS and recruits HELLS to the replication sites through the hemimethylated DNA-binding activity of its C-terminal ZF domain and modulates HELLS-dependent chromatin remodeling activities at the sites associated with maintenance DNA methylation.

Materials and methods

Construction of the plasmids for preparation of the recombinant bacmids

The oligonucleotides used for construction of the plasmids are summarized in Supplementary Table S1. The ligation reactions were performed with Gibson assembly method (24), using Gibson Assembly Master Mix (New England BioLabs).

To construct the plasmid pI-HisSUMO-HELLS, cDNA encoding the fusion protein consisting of (His)6 tag, small ubiquitin-like modifier (SUMO) tag and human HELLS was cloned under the polyhedrin promoter in the baculovirus transfer vector pI-SUMOstar (LifeSensors, Inc.). The vector linearized by PCR using oligonucleotides P1 and P2 as primers was ligated with the fragment amplified by PCR using the plasmid p3xFLAG hHELLS WT (received from Dr Motoko Unoki of the University of Tokyo) (25), carrying the human HELLS, as a template and oligonucleotides P3 and P4 as primers.

To construct the plasmid pI-HisMBP-CDCA7, cDNA encoding the fusion protein consisting of (His)6 tag, Escherichia coli maltose-binding protein (MBP), and human CDCA7 was cloned under the polyhedrin promoter in the baculovirus transfer vector pI-SUMOstar. The vector was linearized by PCR using oligonucleotides P2 and P5 as primers. The fragment encoding the MBP was amplified by PCR using the pMal-c5X vector (New England BioLabs) as a template and oligonucleotides P6 and P7 as primers. For preparation of the fragment encoding the CDCA7, PCR was performed using the plasmid pI-HisSUMO-CDCA7 (see below) as a template and oligonucleotides P8 and P9 as primers. The three fragments were ligated to construct the pI-HisMBP-CDCA7. For construction of the pI-HisSUMO-CDCA7, the linearized vector used for the construction of the pI-HisSUMO-HELLS (see above) was ligated with the fragment amplified by PCR using the plasmid p3xFLAG hCDCA7 WT (received from Dr Motoko Unoki of the University of Tokyo (25), carrying the human CDCA7, as a template and oligonucleotides P9 and P10 as primers.

For construction of the plasmid carrying the deletion mutant of CDCA7, the vector linearized by PCR using the pI-HisMBP-CDCA7 (see above) as a template and oligonucleotides P2 and P11 as primers, was ligated with a fragment encoding a deletion mutant of CDCA7. The mutant DNA fragments were amplified by PCR using the pI-HisMBP-CDCA7 as a template, and oligonucleotides P9 and P12 for the 31–371, P9 and P13 for the 68–371, P9 and P14 for the 108–371, P9 and P15 for the 140–371, P9 and P16 for the 177–371, P9 and P17 for the 218–371, P9 and P18 for the 262–371, P8 and P19 for the 1–261, P8 and P20 for the 1–176, P8 and P21 for the 1–143, P8 and P22 for the 1–107, and P14 and P19 for the 108–261 mutants as primers, respectively. To construct the pI-HisSUMO-wild-type CDCA7-MBP, the vector linearized by PCR using the pI-HisSUMO-CDCA7 (see above) as a template and oligonucleotides P23 and P2 as primers, was ligated with a fragment using the pMal-c5X (New England BioLabs) as a template and oligonucleotides P24 and P25 as primers. For construction of the pI-HisSUMO-CDCA7_68–371-MBP, the linearized vector used for the construction of the pI-HisSUMO-HELLS (see above), was ligated with a fragment using the pI-HisMBP-CDCA7_68–371 (see above) as a template and oligonucleotides P26 and P23 as primers and the fragment used for construction of the pI-HisSUMO-wild-type CDCA7-MBP.

For construction of the expression plasmids for the ICF syndrome mutants and T163A mutant of CDCA7, site-directed mutagenesis was performed by PCR using 1 pg/μl of the pI-HisMBP-CDCA7 (see above) as a template and 0.1 μM each of oligonucleotides P27 and P28 for the R274C, P29 and P30 for the R274H, P31 and P32 for the R304H, and P33 and P34 for the T163A mutant, as primers, respectively. The PCR was performed with 30 cycles of 98°C for 10 s, 55°C for 15 s and 72°C for 40 s, by using the PrimeStarMax (Takara Bio Inc.).

To construct the plasmid pI-HisSUMO-DDM1, cDNA encoding the fusion protein consisting of (His)6 tag, SUMO tag and Arabidopsis thaliana DDM1 was cloned under the polyhedrin promoter in the baculovirus transfer vector pI-SUMOstar. The vector used for construction of the plasmid pI-HisSUMO-HELLS (see above) was ligated with the fragment amplified by PCR using the plasmid pda08628 (26,27), carrying the DDM1, as a template and oligonucleotides P35 and P36 as primers. To construct the plasmid pI-HisMBP-Arabidopsis CDCA7, cDNA encoding the fusion protein consisting of (His)6 tag, MBP tag, and A. thaliana CDCA7 (NCBI accession no. NP_195428) was cloned under the polyhedrin promoter in the baculovirus transfer vector pI-SUMOstar. The vector used for construction of the plasmid carrying the deletion mutant of CDCA7 (see above) was ligated with the fragment amplified by PCR using the plasmid pda19584 (26,27), carrying the A. thaliana CDCA7, as a template and oligonucleotides P37 and P38 as primers.

Construction of the recombinant baculovirus and expression of the recombinant protein in insect cells

The transfer vector carrying the recombinant gene (see above) was introduced into E. coli DH10Bac strain (Thermo Fisher Scientific), and the recombinant bacmid was prepared from the recombinant strain by using Plasmid Midi Kit (QIAGEN). Two microgram of the bacmid was transfected to 2 × 106 of Spodoptera frugiperda (Sf9) cells with 6 μl of the Cellfectin Reagent (Thermo Fisher Scientific) in 2 ml of Sf-900 II SFM medium (Thermo Fisher Scientific) at 27°C for 1 week to prepare the recombinant virus from the supernatant. Then, 0.1 ml of the virus was transfected to a confluent monolayer (0.5−1 × 107 cells) of Sf9 cells grown in 10 ml of Sf-900 II SFM medium containing 10% of fetal calf serum (Equitech-Bio Inc.), 100 units/ml penicillin, and 0.1 mg/ml streptomycin (Thermo Fisher Scientific) at 27°C for 1 week, and then the supernatant was prepared as the secondary recombinant virus.

For expression of the recombinant protein, Sf9 cells were grown in suspension culture in Sf-900 II SFM medium containing 2% fetal calf serum, 100 units/ml penicillin, and 0.1 mg/ml streptomycin at 27°C. When the cell concentration attained 1−2 × 106 cells/ml, the secondary recombinant virus was transfected to the cells (0.5 ml for 108 cells), and continued cultivation for further 72 h.

Preparation of the recombinant proteins

To prepare the HELLS, the recombinant insect cells expressing the HisSUMO-HELLS (wet weight is around 1 g) were resuspended in buffer A (20 mM Tris–HCl, pH 8.0, 0.5 M NaCl) containing 50 mM imidazole and protease inhibitor cocktail (EDTA-free, Nacalai tesque, Inc.), then the cells were disrupted by sonication. The sample was centrifuged at 44 000 × g for 30 min at 4°C, and then the supernatant was filtered by using PVDF membrane (pore size: 0.45 μm), and then applied to a nickel-nitrilotriacetic acid (Ni-NTA) agarose (4 ml) (QIAGEN) column. The column was washed with the buffer A containing 50 mM imidazole, then the bound proteins were eluted with the buffer A containing 0.3 M imidazole. Dithiothreitol (DTT) and the SUMOstar protease (Life Sensors, Inc.) was added to the sample at a concentration of 2 mM and 50 units, respectively, and then dialyzed against buffer B (20 mM Tris–HCl, pH 8.0, 0.1 M NaCl) containing 2 mM DTT. The sample was applied to a Resource S column (1 ml) (Cytiva) preequilibrated with the buffer B. The column was washed with the buffer B, and then the bound protein was eluted with a linear gradient of 0.1 to 0.5 M NaCl in 20 mM Tris–HCl, pH 8.0. The fractions containing the HELLS were collected and dialyzed against the buffer B, and then concentrated with an Amicon Ultra concentrator (10-kDa molecular weight cut-off, Merck). The protein was stored at –80°C in the buffer B containing 10% glycerol and 2 mM 2-mercaptethanol (MET).

The DDM1 was prepared from the recombinant insect cells expressing the HisSUMO-DDM1 by basically the same way as that in the case of the HELLS, except that the buffer used for resuspension of the cells and for washing the Ni-NTA agarose column was the buffer A containing 20 mM imidazole, and the buffer for elution from the column was the buffer A containing 0.2 M imidazole. The fractions containing the DDM1 was collected and dialyzed against buffer C (20 mM Tris–HCl, pH 8.0, 0.15 M NaCl), and then concentrated as described above. The HisSUMO-DDM1 was prepared by basically the same way as that in the case of the DDM1 with no SUMOstar protease treatment. The proteins were stored at –80°C in the buffer C containing 10% glycerol and 2 mM MET.

To prepare the HisMBP-CDCA7 and HisMBP-Arabidopsis CDCA7 proteins, the recombinant insect cells (wet weight is around 1 g) were disrupted, and then the Ni-NTA agarose column chromatography was performed as described in the case of the HELLS except that the buffer used for resuspension of the cells and for washing the column was the buffer A containing 30 mM imidazole. The protein eluted from the column was applied to an amylose resin (3 ml) (New England BioLabs) column preequilibrated with the buffer A. The column was washed with the same buffer, and the bound protein was eluted with the buffer A containing 20 mM maltose. The fractions containing the HisMBP-CDCA7 was collected and dialyzed against the buffer A, and then concentrated as described above. The CDCA7-MBP was prepared from the recombinant cells expressing the HisSUMO-CDCA7-MBP. The cells (wet weight is around 1 g) were disrupted and fractionated by the Ni-NTA agarose column chromatography by basically the same way as that in the case of the DDM1. The fractions containing the protein was collected, and DTT and the SUMOstar protease (Life Sensors, Inc.) was added to the sample at a concentration of 2 mM and 50 units, respectively, and then dialyzed against the buffer A containing 2 mM DTT. The sample was fractionated by the Ni-NTA agarose column, and the flowthrough fraction was collected. The proteins bound to an amylose resin column were collected, dialyzed against the buffer A, and concentrated as described above. The protein was stored at -80°C in the buffer A containing 10% glycerol and 2 mM MET.

To prepare the HisMBP, a HisMBP-CDCA7 protein fractionated by the Ni-NTA agarose column chromatography (see above) was dialyzed against buffer comprising 50 mM Tris–HCl, pH 8.0, 0.1 M NaCl, and 1 mM CaCl2, for 1.5 h at 4°C, and then 15 μg of the factor Xa protease (New England BioLabs) was added to the sample, followed by dialysis against the same buffer at 22°C for 17 h. Then, amylose resin column chromatography was performed as described above, and then the eluted protein was applied to a Ni-NTA agarose column, washed with the buffer A, and the bound protein was eluted with the buffer A containing 75 mM imidazole. The protein was dialyzed against the buffer A, and then concentrated as described above. The protein was stored at –80°C in the buffer A containing 10% glycerol and 2 mM MET.

The HisSUMO was prepared from the HisSUMO-HELLS sample. Flowthrough fraction of the Resource S column chromatography after SUMOstar protease treatment (see above) was applied to a Ni-NTA agarose column (4 ml) preequilibrated with the buffer A, washed with the same buffer, and then eluted with the buffer containing 0.4 M imidazole. The sample was dialyzed against buffer A, and then concentrated as described above. The protein was stored at –80°C in the buffer A containing 10% glycerol and 2 mM MET.

The protein concentration was determined by measuring the absorbance at 280 nm (28) except the HisSUMO which was determined on the gel stained with Coomassie Brilliant Blue R-250 using the HisSUMO-DDM1 as a standard.

Pull-down assay

To investigate the binding of the HELLS to the CDCA7 proteins, the purified HELLS and HisMBP-CDCA7 or HisMBP proteins were mixed in a 40 μl of buffer D comprising 20 mM Tris–HCl, pH 8.0, 250 mM NaCl, and 1 mM MET at a final concentration of 0.2 μM, and then 15 μl amylose resin equilibrated in the same buffer was added to the solution and mixed at room temperature for 30 min. The resin was collected by centrifugation, washed with 0.2 ml of the same buffer three times, and then the bound proteins were eluted with 40 μl of buffer D containing 20 mM maltose. To detect the pulled-down proteins, dot blotting with the anti-HELLS rabbit polyclonal antibody and with the anti-MBP tag mouse monoclonal antibody (see Antibody subsection) was performed. The IRDye secondary antibodies, 680RD goat anti-Rabbit and 800CW goat anti-mouse antibodies (LI-COR, Inc.), were used to image the dots, and then they were quantified with the Odyssey CLx Imaging Systems (LI-COR, Inc.). To investigate the binding of the HELLS and CDCA7-MBP proteins, the pull-down assay was performed as described above except that the HELLS and CDCA7-MBP proteins were mixed at a final concentration of 0.25 μM and 1 μM, respectively. The pulled-down proteins were fractionated on a polyacrylamide gel, followed by staining with Coomassie Brilliant Blue R-250. The bands were quantified by scanning with ImageJ (https://imagej.nih.gov/ij/) (29).

To investigate the binding of the DDM1 to the CDCA7 proteins, the purified HisSUMO-DDM1 or HisSUMO, and HisMBP-CDCA7 or HisMBP were mixed in a 30 μl of buffer D at a final concentration of 1 μM, and then pull-down assay by using amylose resin was performed as described above except that the elution volume was 30 μl. To detect the proteins, dot blotting with the anti-SUMO/SUMOstar chicken polyclonal antibody and with the anti-MBP tag mouse monoclonal antibody (see Antibody subsection) was performed. The Alexa-Fluor 647 goat anti-chicken antibody (Thermo Fisher Scientific) and IRDye secondary antibody, 800CW goat anti-mouse (LI-COR, Inc.), were used to image the dots, and then they were quantified as described above.

ATPase assay

ATPase activity was measured by using the ADP-Glo Kinase Assay kit (Promega). The reaction was performed in 10 μl solution containing 20 mM Tris–HCl, pH 8.0, 0.12 M NaCl, 4 mM MgCl2, 0.4 mM ATP, 1 mM DTT and 0.5 mg/ml bovine serum albumin (BSA) at 37°C for 2 h. After the reaction, 10 μl of the ADP-Glo Reagent was added, followed by incubation at room temperature for 40 min to terminate the kinase reaction and deplete the remaining ATP. Then, 20 μl of the Kinase Detection Reagent was added, followed by incubation at room temperature for 1 h to convert ADP to ATP, which allowed the newly synthesized ATP to be measured using a luciferase/luciferin. The luminescence was measured with 2300 Enspire Multimode Plate Reader (PerkinElmer).

The 280-bp DNA, 27CG (Supplementary Table S2), containing 27 CpG sites on one strand, used for the assay was amplified by PCR using 5 pg/μl pI-SUMOstar vector as a template and 0.1 μM each of oligonucleotides P39 and P40 (Supplementary Table S1). The PCR was performed with 98°C for 2 s, followed by 35 cycles of 98°C for 10 s, 55°C for 5 s and 72°C for 10 s, by using the PrimeStarMax (Takara Bio Inc.). The amplified fragment was purified by QIAquick PCR Purification Kit (QIAGEN), followed by ethanol precipitation. The 24-bp DNAs used for the ATPase assay; TA (0CG), CG (5CG), GC, GG, CC, C, 1CG, 2CG, 3CG (CG/CG), 4CG, MG/MG, or CG/MG (M = 5-methylcytosine, 5mC) (Supplementary Table S2), was prepared by annealing each complementary oligonucleotide.

Nucleosome sliding assay

Restriction enzyme accessibility assay

The mononucleosome was constructed from the human histone octamer and the 193-bp 601 DNA containing the Widom 601 sequence (30) (Supplementary Table S2) as described previously (31). The nucleosome (0.2 μM) was incubated in a reaction buffer (20 mM Hepes-–NaOH, pH 7.5, 135 mM NaCl, 2.5 mM MgCl2, 1 mM DTT, 0.1 mg/ml BSA) containing HELLS and/or HisMBP-CDCA7 in the absence or presence of 1 mM ATP, at 30°C for 1 h. Two units/μl of restriction enzyme MspI (New England BioLabs), 1 unit/μl HpyCH4IV (New England BioLabs), or 1 unit/μl HaeIII (New England BioLabs) was added to the reaction mixture, and then incubated at 30°C for 30 min, followed by addition of 1 μl of the Proteinase K solution [0.5% SDS, 2.5 mg/ml proteinase K (Nacalai tesque, Inc.), 0.1 M EDTA] and incubation for 20 min. DNA was extracted by treatment with phenol/chloroform, and then fractionated on a polyacrylamide gel, followed by staining with SYBR Green I (Takara Bio Inc.) to detect the DNA by UV irradiation. The bands were quantified by scanning with ImageJ (https://imagej.nih.gov/ij/) (29) to determine the ratio of the amount of the cleaved DNA to that of the uncleaved one.

DNA methylation assay

After the mononucleosome was incubated at 30°C for 1 h as described above, 0.1 unit/μL CpG methylase (M.SssI), 1.2 mM S-adenosylmethionine, and the CpG reaction buffer (New England BioLabs) was added to the sample followed by incubation at 30°C for 30 min. DNA was extracted by treatment with phenol/chloroform, and then treated with restriction enzyme HapII or PmaCI (Takara Bio Inc.) at 37°C. The sample was fractionated on a polyacrylamide gel, followed by staining with SYBR Green I (Takara Bio Inc.) to detect the DNA by UV irradiation.

Electrophoretic mobility shift assay

Proteins were incubated with 6-carboxy-fluorescein (FAM)-labeled 13-bp long DNA probes (0.1 μM) in binding buffer [20 mM bis-tris–HCl, pH 6.0, 0.1 M NaCl, 1 mM DTT, 0.1 mg/ml of BSA, and 5% glycerol] for 10 min at 22°C. The dsDNA sequences are TA, C/C, M/C, and M/M (Supplementary Table S2). Samples were load onto 10% (v/v) native polyacrylamide gel in 0.5× TBE (Tris–borate–EDTA) buffer and ran for 40 min at 100 V. Gels were scanned with ChemiDoc imaging system (Bio-Rad).

Mouse embryonic stem cell (mESC) lines

Construction of Cdca7 and Hells KO mESC lines

The Dnmt3bfl/fl mESC derived from EB5 (32) were maintained on gelatin-coated dishes in Glasgow's minimum essential medium (#078-05525, Fujifilm Wako Chemicals) supplemented with 1% fetal bovine serum (BioWest), 10% knock-out serum replacement (Thermo Fisher Scientific), 1 mM sodium pyruvate (Thermo Fisher Scientific), 0.1 mM MET (Nacalai tesque, Inc.), 1× nonessential amino acids (Thermo Fisher Scientific), and leukemia inhibitory factor. The gRNA vector for construction of Cdca7 KO cells was constructed by inserting linkers, oligonucleotides P41/P42 and P43/P44 (Supplementary Table S1) into the Bbs I and Bsa I sites of the plasmid PX330-BB carrying a cas9 gene (Addgene), respectively. To construct the gRNA vector for construction of Hells KO cells, linkers, oligonucleotides P45/P46 and P47/P48 (Supplementary Table S1) were inserted into the PX330-BB as described above. The gRNA vector (1.5 μg) and plasmid pCAG-FLAG-IRESpuro (NRI) (0.5 μg), carrying a puromycin resistant gene, were co-transfected into the mESCs (5 × 105 cells in 2 ml medium) with 0.2 ml Opti-MEM Reduced Serum Medium (Thermo Fisher Scientific) containing 4 μl Lipofectamine 2000 (Thermo Fisher Scientific). After one day, puromycin was added at a concentration of 1.5 μg/ml, and then further cultured for two days. The Cdca7 and Hells KO cells were screened from several puromycin resistant cells by genomic PCR.

Establishment of Cdca7 KO mESCs expressing G196 epitope-tagged CDCA7 wild type or R285C mutant

The pyggyBac-CAG-ires-puro vector (33) linearized by NdeI and XhoI was ligated with the linker, oligonucleotides P49/P50, and the fragment amplified by PCR using oligonucleotides P51 and P52 as primers and the mouse full-length Cdca7 (mCdca7) cDNA clone (FANTOM3 clone ID: 2310021G01) (34) as a template to construct the pB-G196-mCDCA7. To construct the expression plasmid for R285C mutant of mouse CDCA7, the vector linearized by PCR using the pB-G196-mCDCA7 as a template and P53 and P54 as primers was ligated with a fragment amplified by PCR using the pB-G196-mCDCA7 as a template and P55 and P52 as primers. The expression plasmid of the mouse CDCA7 (0.5 μg) and the pyggyBac Transposase Expression plasmid (33) (0.5 μg) were cotransfected into the Cdca7 KO mESCs (1 × 106 cells in 2 ml medium) with 0.2 ml Opti-MEM Reduced Serum Medium (Thermo Fisher Scientific) containing 4 μl Lipofectamine 2000 (Thermo Fisher Scientific). After one day, puromycin was added at a concentration of 1.5 μg/ml, and further cultured to select the stable transfectants.

Establishment of Dnmt1, Dnmt3a, and Dnmt3b triple KO (Dnmt TKO) mESCs

We obtained Dnmt1, Dnmt3a, and Dnmt3b triple conditional KO (Dnmt cTKO) mESC line from Dr Haruhiko Koseki of RIKEN Center for Integrative Medical Science. The Dnmt cTKO mESC line was established from the blastocyst cells of quadruple transgenic background [Dnmt1tm2Jae (35), B6.129S4-Dnmt3atm3.1Enl (36), B6;129S4-Dnmt3btm5.1Enl (37) and C57BL/6-Gt(ROSA)26Sortm9(Cre/ESR1)Arte (CreERT2, Taconic Biosciences, Inc.)]. We established Dnmt TKO mESC line, 4OHT-2 from Dnmt cTKO mESC line, 4D-1 by the 4-OHT (4-hydroxytamoxifen) treatment (800 μM) and subcloning (Supplementary Figure S14A). Loss of DNA methylation in 4OHT-2 was validated by DNA methylation-sensitive and-resistant restriction enzyme digestion (Supplementary Figure S14B).

Western blotting

Cells (1 × 105) were washed with Dulbecco's phosphate buffered saline (PBS) and disrupted by sonication in an SDS-PAGE sample buffer. The sample was separated by SDS-PAGE and transferred onto PVDF membrane (Millipore). The membrane was blocked by 5% skim milk in TBS-T buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.2% Tween 20) at room temperature for 1 h, followed by incubation with the primary antibody in TBS-T with 5% skim milk at 4°C for 16 h. After the membrane was washed with TBS-T, the membrane was incubated with horseradish peroxidase-linked anti-rabbit (#NA934-1ML, Cytiva) or anti-mouse (#NA931-1ML, Cytiva) IgG at room temperature for 1 h in TBS-T with 5% skim milk. After the membrane was washed with TBS-T, bands were detected by chemiluminescence reaction using the Western Lightning Plus ECL (PerkinElmer, Inc.) on X-ray film (Fujifilm).

Immunofluorescence (IF) microscopy

Cells (5 × 104 cells in 0.2 ml medium) were cultured for 16 h on a well of the 8 well-chamber slides (Watson Bio Lab) coated with 10 μg/ml laminin. The cells were labeled with 5-ethynyl-2′-deoxyuridine (EdU) of the Click-iT EdU Imaging Kits (Thermo Fisher Scientific) for 30 min and then fixed in 4% paraformaldehyde at 37°C for 10 min. The fixed cells were washed twice with PBS containing 3% BSA, and then permeabilized with PBS containing 0.5% TritonX-100 at room temperature for 20 min, and then the cells labeled with EdU were detected using the Click-iT EdU Imaging Kits (Thermo Fisher Scientific). The cells were washed with PBS containing 3% BSA, and then incubated at room temperature for 30 min in the same buffer. Then, the cells were incubated with primary antibodies diluted in 4× SSC buffer (0.6 M NaCl, 60 mM sodium citrate, pH 7.0) containing 1% BSA and 0.2% Tween 20 at 4°C for 16 h, washed three times with 4× SSC, and incubated with secondary antibodies conjugated with Alexa568 or 647 Fluor (Thermo Fisher Scientific) diluted in 4× SSC containing 1% BSA, 0.2% Tween20 and 1 μg/ml DAPI at room temperature for 1 h. The cells were observed under a confocal microscope FV3000 (Olympus). Only in case for H4K20me1 staining, we used Cy3 direct-conjugated anti-H4K20me1 antibody (see Antibody subsection).

DNMT1 inhibitor, GSK-3484862 treatment

The mESCs were cultured in the presence of the DNMT1 inhibitor, GSK-3484862 (#2170136–65-7, MedChemExpress) at 5 μM concentration for 24 h prior to the IF and western blot analysis.

Whole genome bisulfite sequencing (WGBS) analysis

Preparation of genomic DNA

The cells were suspended in a buffer comprising 20 mM Tris–HCl, pH 8.0, 0.4 M NaCl, 10 mM EDTA, 0.5% SDS and 0.1 mg/ml RNase A, and then incubated at 37°C for 1 h. Proteinase K was added to the sample at a concentration of 1 mg/ml, and then incubated at 56°C for overnight. The sample was treated with phenol/chloroform followed by ethanol precipitation. The genomic DNA was dissolved in TE buffer.

Preparation of the library

The genomic DNA (1 μg) was sheared by sonication to 100–500 bp using a Covaris S220 and purified by AMPure XP beads (Agencourt Bioscience Corp.). Then, genomic DNA libraries were constructed by KAPA Hyper Prep Kit (Kapa Biosystems) and xGen Stubby Adapter and UDI Primers Pairs kit (Integrated DNA technologies) according to the manufacturer's instructions. After adapter ligation, the DNA was treated with sodium bisulfite using EZ DNA Methylation GOLD kit (Zymo Research) following the manufacturer's instructions. Enrichment for adapter-ligated DNA was carried out through 17 PCR cycles using KOD One PCR Master mix (Toyobo). The concentration of the WGBS library was quantified by KAPA Library quantification kit (Kapa Biosystems). Paired-end DNA sequencing (2 × 150 bp) was then performed using the Illumina NovaSeq X plus (Illumina). We performed two technical replicates for WGBS and correlation between replicates was described in Supplementary Table S33. Each library was sequenced with 21–24 M read pairs.

Analysis

Adaptor sequences and low-quality bases in reads were trimmed using Trim Galore version 0.6.10 (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). The trimmed reads were mapped to the mouse GRCm38 genome assembly using Bismark v0.24.1 with default parameters (38). The methylation level of each CpG site was calculated as follows: number of methylated reads/number of total reads. Replication timing for every 80-kb in mESC was obtained using GSE108556 (39). To calculate DNA methylation levels in each 80-kb bin, we averaged the DNA methylation levels of each CpG site within a bin. The methylation levels of Major satellite and Minor satellite were calculated by mapping to the GSAT_MM and SATMIN DNA sequences registered in RepBase (40) using Bismark.

Antibody

The anti-MBP tag mouse monoclonal antibody (#66003-1-1g, Proteintech Group, Inc.), anti-G196 mouse monoclonal antibody (#R-G-001, mAbProtein Co., Ltd), anti-CDCA7 rabbit polyclonal antibody (#15249–1-AP, Proteintech Group, Inc.), anti-HELLS rabbit polyclonal antibody (#11955-1-AP, Proteintech Group, Inc.), anti-UHRF1 mouse monoclonal antibody (#ab46187, Abcam), anti-Dnmt1 rabbit polyclonal antibody (#sc-20701, Santa Cruz), anti-H4K20me1 mouse monoclonal antibody (41) which was directly conjugated with Cy3, and anti-SUMO/SUMOstar chicken polyclonal antibody (#AB7002, LifeSensors) were used as the primary antibodies.

Results

Preparation of the recombinant proteins

Human CDCA7 consists of 371 amino acid residues, with a C-terminal 4CXXC-type ZF domain (amino acid residues 262–371) and a binding region for c-Myc and 14–3–3 proteins (amino acid residues 146–170) (Figure 1A). According to the three-dimensional structure predicted by AlphaFold2 (https://alphafold.ebi.ac.uk/entry/Q9BWT1) (42,43), the structure of CDCA7 has not been confidently predicted, except for the C-terminal domain and amino acid residues 114–140, which are conserved among several homologs (Supplementary Figure S1). To investigate the function of the CDCA7 domain in HELLS, recombinant human HELLS, wild-type, and various deletion mutants of CDCA7 were prepared from baculovirus-infected insect cells (Figure 1A and B). In addition, we prepared three ICF syndrome mutants of CDCA7: CDCA7_R274C, R274H and R304H (Figure 1A and B) out of the four detected in patients with ICF3-type ICF syndrome to date (18). In the case of CDCA7 proteins, (His)6-MBP (HisMBP) tags were fused at the N-terminus of the proteins to increase protein solubility. We investigated the biochemical properties of CDCA7 proteins. The results are summarized in Figure 1A.

Figure 1. The recombinant HisMBP-CDCA7, HELLS, DDM1 and HisMBP-CDCA7 proteins. (A) Schematic representation of the wild-type and mutants of the HisMBP-CDCA7 proteins. The 4CXXC-type ZF domain (), binding region of the c-Myc and 14–3–3 proteins (), and an α-helix () predicted by AlphaFold2 program (https://alphafold.ebi.ac.uk/entry/Q9BWT1) (42,43) (Supplementary Figure S1B) are indicated. The asterisks in the R274C, R274H and R304H represent the position of the amino acid substitutions. Of the CDCA7 proteins, propensities of binding ability to the HELLS (HELLS binding) (Figure 2B, Supplementary Table S3), enhancement of the ATPase activity of the HELLS (Enhancement of HELLS ATPase) (Figure 3A and B, Supplementary Table S5), enhancement of the nucleosome sliding activity of the HELLS by using the canonical nucleosome (see below) (Nucleosome sliding to the 3′ and to the 5′) (Figure 4A, C and E, Supplementary Table S8 and S9), and preference to the hemimethylated DNA for activation of the ATPase activity of the HELLS (Enhancement of HELLS ATPase by CG/5mCG) (Figure 7 and Supplementary Table S15), which we determined from their measurement values, were indicated. (B) SDS-PAGE analysis of the recombinant proteins purified from the baculovirus-infected insect cells. Lane 2; the HisMBP-wild-type CDCA7, lanes 3−6 and 8−15, the deletion mutants of the HisMBP-CDCA7 proteins; lanes 16−18, the ICF syndrome mutants of the HisMBP-CDCA7 proteins; lane 19, HisMBP; lane 21, the HELLS; lane 22, the DDM1; lane 24, the HisMBP-Arabidopsis CDCA7 (NP_195428). The samples were analyzed on 10% polyacrylamide gel, which was stained with Coomassie Brilliant Blue R-250. Lanes 1, 7, 20 and 23; molecular mass markers.

Binding of the CDCA7 proteins to HELLS

We performed a pull-down assay to investigate the binding of HisMBP-CDCA7 to HELLS. Purified HELLS were mixed with each HisMBP-CDCA7 protein, and the sample was pulled down with amylose resin. The amounts of HELLS and HisMBP-CDCA7 eluted from the resin were quantified by dot blotting using anti-HELLS and anti-MBP antibodies that specifically recognize cognate proteins (Figure 2A, Supplementary Figures S2A and S2B). The ratio of HELLS to HisMBP-CDCA7 was determined as the binding ability to HELLS and then compared with that of HisMBP-CDCA7 proteins (Figure 2B and Supplementary Table S3). The binding ability of HisMBP, which was used as a negative control, was significantly lower than that of HisMBP-wild-type CDCA7, indicating that HELLS preferentially binds to CDCA7. HisMBP-CDCA7_1–143 showed a binding ability similar to that of the wild-type. When the C-terminal deletion was extended to residue 108, the mutant CDCA7 protein barely bound to HELLS. Furthermore, the binding ability of HisMBP-CDCA7_140–371 was significantly reduced compared with that of the wild type. These results indicate that residues 108–143, containing the conserved region predicted to form an α-helix (Supplementary Figure S1), are critical for binding to HELLS. The HisMBP-CDCA7_31–371 and HisMBP-CDCA7_68–371 proteins tended to show a higher degree of binding than that of the wild type, although significant differences were not observed (Supplementary Table S19). To validate the effects of the MBP tag fused to the N-terminus of CDCA7 on binding to HELLS, we investigated the binding of HELLS to CDCA7 proteins using C-terminal MBP tags (Supplementary Figures S3A−C, Supplementary Table S4). This case also revealed that CDCA7_68–371 tended to show a higher degree of binding than that of the wild type, although no significant differences were observed (P= 0.10, Student's t-test). Thus, the effects of the N-terminal MBP tag of CDCA7 on binding to HELLS may be negligible. The three ICF syndrome mutants bound to HELLS to the same extent as the wild type, as previously indicated (25).

Figure 2. Binding ability of the CDCA7 proteins to the HELLS and DDM1. (A) Mixtures of the purified HELLS and wild-type, truncated mutant, or ICF syndrome mutant of HisMBP-CDCA7 or HisMBP were pulled down with amylose resin, followed by elution with maltose. Dot blotting of the input (0.4 μl) and the eluted (2 μl) samples was performed with the rabbit anti-HELLS and the mouse anti-MBP antibodies, which was detected by using the IRDye secondary antibodies and the Odyssey CLx Imaging Systems (LI-COR, Inc.). The MBP tag and the HELLS were detected by emission wavelengths at 812–832 nm (channel 800) (upper panels) and 710–730 nm (channel 700) (lower panels), respectively. See also Supplementary Figure S2B. (B) Binding ability of each mutant CDCA7 relative to that of the HisMBP-wild-type CDCA7. The fluorescent intensities corresponding to the HELLS and HisMBP-CDCA7 or HisMBP proteins pulled down together were quantified, and the value of the HELLS relative to that of the HisMBP-CDCA7 was calculated, which was defined as the relative binding ability to the HELLS (Supplementary Table S3). The experiments were performed five times (see also Supplementary Figure S2B), and each value relative to one of those in the case of the wild type (Supplementary Table S3) was plotted with dot. Bars and error bars represent the average values and ± standard errors, respectively. RM 1-way ANOVA was performed using the Prism version 10 (Supplementary Table S19). **P< 0.01 and *P< 0.05 denote the significant difference between the wild type and the others. (C) Mixtures of the purified HisSUMO-DDM1 (Supplementary Figure S2C) and HisMBP-human CDCA7, Arabidopsis CDCA7, or HisMBP were pulled down with amylose resin, followed by elution with maltose. As a control, the purified HisSUMO fragment (Supplementary Figure S2C) was used instead of the HisSUMO-DDM1. Dot blotting of the input (0.5 μl) and the eluted (2 μl) samples was performed with the chicken anti-SUMO and the mouse anti-MBP antibodies, which was detected by using the fluorescence-labeled secondary antibodies and the Odyssey CLx Imaging Systems (LI-COR, Inc.). The MBP and the SUMO tags were detected by emission wavelengths at 812–832 nm (channel 800) (upper panels) and 710–730 nm (channel 700) (lower panels), respectively. See also Supplementary Figure S2E. (D) Binding ability of the human or Arabidopsis CDCA7 proteins to the DDM1. The ratio of the HisSUMO-DDM1 (left panel) or the HisSUMO (right panel) pulled down with the amylose resin in the absence and presence of the CDCA7 proteins or HisMBP were calculated from the fluorescent intensities (Supplementary Table S17). The experiments were performed three times (see also Supplementary Figure S2E), and each value (Supplementary Table S17) was plotted with dot. Bars and error bars represent the average values and ± standard errors, respectively. RM 1-way ANOVA followed by Tukey's multiple comparison test were performed using the Prism version 10 (Graph Pad) (Supplementary Table S29). *P< 0.05 denote the significant difference between the values in the absence (none) and in the presence of the CDCA7 proteins. n.s., not significant. :P< 0.05 represents the significant difference between the samples indicated by bars.

Identification of the domain of CDCA7 important for enhancement of the ATPase activity of HELLS

To investigate the region of CDCA7 critical for the enhancement of the DNA-dependent ATPase activity of HELLS, the effects of various mutant HisMBP-CDCA7 proteins on the ATPase activity of HELLS were measured (Figure 3 and Supplementary Table S5). The ATPase activity of HELLS was significantly enhanced in the presence of both DNA and HisMBP wild-type CDCA7, as previously reported (23), whereas HisMBP had no effect. HisMBP-CDCA7_1–261 and HisMBP-CDCA7_1–176 kept their activity, although the activity was significantly reduced compared with that of the wild type. The activities of the three ICF syndrome mutants were also significantly weaker (Figure 3B). Thus, the C-terminal domain may be important for full activity. The enhancing activity of HisMBP-CDCA7_1–143 was extremely low, and that of HisMBP-CDCA7_1–107 was hardly observed. In the case of the N-terminal deletion mutants, enhanced activity was hardly observed when the deletion was extended to 139 residues from the N-terminus. These results indicate that residues 108–176, consisted of the conserved domain predicted to form an α-helix and the c-Myc and 14–3–3-binding region, are critical for activation of the ATPase activity of HELLS. c-Myc and 14–3–3 proteins bind to CDCA7 depending on the phosphorylation of the Thr163 residue of CDCA7 (19). We observed that the HisMBP-CDCA7_T163A mutant enhanced the ATPase activity of HELLS to the same extent as the wild type (Supplementary Figure S4, Supplementary Tables S6 and S22), indicating that phosphorylation of Thr163 is not necessary for HELLS activation.

Figure 3. ATPase activity of the HELLS in the presence of the wild-type or mutant CDCA7. The ATPase activity was measured in the absence or presence of 25 μg/ml 27CG DNA (Supplementary Table S2) and in the absence or presence of 0.25 μM (A,B) or 1 μM (C) of each protein denoted below the panel. Triplicate experiments were performed and each luminescence intensity relative to one of that in the presence of the HELLS and the HisMBP-wild-type CDCA7 without DNA (A, B) or that in the presence of the HELLS and DNA (C) was shown with dot. Bars and error bars represent the average values and ± standard errors, respectively (Supplementary Table S5). Tukey's multiple comparison test was performed using R version 4.2.3 (https://www.R-project.org/) (Supplementary Table S20). ***P< 0.001 and **P< 0.01 denote the significant difference between the HELLS plus HisMBP-wild-type CDCA7 (+ DNA) and the others. :::P< 0.001 represents the significant difference between the samples indicated by bars.

The effects of HisMBP-CDCA7_31–371 and HisMBP-CDCA7_68–371 were more than those of the wild type, indicating that the N-terminal 67 residues negatively control ATPase activity. In the case of HisMBP-CDCA7_108–371, the activity was significantly but slightly higher than or equal to that of the wild type and significantly lower than that of HisMBP-CDCA7_68–371 (Figure 3A, 6I, and 7H). At a protein concentration four times higher, the effect of the N-terminal deletion mutant (HisMBP-CDCA7_68–371) on ATPase activity was almost the same as that of the wild type, which may be due to saturation of the reaction at this protein concentration (Figure 3C). Since the N-terminal MBP tag may affect the function of the N-terminal domain of CDCA7, we also investigated the effects of CDCA7-MBP proteins (Supplementary Figure S3A) on the ATPase activity of HELLS. CDCA7_68–371-MBP significantly enhanced the ATPase activity of HELLS more than that of the wild type CDCA7-MBP (Supplementary Figure S3D, Supplementary Table S7 and S21), as seen in the case of CDCA7 proteins with N-terminal MBP tags. Thus, once again, the effects of the N-terminal MBP tag of CDCA7 on the activation of HELL ATPase activity may be negligible.

Nucleosome sliding activity of HELLS-CDCA7

Subsequently, we performed a restriction enzyme accessibility assay for the HELLS-CDCA7-mediated nucleosome sliding activity using mononucleosome constructed from 193-bp 601 DNA and a histone octamer as reported previously (23). At the beginning of this study, we used a restriction enzyme HaeIII for monitoring of nucleosome positioning (Supplementary Figure S5A and B). The 601 DNA contains three HaeIII sites. The nucleosome at central position (C) limits the access of HaeIII to the central HaeIII site of the 601 DNA; thus, 146-bp fragment is generated by the HaeIII treatment. If the histone octamer slides to the 3′ terminal (right position, R), the HaeIII site near the 3′ terminal is protected, and the DNA is cleaved at remaining two sites to generate the 149-bp band. If it slides to the 5′ terminal (left position, L), the HaeIII site near the 5′ terminal is protected, and the DNA is cleaved at remaining two sites to generate the 169-bp band (Supplementary Figure S5A). We observed that if CDCA7 and HELLS were added in the presence of ATP, the 146-bp fragment disappeared and the 149-bp fragment dominantly emerged which is generated if the nucleosome shifted to the right position. But, the 169-bp fragment was also detected which is generated if the nucleosome shifted to the left position, although weakly. However, the 169-bp fragment was also generated to some extent by CDCA7-HELLS in the absence of ATP (Supplementary Figure S5B). These data indicate that the CDCA7-HELLS complex could slide the nucleosome at least to the right direction in the presence of ATP, but also weakly protected the 5′ HaeIII site from the enzyme digestion without sliding the nucleosome to the right direction if ATP was absent.

Having found that this assay seemed to be able to assess ATP-dependent chromatin sliding activity, we proceeded to examine it in more detail by using different restriction enzymes, MspI and HpyCH4IV (Figure 4A). If the histone octamer slides in the direction of the 3′ terminal of the DNA (R position), the MspI site is exposed, resulting in an increase of the 159-bp DNA by treatment with MspI (Figure 4B and Supplementary Figure S5C). To evaluate the sliding activity, the ratio of the amount of 159-bp DNA to that of the undigested DNA was determined (Figure 4C and Supplementary Table S8). We observed that HELLS or HisMBP-CDCA7 alone had no impact on the ratio of cleaved nucleosomal DNA after the restriction enzyme digestion. By contrast, in the presence of both HELLS and HisMBP-wild-type CDCA7, the ratio of cleaved DNA increased with ATP. These results indicate that both HELLS and CDCA7 are necessary for ATP-dependent nucleosome sliding, as previously reported (23). HisMBP-CDCA7_31–371, HisMBP-CDCA7_68–371, or HisMBP-CDCA7_108–371 showed similar effects to that of HisMBP-wild-type CDCA7, indicating that the N-terminal 107 residues of CDCA7 are not necessary for this sliding activity, as in the case of DNA-dependent ATPase activity (Figure 3). HisMBP-CDCA7_1–261, HisMBP-CDCA7_1–176, HisMBP-CDCA7_R274C, HisMBP-CDCA7_R274H, and HisMBP-CDCA7_R304H were also effective for this sliding activity, but their efficiency, except CDCA7_R274H, was approximately 60–80% of that of HisMBP-wild-type CDCA7, indicating that the C-terminal domain of CDCA7 plays some role in full activity, as in the case of DNA-dependent ATPase activity. The effect of HisMBP-CDCA7_1–143 was significantly low, and those of the HisMBP-CDCA7_1–107 and HisMBP-CDCA7_177–371 were at background levels, indicating that residues 108–176 are also important for this sliding activity, as in the case of DNA-dependent ATPase activity. HisMBP-CDCA7_140–371, HisMBP-CDCA7_177–371, and HisMBP-CDCA7_218–371 were slightly effective for sliding activity, although their contribution to DNA-dependent ATPase activity was hardly observed, even at higher protein concentrations (Figure 3C). These mutants bound slightly to HELLS (Figure 2A−C, Supplementary Figure S2B), which may have modulated the sliding activity of HELLS-CDCA7.

Figure 4. Nucleosome sliding activity of the HELLS with the wild-type or mutant CDCA7. (A) Schematic representation of the restriction enzyme accessibility assay for panels B–E. Msp and Hpy denote the positions of the restriction enzyme MspI and HpyCH4IV sites, respectively. If the histone octamer slides to the 3′ terminal, the DNA is cleaved by MspI to generate the 159-bp band. If it slides to the 5′ terminal, it is cleaved by HpyCH4IV to generate the 146-bp band. The mononucleosome was treated with restriction enzyme MspI (B) or HpyCH4IV (D) in the absence (lane 4) or presence of 1 μM HELLS (lane 5), 1 μM HisMBP-wild-type CDCA7 (lane 6), the HELLS plus the wild type or the mutants of the CDCA7 (lanes 7−10, 12−19, 21−23), or the HELLS plus the HisMBP (lane 24). The DNA extracted from the mononucleosome was analyzed on 6% (B) or 8% (D) polyacrylamide gel. Lane 2, the 601 DNA; lane 3, the 601 DNA treated with MspI (B) or HpyCH4IV (D); lanes 1, 11, and 20, DNA size markers. The ratio of the 159-bp band to the 193-bp band appeared by the assay with MspI (C) or that of the 146-bp band to the 193-bp band with HpyCH4IV (E) was determined (Supplementary Table S8 and S9) and shown as digestion (%). For each assay, triplicate experiments were performed, and each value is shown with dot. Bars and error bars represent the average values in the absence (white) or presence (grey) of ATP and ± standard errors, respectively. Tukey's multiple comparison test was performed using R version 4.2.3 (https://www.R-project.org/) (Supplementary Table S23). ***P< 0.001, **P< 0.01 and *P< 0.05 denote the significant difference between the HELLS plus HisMBP-wild-type CDCA7 [ATP (+)] and the others. :::P< 0.001 and :P < 0.05 represent the significant difference between the samples indicated by bars. (F) Schematic representation of the mononucleosomes used for the restriction enzyme accessibility assay for panels G–I. The three CpG sites of the 5′ linker region of the canonical 601 DNA were replaced by three TpA sites to construct the 5′-TA 601 DNA. The two CpG sites of the 3′ linker region of the canonical 601 DNA were replaced by two TpA sites to construct the 3′-TA 601 DNA. Msp and Hpy denote the positions of the restriction enzyme MspI and HpyCH4IV sites, respectively. The mononucleosome containing the canonical 601 DNA (can.), the 5′-TA 601 DNA (5′-TA), or the 3′-TA 601 DNA (3′-TA) was treated with restriction enzyme MspI (G) or HpyCH4IV (H) in the absence (lanes 2−4) or presence of the HELLS plus the HisMBP-wild-type CDCA7 (lanes 5−7) or the HELLS plus HisMBP-CDCA7_R274C (lanes 8−10). The DNA extracted from the nucleosome was analyzed on 8% polyacrylamide gel. Lane 1, DNA size markers. (I) The ratio of the 159-bp band to the 193-bp band appeared by the assay with MspI (upper panel) or that of the 146-bp band to the 193-bp band with HpyCH4IV (lower panel) was determined (Supplementary Table S10 and S11) and shown as digestion (%). For each assay, triplicate experiments were performed, and each value is shown with dot. Bars and error bars represent the average values in the absence (white) or presence (grey) of ATP and ± standard errors, respectively. Tukey's multiple comparison test was performed using R version 4.2.3 (https://www.R-project.org/) (Supplementary Table S24). ::P< 0.01 represents the significant difference between the samples indicated by bars. n.s., not significant.

Subsequently, we investigated the sliding in the opposite direction. If the histone octamer slides to the 5′ terminal (L position), one of the two HpyCH4IV sites is exposed, resulting in an increase of the 146-bp DNA by treatment with HpyCH4IV (Figure 4A, D, and E, Supplementary Figure S5D, and Supplementary Table S9). We observed that even in the presence of both HELLS and the HisMBP-wild-type CDCA7, the accessibility of the HpyCH4IV was hardly changed as compared with that in the absence of these proteins or in the presence of the HELLS, the HisMBP-CDCA7, or the HisMBP alone, indicating that the histone octamer hardly slides to the 5′ terminal of the DNA. HisMBP-CDCA7_1–261, HisMBP-CDCA7_1–176, HisMBP-CDCA7_R274C and HisMBP-CDCA7_R304H effectively generated 146-bp DNA depending on ATP. This effect was not observed in the N-terminal deletion mutants of CDCA7. These results indicate that the defects in the C-terminal ZF domain of CDCA7 cause the enhancement of the sliding to the 5′ terminal of the DNA.

Since the ZF domain of CDCA7 preferentially recognized CpG sites (see below), the HELLS-CDCA7 possibly binds the 5′ or 3′ linker region of the nucleosome via the ZF domain of the CDCA7. To investigate this, we examined the nucleosome sliding activity using the nucleosome constructed from the 193-bp 5′-TA 601 DNA or 3′-TA 601 DNA (Figure 4F, Supplementary Table S2). In these DNAs, three CpG sites in the 5′-linker region or two CpG sites in the 3′-linker region of the 193-bp 601 DNA were replaced with TpA. We observed that the HELLS-HisMBP-CDCA7 could also slide the 5′-TA and 3′-TA nucleosomes in an ATP-dependent manner, respectively (Figure 4G and H, Supplementary Figures S5E and F). As for the sliding of the 5′-TA nucleosome in the presence of the HELLS-HisMBP-wild type CDCA7, the sliding to the 3′ terminal was weak and that to the 5′ terminal frequently occurred as compared with that of the canonical and the 3′-TA nucleosomes (Figure 4I, Supplementary Tables S10 and S11). These results indicate that the three CpG sites in the 5′ linker region rather than the two CpG sites in the 3′ linker region of the canonical nucleosome are important for the HELLS-HisMBP-wild-type CDCA7 to efficiently slide the nucleosome to the 3′ terminal. For HELLS-HisMBP-CDCA7_R274C, the direction of sliding was almost identical among the three nucleosomes (Figure 4I, Supplementary Tables S10 and S11). These results indicate that the defect in the C-terminal ZF domain of the CDCA7 lacks selective recognition of the three CpG sites in the 5′ linker region of the canonical nucleosome.

To investigate the relation between nucleosome sliding and DNA methylation, we performed a nucleosome sliding reaction followed by a DNA methylation assay using CpG methylase. After the reaction, 601 DNA molecules were extracted from the nucleosome, and DNA methylation was assessed using the restriction enzymes HapII and PmaCI, which are sensitive to DNA methylation (Figure 5A). In the absence of HELLS-CDCA7 proteins or in the presence of HELLS alone, the 601 bp DNA from the nucleosome was mostly cleaved by the restriction enzymes, independent of the DNA methylation reaction (Figure 5B, lanes 5−7 and 9−11; Figure 5C; Supplementary Figure S5G, lanes 5−7 and 9−11; Supplementary Tables S12 and S13), suggesting that both restriction sites are protected by histones and that histones hardly slide. In the presence of HELLS alone, resistance to restriction enzymes tended to be lower than that in the absence of HELLS-CDCA7 (Figure 5C). It is possible that HELLS bound to the nucleosome limits access to restriction enzymes. In the presence of HELLS plus HisMBP-wild-type CDCA7, the 601 DNA from the nucleosome was mostly resistant to HapII and cleaved by PmaCI (Figure 5B, lanes 12−14; Figure 5C; Supplementary Figure S5G, lanes 12−14; and Supplementary Tables S12 and S13) in an ATP-dependent manner, suggesting that the histone slides to the 3′ terminal (R position) and the HapII site was exposed, and then the CpG site was methylated. In the presence of HELLS plus HisMBP-CDCA7_R274C, the 601 DNA from the nucleosome was cleaved by both HapII and PmaCI, suggesting that the histone slides to both the 5′ and 3′ terminal sides in an ATP-dependent manner (Figure 5B, lanes 15−17; Figure 5C; Supplementary Figure S5G, lanes 15−17; and Supplementary Tables S12 and S13). These results were consistent with those of the restriction enzyme accessibility assay described previously. These results indicate that the C-terminal ZF domain recognizes CpG binding sites on the nucleosome for unidirectional sliding. The results of the DNA methylation assay also suggest that the nucleosome limits the access of the DNA methyltransferase, and when the nucleosome slides due to the action of HELLS-CDCA7, the DNA methyltransferase can introduce methyl groups to the CpG sites exposed from the nucleosome, as indicated previously (23,44–46).

Figure 5. Chromatin remodeling-coupled DNA methylation assay. (A) Schematic representation of the DNA methylation assay for panels B and C. If the histone octamer slides to the 3′ terminal, the HapII site is methylated by the CpG methyltransferase to be HapII resistant. If it slides to the 5′ terminal, the PmaCI site is methylated to be PmaCI resistant. (B) After the 601 DNA (lanes 2–4) or the mononucleosome (lanes 5–7, 9–17) were incubated with (lanes 2, 3, 5, 6, 9, 10, 12, 13, 15 and 16) or without (lanes 4, 7, 11, 14 and 17) ATP in the absence (lanes 2–7) or presence of the HELLS (lanes 9–11), the HELLS plus the HisMBP-wild-type CDCA7 (lanes 12–14), or the HELLS plus the HisMBP-CDCA7_R274C (lanes 15–17), the samples were treated with (lanes 3, 4, 6, 7, 10, 11, 13, 14, 16 and 17) or without (lanes 2, 5, 9, 12 and 15) the CpG methyltransferase. The DNA extracted from the nucleosome was treated with restriction enzyme HapII (upper panel) or PmaCI (lower panel), and then analyzed on 8% polyacrylamide gel. Lanes 1 and 8, DNA size markers. (C) The ratio of the DNA resistant to HapII (upper panel) or PmaCI (lower panel) was determined (Supplementary Table S12 and S13) and is shown as digestion resistance (%). For each assay, triplicate experiments were performed, and each value is shown with dot. Bars and error bars represent the average values in the absence (white) or presence (grey) of ATP and ± standard errors, respectively. Tukey's multiple comparison test was performed using R version 4.2.3 (https://www.R-project.org/) (Supplementary Table S25). ***P< 0.001 represents the significant difference between the samples indicated by bars. n.s., not significant.

Effect of DNA sequence on the DNA-dependent ATPase activity of the HELLS-CDCA7 proteins

To investigate the DNA sequence specificity of HELLS-HisMBP-CDCA7 proteins, we performed a DNA-dependent ATPase activity assay in the presence of various 24-bp DNA fragments (Figure 6; Supplementary Tables S2 and S14). The DNA fragments comprised only T and A (TA) and those containing five CpG (CG), GpC (GC), GpG (GG), CpC (CC) and C (C) sites on each strand. Among the six DNA fragments, CG DNA was the most effective in enhancing the ATPase activity of HELLS-HisMBP-wild-type CDCA7 under these reaction conditions (Figure 6A). Furthermore, the ATPase activity increased with the number of CpG sites (Figure 6B). These results indicate that HELLS-HisMBP-wild-type CDCA7 preferentially recognizes CpG sites. HisMBP-CDCA7_108–371 also induced a similar preference for DNA sequences (Figure 6C). However, the preference for the CG DNA sequence was lost due to the lack of the C-terminal ZF domain (Figure 6D and E) and by the ICF syndrome mutation (Figure 6F–H). Defects in the ZF domain of CDCA7 also resulted in decreased DNA-dependent ATPase activity in the presence of CG DNA (Figure 6I). These results indicated that the C-terminal ZF domain of CDCA7 was involved in the preferential recognition of CpG sites by HELLS-CDCA7 to enhance ATPase activity.

Figure 6. Effect of DNA sequence on the DNA-dependent ATPase activity of the HELLS-CDCA7. The ATPase activity of the 0.25 μM HELLS plus 0.25 μM wild-type (A,B) or mutant (C−H) HisMBP-CDCA7 was measured in the absence or presence of 40 μg/ml 24-bp TA (0CG), CG (5CG), GC, CC, GG, C, 1CG, 2CG, 3CG or 4CG DNA (Supplementary Table S2) as denoted. The ATPase activity of the 0.25 μM HELLS in the presence of 40 μg/ml CG DNA with or without 0.25 μM HisMBP-wild-type CDCA7, 108–371, 1–261, 108–371, R274C, R274H or R304H was also measured (I). Triplicate experiments were performed and each luminescence intensity relative to one of that in the absence of DNA (A−H) or in the absence of HisMBP-CDCA7 (I) (Supplementary Table S14) was shown with dot. Bars and error bars represent the average values and ± standard errors, respectively. Tukey's multiple comparison test was performed using R version 4.2.3 (https://www.R-project.org/) (Supplementary Table S26). ***P< 0.001, **P< 0.01 and *P< 0.05 denotes the significant difference between the CG DNA and the others (A, C–H) or between the HisMBP-wild-type CDCA7 and the others (I). :::P< 0.001 and n.s. (not significant) between the samples indicated by bars were shown.

Effect of DNA containing C5 methylated cytosine on the DNA-dependent ATPase activity of the HELLS-CDCA7 proteins

Subsequently, we investigated the effect of DNA containing 5mC at the CpG sites on the DNA-dependent ATPase activity of HELLS-HisMBP-CDCA7 (Figure 7 and Supplementary Table S15). Based on the 24-bp 3CG DNA containing three CpG sites on one strand, we prepared non-methylated DNA (CG/CG), DNA methylated at all six CpG sites on both strands (5mCG/5mCG), and hemimethylated DNA (CG/5mCG), which was methylated at all three CpG sites on one strand (Supplementary Table S2).

Figure 7. Effect of the DNA containing 5-methylcytosine at CpG site on the ATPase activity of the HELLS-CDCA7. The ATPase activity of the 0.25 μM HELLS plus 0.25 μM wild-type (A) or mutant (B−G) HisMBP-CDCA7 was measured in the absence or presence of 40 μg/ml 24-bp non-methylated DNA (CG/CG), the DNA methylated at the 5th carbon of the cytosine of the CpG sites on both strand (5mCG/5mCG), or the hemimethylated DNA (CG/5mCG) (Supplementary Table S2) as denoted. The ATPase activity of the 0.25 μM HELLS in the presence of 40 μg/ml CG/5mCG DNA with or without 0.25 μM HisMBP-wild-type CDCA7, 108–371, 1–261, 108–371, R274C, R274H or R304H was also measured (H). Triplicate experiments were performed and each luminescence intensity relative to one of that in the absence of DNA (A−G) or in the absence of HisMBP-CDCA7 (H) (Supplementary Table S15) was shown with dot. Bars and error bars represent the average values and ± standard errors, respectively. Tukey's multiple comparison test was performed using R version 4.2.3 (https://www.R-project.org/) (Supplementary Table S27). ***P< 0.001 and **P< 0.01 denotes the significant difference between the CG/5mCG DNA and the others (A–G), or between the HisMBP-wild-type CDCA7 and the others (H). :::P< 0.001, :P< 0.05 and n.s. (not significant) between the samples indicated by bars were shown.

Among the three DNA fragments, CG/5mCG was the most effective and 5mCG/5mCG was the least effective for HisMBP-wild-type CDCA7 in enhancing ATPase activity under these reaction conditions (Figure 7A). These results indicate that HELLS-CDCA7 preferentially recognizes hemimethylated CpG sites over non-methylated sites and sites methylated on both strands. HisMBP-CDCA7_108–371 also showed similar selectivity to that of the wild-type (Figure 7B), whereas HisMBP-CDCA7_1–261, HisMBP-CDCA7_108–261, and the three ICF syndrome mutants showed little or no selectivity (Figure 7C–G). Defects in the ZF domain of CDCA7 also resulted in decreased DNA-dependent ATPase activity in the presence of CG/5mCG DNA (Figure 7H). These results indicated that the C-terminal ZF domain of CDCA7 is involved in the preferential recognition of hemimethylated CpG sites.

Effects of the human and Arabidopsis CDCA7 proteins on DDM1

DDM1 is a plant homolog of HELLS that shows DNA-dependent ATPase activity in vitro (47,48). Apart from the physiological implications, we determined whether human CDCA7 affects DDM1. We investigated the effects of CDCA7 on the DNA-dependent ATPase activity of recombinant A. thaliana DDM1 prepared from baculovirus-infected insect cells (Figure 1B, Figure 8, and Supplementary Table S16). DDM1 alone exhibited DNA-dependent ATPase activity (Figure 8A), as previously reported (48). DDM1 did not show a preference for the CpG or hemimethylated CpG site for activity (Figure 8A and E), unlike in the case of HELLS-HisMBP-wild-type CDCA7. Interestingly, in the presence of HisMBP-wild-type CDCA7, DDM1 showed a preference for both CpG and hemimethylated CpG sites (Figure 8B, F, I, and J), as seen in HELLS. Furthermore, the HisMBP-CDCA7_1–261 and HisMBP-CDCA7_R274C proteins lost these effects (Figure 8C, D, G, H, I, and J). In addition, defects in the ZF domain of CDCA7 had no significant effect on the DNA-dependent ATPase activity of DDM1 in the presence of CG or CG/5mCG DNA (Figure 8I and J).

Figure 8. Effect of the human and Arabidopsis CDCA7 proteins on the DNA-dependent ATPase activity of the DDM1. The ATPase activity of the 0.25 μM DDM1 in the absence (A,E) or presence of the 0.25 μM HisMBP-wild-type CDCA7 (B,F), 1–261 (C, G) or R274C (D,H), or 0.5 μM Arabidopsis CDCA7 (NP_195428) (K,L) was measured in the absence or presence of 40 μg/ml 24-bp DNA; TA, CG, GC, CC, GG, or C (A−D, K) or 24-bp non-methylated DNA (CG/CG), the DNA methylated at the 5th carbon of the cytosine of the CpG sites on both strand (5mCG/5mCG), or the hemimethylated DNA (CG/5mCG) (E−H, L) (Supplementary Table S2). The ATPase activity of the 0.25 μM DDM1 in the absence or presence of 0.25 μM HisMBP-wild-type CDCA7, 1–261 or R274C was measured in the presence of 40 μg/ml 24-bp DNA; CG (I) or CG/5mCG (J). Triplicate experiments were performed and each luminescence intensity relative to one of that in the absence of DNA (A−H, K, L) or in the absence of HisMBP-CDCA7 (I, J) (Supplementary Table S16) was shown with dot. Bars and error bars represent the average values and ± standard errors, respectively. Tukey's multiple comparison test was performed using R version 4.2.3 (https://www.R-project.org/) (Supplementary Table S28). ***P< 0.001, **P< 0.01 and *P< 0.05 denote the significant difference between the CG DNA and the others (A–D, K), between the CG/5mCG DNA and the others (E–H, L), or between the DDM1 alone and the others (I, J).

Recently, three classes of proteins containing 4CXXC-type ZF domains have been identified in A. thaliana (49). The class I proteins, NP_179934 and NP_195428, appear to be the most closely related to human CDCA7 among the three classes because not only the CXXC motifs but also the three amino acid residues that are mutated in patients with ICF syndrome are conserved in the proteins (49) (Supplementary Figure S6). In addition, the long α-helix structure predicted in the human CDCA7, which is important for binding to HELLS and activation of the ATPase and nucleosome sliding activities of HELLS, is also found in both the predicted three-dimensional structures of the NP_179934 and NP_195428 proteins (Supplementary Figures S6 and S7). We observed that DDM1 showed a preference for CpG and hemimethylated CpG sites in the presence of recombinant NP_195428 prepared from baculovirus-infected insect cells (HisMBP-Arabidopsis CDCA7) (Figure 1B, Figure 8K, L, and Supplementary Table S16).

Subsequently, we investigated the binding of human and Arabidopsis CDCA7 to HELLS. Purified HisSUMO-DDM1 (Supplementary Figure S2C) was pulled down using amylose resin in the absence or presence of HisMBP-human or Arabidopsis CDCA7. The proteins were detected by dot blotting using anti-SUMO and anti-MBP antibodies, which specifically recognize the cognate proteins (Figure 2C, Supplementary Figure S2D and E), and the ratio of HisSUMO-DDM1 pulled down with the resin was determined (Figure 2D and Supplementary Table S17). As a result, the HisSUMO-DDM1 was significantly pulled down in the presence of the human and/or Arabidopsis CDCA7, but not in the presence of the HisMBP. As a control, we performed pull-down assays using the HisSUMO fragment (Figure 2C, Supplementary Figure S2C–E). HisSUMO alone was pulled down to some extent, but was not significantly increased in the presence of CDCA7 proteins (Figure 2D and Supplementary Table S17). These results indicate that DDM1 can bind to both human and Arabidopsis CDCA7 proteins and that the binding of CDCA7 to DDM1 confers a preference for CpG or hemimethylated CpG, as in the case of HELLS in mammals.

DNA binding ability of CDCA7

We observed that the C-terminal ZF domain of mammalian CDCA7 is involved in the recognition of DNA sequences, preferring the hemimethylated CpG site. To further clarify whether this phenomenon was due to the high affinity and selectivity for hemimethylated DNA, we performed an electrophoretic mobility shift assay (Figure 9). We tested binding to the FAM-labeled 13-bp DNA probes, which contained three CpG sites with either no C methylation or one hemi- or full-methylation and one that only consisted of TA sequences (C/C, M/C, M/M and TA) (Supplementary Table S2). Among the probes examined, HisMBP-wild-type human CDCA7 preferentially bound to hemimethylated DNA (M/C) (Figure 9). As the concentration of CDCA7 increased, the C/C and M/M probes showed a weak mobility shift compared with that of the TA probe. Such a hemimethylated CpG DNA-binding preference of CDCA7 was not observed in the case of CDCA7 lacking the ZF domain (HisMBP-CDCA7_1–261) and the ICF syndrome mutants of CDCA7 (HisMBP-CDCA7_R274C, HisMBP-CDCA7_R274H, and HisMBP-CDCA7_R304H). We also examined the DNA binding of HELLS and DDM1 and found that all types of DNA containing CpG sites showed similar band shifts but not the TA probe. These results indicate that the preference for hemimethylated CpG DNA for the DNA-dependent ATPase activity of HELLS-CDCA7 or DDM1-CDCA7 (Figures 7 and 8) is attributed to CDCA7, which preferentially binds to hemimethylated DNA via the C-terminal ZF domain. Several results of this assay were inconsistent with those of the ATPase assay, that is, binding to the TA probe was not observed, whereas TA DNA enhanced the ATPase activity of HELLS-CDCA7 (Figure 6). The length of the DNA used differed between the two assays, and the protein contained in the DNA-binding assay was only CDCA7, whereas that in the ATPase assay was HELLS-CDCA7. These differences may reflect the inconsistent results.

Figure 9. DNA biding ability of the CDCA7, HELLS, and DDM1 proteins. Electrophoretic mobility shift assay was performed using the 0.1 μM FAM-labeled 13-bp DNA composed of T and A (TA), containing unmethylated CpG (C/C), a hemimethylated site (M/C), or a methylated CpG site on each strand (M/M) as a probe (Supplementary Table S2). (A) The assay was performed without (-) or with 0.125, 0.25, 0.5, 1, 2 and 4 μM HisMBP-wild-type CDCA7. (B) The assay was performed without (no protein) or with 4 μM HisMBP-CDCA7_wild type, 1–261, R274C, R274H, R304H, HELLS, DDM1 or HisMBP-Arabidopsis CDCA7. The samples were analyzed on 10% polyacrylamide gel and the bands were detected by excitation and emission wavelengths at 488 and 536 nm, respectively.

We observed that HisMBP-CDCA7_1–176, but not HisMBP-CDCA7_1–261 and HisMBP-CDCA7_1–143, bound to all types of DNA probes, except TA (Supplementary Figure S8). These results suggest the possibility that residues 144–176, which are essential for the activation of HELLS but not for binding to HELLS (Figure 1A), have intrinsic DNA-binding ability, although they are blocked by the following C-terminal residues.

We observed that HisMBP-Arabidopsis CDCA7 also bound to all probes, except for TA (Figure 9B). Binding to the M/M probe was weaker than that to the C/C or M/C probes, and preferential binding to the M/C probe was not observed. The hemimethylated DNA preference of the Arabidopsis CDCA7 may be weaker than that of human CDCA7.

Replication-dependent heterochromatin foci formation of CDCA7 in mESCs

Finally, we examined the relation between the hemimethylated DNA-binding preference of CDCA7 and the subcellular localization of CDCA7 and HELLS, particularly their replication-dependent foci formation in DAPI-dense regions (PHC foci formation) in mice. First, we analyzed the localization of endogenous CDCA7 and HELLS in mESCs. We observed the formation of CDCA7 and HELLS PHC foci in the nucleus of a certain percentage of cells (Figure 10A and Supplementary Figure S9A). Subsequently, we analyzed the cell cycle stage in which these PHC foci were formed. Specifically, we wanted to correlate this with the late S phase in which the DAPI-dense region replicates, so we examined the situation in the mid-late S phase and before and after the mid-late S, early S and G2 phases. S-phase cells were detected by EdU incorporation, early and mid-late S were further distinguished by differences in their short-term labeled nuclear localization patterns (50), and G2-phase cells were determined by strong staining with an anti-H4K20me1 antibody (51). We found that the ratio of cells with CDCA7 and HELLS PHC foci formation was highest in the mid-late S phase (M-L) (73.7 ± 8.4 and 58.9 ± 5.6% for CDCA7- and HELLS-foci-positive cells, respectively), while most cells in early S (E) and G2 phase were foci negative (5.0 ± 1.1% and 0% in early S and 20.4 ± 2.8% and 22.3 ± 2.3% in G2 for CDCA7- and HELLS-foci-positive cells, respectively) (Figure 10B and Supplementary Figure S9A, upper and middle panels). This indicates that mouse CDCA7 and HELLS PHC foci formation is replication-dependent (52). We also checked PHC foci formation of another hemimethyl CpG binding molecule UHRF1 and confirmed enrichment of UHRF1 PHC foci formation in mid-late S phase as reported (53) (Supplementary Figure S9A, bottom panel, and Supplementary Figure S9B).

Figure 10. Replication-dependent PHC foci formation of mouse CDCA7 and HELLS in mESCs. (A) CDCA7 and HELLS foci formation at DAPI-dense regions (PHC foci formation) in wild-type mESCs. (B) CDCA7 and HELLS PHC foci positive cells in early S (E), mid-late S (M-L), and G2 phases were counted. Values are means ± SD of three independent experiments (n = 16−61 for each cell cycle in each experiment). S-phase cells were detected by EdU incorporation, and early and mid-late S were further distinguished by differences in the Edu nuclear localization pattern, and G2-phase cells were determined by strong staining with anti-H4K20me1 as shown in Supplementary Figure S9A. ****P< 0.0001, ***P< 0.001, **P< 0.01, n.s., not significant, one-way ANOVA (Prism version 10) (Supplementary Table S30). (C) IF analysis of exogenous G196 epitope-tagged mouse CDCA7 (G196-CDCA7) and endogenous HELLS in Cdca7 KO mESCs, or Cdca7 KO mESCs expressing G196-CDCA7 wild type or R285C mutant (G196-CDCA7 R285C) after 30 min Edu labeling. Upper panel: The arrowheads point to the example of G196-CDCA7 PHC foci positive cells in late S phase judged by the Edu staining pattern, and an enlarged image of one of them (pointed by arrowhead) is shown in the corner. Cener and lower panel: The arrowheads point to the example of cells in mid-late S phase, and an enlarged image of one of them (pointed by arrowhead) is shown in the corner. (D) G196-CDCA7 PHC foci positive cells in Cdca7 KO mESCs expressing wild type or R285C mutant CDCA7 in mid-late S (M-LS) phase were counted. Values are means ± SD of three independent experiments (n = 44−72 in each experiment and shown in Supplementary Table S18). ****P< 0.0001, t-test (Prism version 10) (Supplementary Table S31).

Furthermore, to test whether the 4CXXC-type ZF domain-dependent DNA hemimethylation recognition ability is important for the replication-dependent PHC foci formation of CDCA7, we first generated Cdca7 KO mESCs and then complemented them with the G196 epitope-tagged mouse CDCA7 wild type or R285C mutant (corresponding to the human ICF mutation R274C) (Supplementary Figure S10). We also generated Hells KO mESCs (Supplementary Figure S10). Before proceeding with the desired experiments, we performed WGBS analysis to determine the extent to which DNA methylation was affected in Cdca7 KO mESCs and, if so, in what regions. WGBS analysis of wild-type, Cdca7 KO, and CDCA7 wild-type or R285C rescued Cdca7 KO mESCs showed that the genome-wide DNA methylation level was reduced by approximately 10% in Cdca7 KO cells compared with that in wild-type cells (Supplementary Figure S11). In the satellite repeat regions, where DNA methylation levels are decreased in patients with the ICF syndrome and CDCA7 mutated cells (18,21,25,54,55), significant DNA hypomethylation was observed in minor satellite repeats, whereas no clear reduction was observed in major satellite repeats (Supplementary Figure S11A and B). This result is consistent with recent DNA methylation analyses of ICF-mutant CDCA7 knock-in mice (56). Furthermore, using information on the early and late replication regions obtained from mESC (39), we examined the impact of Cdca7 KO on these two chromatin regions and found significantly lower DNA methylation levels in the late replication regions than in the early replication regions (Supplementary Figure S11D). In addition, these DNA hypomethylation phenotypes were complemented in all wild type CDCA7-expressing cells, whereas the restorative effect was very poor in the ICF mutant R285C (Supplementary Figure S11).

We then analyzed the intracellular localization of exogenous CDCA7 wild type or R285C and endogenous HELLS. The results showed that exogenous G196-CDCA7 wild type expressed in Cdca7 KO mESCs was localized in the nucleus, and foci-positive cells were enriched in the mid-late S phase with a similar ratio (more than 70%) (Figure 10D and Supplementary Table S18) to those of endogenous cells. Furthermore, co-foci formation in HELLS was observed in almost all CDCA7 foci-positive cells (Supplementary Table S18). By contrast, in Cdca7 KO mESCs, although nuclear localization of HELLS was observed, clear foci formation was almost absent (HELLS foci-positive cells were occasionally observed, but their signal intensities were much weaker) (Figure 10C, lower panel). Importantly, in cells expressing the mouse version of the CDCA7 ICF syndrome mutant R285C, although the nuclear localization of CDCA7 was confirmed, clear foci formation almost disappeared, and HELLS PHC foci formation was almost completely absent, as in Cdca7 KO mESCs (Figure 10C, center panel,10D, and Supplementary Table S18). We further examined whether CDCA7 PHC foci formation is HELLS-dependent. As shown in Supplementary Figure S12A and B, CDCA7 foci formation was maintained in Hells KO mESCs. These results indicate that CDCA7 and HELLS induce replication-dependent PHC foci formation, which correlates with the hemimethylated DNA-binding preference of CDCA7; HELLS foci formation is CDCA7-dependent, but CDCA7 foci formation is independent of HELLS.

To further verify the correlation between CDCA7 hemimethylated DNA binding preference and foci formation, we analyzed the foci formation of CDCA7, HELLS and UHRF1 after a 1-day treatment with GSK-3484862, an inhibitor of DNMT1 that prevents full methylation of hemimethylated DNA by DNMT1 after replication. First, we observed that the amount of DNMT1 was significantly reduced in cells treated with GSK-3484862 for 1 day (57), indicating that the inhibitor treatment was effective (Supplementary Figure S13). When treated with GSK-3484862, the percentage of CDCA7 foci-positive cells increased, and the signal intensities of CDCA7 and HELLS replication-dependent PHC foci increased in Cdca7 KO mESCs expressing wild-type CDCA7 (Figure 11A and B). In Cdca7 KO mESCs, UHRF1 foci formation was also enhanced by GSK-3484862 treatment, and HELLS foci-positive cells were detected, although their signals were weaker (Supplementary Figure S12C). As it has been reported that HELLS physically and functionally interacts with UHRF1 and that UHRF1 induces replication-coupled PHC foci formation in HELLS (14), we speculated that the formation of HELLS foci observed in Cdca7 KO mESCs treated with GSK-3484862 is UHRF1 dependent. A definitive answer awaits the results of the analysis of whether HELLS PHC foci formation can no longer be observed after the GSK-3484862 treatment in Cdca7 KO cells with further knockdown or KO of Uhrf1.

Figure 11. Hemimethylated DNA dependent CDCA7 and HELLS PHC foci formation. (A) IF analysis of G196-CDCA7 and HELLS in Cdca7 KO mESCs expressing G196-CDCA7 wild type treated with (+) or without (–) a DNMT1 specific inhibitor, GSK-3484862 (GSK) (5 μM) for 24 h before the Edu labeling. The arrowheads point to the example of G196-CDCA7 PHC foci positive cells, and an enlarged image of one of them (pointed by yellow arrowhead) is shown in the corner. (B) G196-CDCA7 PHC foci positive cells were counted in Cdca7 KO mESCs expressing wild type CDCA7 with/without treatment of GSK for 24 hours. Values are means ± SD of three independent experiments (n = 100 in each experiment and shown in A). ***P< 0.001, t-test (Prism version 10) (Supplementary Table S31). (C) CDCA7 and HELLS PHC foci formation in Dnmt1, Dnmt3a, and Dnmt3b triple KO (Dnmt TKO) mESC line, 4OHT-2. White, yellow, and orange arrowheads indicate one example of cells in early S, mid-late S and G2 phase, respectively. (D) CDCA7 and HELLS PHC foci positive cells in early S (E), mid-late S (M-L) and G2 phases were counted in Dnmt TKO mESCs as shown in C. Values are means ± SD of three independent experiments (n = 17−42 for each cell cycle in each experiment). *P< 0.05, n.s., not significant, one-way ANOVA (Prism version 10) (Supplementary Table S32).

Finally, to test whether the replication-dependent PHC foci formation of CDCA7 is dependent on hemimethylated DNA from a different angle, we examined the nuclear localization of CDCA7 in Dnmt1, Dnmt3a, and Dnmt3b triple KO (Dnmt TKO) mESCs (Supplementary Figure S14), and examined the nuclear localization of CDCA7 and HELLS in Dnmt TKO mESCs (Figure 11C). Although replication-dependent foci formation of CDCA7 and HELLS in DAPI-dense regions was no longer enhanced, more than 60% of cells showed PHC foci formation of CDCA7 and HELLS not only in the mid-late phase (M-L), but also in the early S (E) and G2 phases (Figure 11C and D). Although these results do not rule out the possibility that hemimethylated DNA contributes to CDCA7/HELLS chromatin (heterochromatin) binding in wild type cells, in which high levels of CpG methylation of DNA are introduced, at least in the context of the complete absence of CpG methylation, including hemimethylation, it is suggested that CDCA7/HELLS can take on different chromatin binding modes. We await further analyses.

In conclusion, our results suggest that the hemimethylated DNA status induced after replication contributes to the formation of heterochromatin foci in CDCA7, HELLS, and UHRF1. Furthermore, these results suggest that the hemimethylated DNA preference of both CDCA7 and UHRF1 contributes to the formation of heterochromatin foci in HELLS, although the effect of CDCA7 seems to be dominant. As described previously, there was no clear difference in the expression of HELLS and UHRF1 between Cdca7 KO and Cdca7 KO mice rescued with wild type CDCA7 or R285C mESCs (Supplementary Figure S10).

Discussion

By examining the activities of purified recombinant human HELLS and wild-type and various mutant CDCA7 proteins, we identified the domain function of CDCA7 in its activity with HELLS (summarized in Figure 12). We also found that CDCA7 possesses a hemimethylated DNA-binding preference dependent on the C-terminal ZF domain and recruits HELLS to replication-dependent PHC loci through CDCA7’s hemimethylated DNA-binding potential.

Figure 12. Schematic representation of the domain function of CDCA7 for the activity with HELLS. The 4CXXC-type ZF domain (), binding region of the c-Myc and 14–3–3 proteins (), and an α-helix () predicted by AlphaFold2 (https://alphafold.ebi.ac.uk/entry/Q9BWT1) (42,43) (Supplementary Figure S1B), are indicated.

We confirmed that HELLS alone did not exhibit ATPase activity in the absence of CDCA7, despite the presence of an ATPase domain. We found that the mutant CDCA7 lacking the 31 or 68 N-terminal residues showed a higher degree of enhancement of the ATPase activity of HELLS in reaction conditions, as compared with that of the wild-type CDCA7. Thus, the N-terminal region may be involved in the negative regulation of HELLS activation. The residues 108–140 of CDCA7 are critical for binding to HELLS, and residues 108–176 are essential for the ATPase and nucleosome sliding activities of HELLS-CDCA7. The residues 108–176 contain a conserved region (around residues 110–140) among several species, whose three-dimensional structure has been confidently predicted as an α-helix (Supplementary Figure S1B), suggesting the importance of this region for binding to HELLS and the ATPase and nucleosome sliding activities of HELLS-CDCA7. It was suggested that HELLS has both autoinhibitory and CDCA7-binding domains at their N-termini (47,58). It is possible that the autoinhibitory domain interacts with the ATPase domain to inhibit its activity, which may be released by binding to CDCA7. The N-terminal portion of CDCA7 may interact with the central portion of CDCA7 or the N-terminal portion of HELLS to negatively regulate its activity.

It has been reported that c-Myc or 14–3–3 proteins bind to residues 146–170 of CDCA7, including the essential domain for the activation of HELLS, which depends on the phosphorylation of Thr163 of CDCA7 by AKT kinase (19). c-Myc binds to a region independent of phosphorylation to induce the apoptosis or transformation of a cell line. 14–3–3 competitively binds to its phosphorylated region and sequesters CDCA7 in the cytoplasm. Therefore, although the Thr163 phosphorylation of CDCA7 is not necessary for the ATPase activity of HELLS in vitro, it potentially controls HELLS’s nuclear function through modulation of CDCA7 intracellular localization. Additionally, the interaction of CDCA7 with HELLS may compete with c-Myc and/or 14–3–3 proteins in cells. CDCA7 transcription is regulated by c-Myc (15). Because the c-Myc- and 14–3–3-binding regions of CDCA7 are also involved in the binding and activation of HELLS, HELLS-CDCA7 is most likely involved in the phosphoinositide-3-kinase-AKT signaling network. If so, the regulation of DNA methylation is highly correlated with cellular activities in the phosphoinositide-3-kinase-AKT signaling network, including transcription and translation, as well as cell growth, development, proliferation, apoptosis, and aging (19,59,60).

We found that DNA containing hemimethylated CpG sites was most effective in enhancing the ATPase activity of HELLS-CDCA7, depending on the C-terminal ZF domain. In addition, CDCA7, but not HELLS, preferentially binds to hemimethylated DNA, depending on the C-terminal ZF domain. Furthermore, replication-dependent PHC foci formation in HELLS is strongly dependent on the hemimethylated DNA-binding preference of CDCA7. In mESCs, the hemimethylated DNA-binding preference of CDCA7 serves as the primary driving force for replication-dependent PHC foci formation in HELLS; however, when GSK-3484862 treatment creates an excess hemimethylated state, HELLS foci formation is also observed in Cdca7 KO cells (Supplementary Figure S12C), suggesting that hemimethylated DNA uses two reader molecules, UHRF1 and CDCA7, to recruit HELLS to hemimethylated DNA sites. These results support the previous finding that HELLS is involved in replication-uncoupled maintenance DNA methylation in late-replicating regions, which occurs after hemimethylated DNA wraps around histones (13,14,61). CDCA7 may be targeted to hemimethylated sites by the C-terminal ZF domain, followed by the recruitment of HELLS and induction of the chromatin remodeling function of the HELLS-CDCA7 complex. Indeed, the accumulation of CDCA7 and HELLS at replication-dependent PHC foci, depending on the ZF domain of CDCA7, was observed in mESCs (Figure 10). Defects in the ZF domain of CDCA7 reduce the ATPase activity of HELLS-CDCA7, as well as the binding of CDCA7 to DNA; thus, the ZF domain may be involved in not only targeting DNA but also activating HELLS-CDCA7. It is possible that when CDCA7 binds to hemimethylated sites via the ZF domain, conformational changes occur to enhance the binding of CDCA7 to HELLS and increase the ATPase and chromatin remodeling activities of HELLS. However, CDCA7_1–261 lacking the ZF domain could also weakly enhance the activities of HELLS, which may be induced by binding of the central domain of CDCA7 to HELLS. It is possible that binding of the central domain of CDCA7 to HELLS releases autoinhibition of HELLS, as discussed above. It was also observed that residues 144–176 of CDCA7, which are essential for the activation of HELLS, have intrinsic DNA-binding ability, although it might be blocked by the following residues without HELLS. CDCA7’s enhancing activity for HELLS’s DNA-dependent ATPase function was almost the same for HisMBP-CDCA7_1–261 and HisMBP-CDCA7_1–176. Therefore, it is possible that the conformational change in HisMBP-CDCA7_1–261 is induced by binding to HELLS, which allows residues 144–176 to interact with DNA, resulting in enhanced HELLS activity. Activation of the DNA-bound ZF domain of CDCA7 may be an additional mechanism that further enhances the activity of HELLS-CDCA7, the molecular mechanism of which should be clarified.

HELLS-CDCA7 also effectively recognized DNA fragments containing unmethylated CpG sites compared to other sites, although the effect was less than that of hemimethylated DNA, depending on the C-terminal ZF domain of CDCA7, to enhance the ATPase activity of HELLS-CDCA7. HELLS-CDCA7 also exhibited nucleosome-sliding activity in mononucleosomes containing unmethylated DNA. The sliding activity of HELLS-wild-type CDCA7 for canonical nucleosomes with the 601 sequence was biased in one direction. We found that the nucleosome containing three TpA sites instead of the three CpG in the 5′ linker region of the DNA increased sliding to the opposite direction. Such an effect was not observed in the case of nucleosomes containing two TpA sites instead of the two CpG in the 3′ linker region of the DNA. Thus, HELLS-CDCA7 probably efficiently recognized the three CpG in the 5′ linker region rather than the two CpG in the 3′ linker region to slide the nucleosome. Interestingly, CDCA7 defects in the C-terminal ZF domain induced nucleosome sliding in both directions almost equally for the three DNA sequences we tested. Thus, the ZF domain may preferentially guide HELLS-CDCA7 to the three CpG sites in the 5′ linker region of the canonical 601 DNA. It is possible that the ZF domain preferentially recognizes unmethylated CpG sites in the absence of hemimethylated DNA. If so, HELLS-CDCA7 may be involved in de novo DNA methylation of CpG sites, as indicated previously (11,12). Indeed, we observed that a CpG methyltransferase can access CpG sites for methylation when nucleosome sliding occurs via HELLS-CDCA7. In addition, CDCA7 localized to the PHC of Dnmt TKO mESCs, in which full- and hemimethylated DNA are absent. Furthermore, CDCA7/HELLS has been reported to be associated with factors that contribute to non-homologous end joining and DNA double-stranded break repair (25). If the CDCA7-HELLS complex can be recruited to chromatin via CpG sequence preference by the ZF domain, chromatin recruitment in addition to the chromatin remodeling activity of HELLS may contribute to the non-homologous end joining double-stranded break repair function. In patients with ICF syndrome with single amino acid substitutions in the ZF domain of CDCA7, a reduced affinity to hemimethylated sites and incorrect recognition of DNA/nucleosomes by HELLS-CDCA7 may occur, which might cause aberrant DNA methylation.

In A. thaliana, the ratio of non-CpG (CHG and CHH) methylation to CpG methylation is higher than that in vertebrates with HELLS-CDCA7 (62,63). DDM1 is involved in methylation of all sequence contexts (45,64), which might reflect our finding that DDM1 alone had DNA-dependent ATPase activity with no preference for the CpG or hemimethylated CpG site, unlike in the case of HELLS-CDCA7. Interestingly, in the presence of human CDCA7, DDM1 showed a preference similar to that of HELLS-CDCA7 depending on the function of the C-terminal ZF domain of CDCA7. We also observed that the Arabidopsis CDCA7 homolog showed similar activity on DDM1 to that of human CDCA7 in HELLS, although preferential recognition of hemimethylated DNA by Arabidopsis CDCA7 appeared weak compared with that of human CDCA7. We confirmed that DDM1 bound to both human and Arabidopsis CDCA7 proteins. The Arabidopsis CDCA7 homolog may function when CpG methylation is required. Thus, the functions of CDCA7 may be conserved across species.

Our results suggest that the chromatin-remodeling enzyme HELLS-CDCA7 induces nucleosome sliding in an ATP-dependent manner and that CpG sites exposed from the nucleosome are methylated by DNA methyltransferase, as indicated previously (23,44–46). CDCA7 is most likely a factor in the regulation of CpG DNA methylation, especially for the maintenance DNA methylation in late-replicating regions, by modulating the targeting and chromatin remodeling function of HELLS. WGBS analysis of Cdca7 KO mESCs supports this hypothesis.

Note: While preparing this manuscript, two related studies were reported in bioRxiv (58,65). Both studies also showed a C-terminal ZF domain-dependent hemimethylated DNA-binding preference for CDCA7 and an essential region of CDCA7 (110–140 of CDCA7) for HELLS binding. Replication-dependent CDCA7 PHC foci formation was described in Hardikar's work (65), all of which strongly support our findings. It should be pointed out that the results of all analyses, including our findings, contain unique data and complement each other to a certain extent with respect to the molecular function of CDCA7 in HELLS regulation.

Supplementary Material

gkae677_Supplemental_Files

Acknowledgements

We thank Drs Akihisa Osakabe for helpful support for the nucleosome sliding assay, Pierre-Antoine Defossez and Kosuke Yamaguchi for their valuable comments, Kazuyuki Ohbo for providing anti-UHRF1 antibody (ab46187), Hiroshi Kimura for providing anti-H4K20me1 antibody (41), Motoko Unoki for providing human CDCA7 and HELLS cDNAs, and Haruhiko Koseki and Sharif Jafar for providing Dnmt cTKO mESC line. We also thank the staff of the Support Unit for Bio-Material Analysis (BMA) at the RIKEN Center for Brain Science (CBS) Research Resources Division (RRD) for DNA sequencing, RIKEN Center for Sustainable Resource Science (CSRS) for use of equipment, and colleagues at Shinkai laboratory for their support and valuable comments. The A. thaliana full-length clones (pda08628 and pda19584 which encode A. thaliana DDM1 and CDCA7, respectively) used in this research was developed by the plant genome project of RIKEN Genomic Sciences Center.

Data availability

All reads from the WGBS experiments generated in this study have been submitted to the Gene Expression Omnibus under the accession numbers GSE270227.

Supplementary data

Supplementary Data are available at NAR Online.

Funding

Japan Ministry of Education, Culture, Sports, Science and Technology Grant-in-Aid for Scientific Research [18H05530, 18H03991 to Y.S., 18H05534, 23H05475, in part to H.K.]; Japan Science and Technology Agency ERATO (JPMJER1901 to H.K.); RIKEN internal research funds (Pioneering project ‘Genome building from TADs’ to Y.S.); National Institute of Allergy and Infectious Diseases (NIAID) grants from the National Institutes of Health (NIH) [R01 AI165840 to E.W.D.]; Research Support Project for Life Science and Drug Discovery (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED [JP23ama121009]. Funding for open access charge: Japan Society for the Promotion of Science.

Conflict of interest statement. None declared.
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