Jenness C , Giunta S , Müller MM , Kimura H , Muir TW , Funabiki H .

R01 GM075249 NIGMS NIH HHS , R01 GM107047 NIGMS NIH HHS , R37 GM086868 NIGMS NIH HHS

             DNA methylation
             nucleosome organization

           IMMUNODEFICIENCY-CENTROMERIC INSTABILITY-FACIAL ANOMALIES SYNDROME 4; ICF4

	Fig. 1. Proteomic profiling of chromatin regulation by the cell cycle, the CPC, and H3K9me3. (A) Schematic of the procedure to identify chromatin-bound proteins in Xenopus egg extracts. Beads coated with unmodified or H3K9me3 nucleosome arrays were incubated in M phase or interphase Xenopus egg extracts subject to CPC or mock depletion. Beads were recovered, washed, and bound proteins were analyzed by LC-MS/MS. (B–F) Abundance of condensin (B), cohesin (C), Ku70–Ku80 (D), MSH2–6 (E), and MCM2–7 (F), identified by LC-MS/MS on the indicated chromatin beads. Abundance (arbitrary units) was calculated by integrating LC-MS signals for each peptide, as described in Methods.
	Fig. 2. Chromatin binding of HELLS–CDCA7e is coregulated by the CPC and nucleosomes. (A) Unsupervised clustering of chromatin-associated proteins. A heatmap of each protein’s normalized profile is displayed, with proteins enriched on a given chromatin sample colored yellow. Examples of known stoichiometric chromatin-associated complexes cluster together and are indicated. Fine-grained protein complex clusters (Left) and coarse-grained cell-cycle clusters (Right) are labeled. (B) LC-MS/MS analysis HELLS and CDCA7e copurified with unmodified or H3K9me3 nucleosome beads recovered from M phase or interphase mock or CPC-depleted extracts. (C) Western blot analyses of HELLS and CDCA7e copurified with nucleosome beads recovered from M phase or interphase mock or CPC-depleted extracts. Representative of n = 2 independent experiments. (D) Western blot analyses of proteins copurified with nucleosome beads recovered from M phase mock or CPC-depleted extracts. Nucleosomes containing wild-type H3 (SS), H3S10AS28A (AA), or H3S10DS28D (DD) were used. (E) Western blot analyses of proteins copurified with nucleosome or DNA beads recovered from M phase or interphase H3–H4-depleted extracts. (F) Abundance of HELLS and CDCA7e on nucleosome or DNA beads recovered from M phase H3–H4-depleted extracts quantified by LC-MS/MS. Data from ref. 28 (originally missing CDCA7e) was reanalyzed using an mRNA reference database containing CDCA7e (34).
	Fig. 3. CDCA7e directly recruits HELLS to chromatin. (A and B) Western blot analysis of CDCA7e (A) and HELLS (B) immunoprecipitation from M phase Xenopus extracts. Preimmune rabbit IgG was used to control for nonspecific binding. Representative of n = 3 independent experiments. (C) Coomassie-stained gel of purified HELLS–CBP coimmunoprecipitation with MBP–CDCA7. Purified HELLS–CBP was incubated with MBP–CDCA7e or MBP alone and immunoisolation was performed using beads coupled with anti-MBP antibodies or control IgG. Representative of n = 2 independent experiments. (D) Western blot analyses of proteins copurified with nucleosome beads recovered from interphase extracts mock depleted or depleted of HELLS or CDCA7e. Representative of n = 4 independent experiments. (E) Abundance of HELLS and CDCA7e on nucleosome beads recovered from interphase or M phase extracts mock depleted or depleted of HELLS or CDCA7e, quantified by LC-MS/MS. (F) Coomassie-stained gel of a pulldown of nucleosome or DNA beads incubated with MBP–CDCA7e. Uncoupled beads were used to control for nonspecific binding. Representative of n = 2 independent experiments.
	Fig. 4. CDCA7e is an essential stimulator of HELLS nucleosome remodeling activity. (A) Restriction enzyme accessibility nucleosome remodeling assay with HELLS–CBP and MBP–CDCA7e. The 601-positioned mononucleosomes with a PstI site engineered 15 bp into the nucleosome with 20 bp flanking DNA on each end were incubated with the indicated remodeling proteins and PstI. Productive nucleosome sliding exposes the PstI site, resulting in cleaved DNA (arrow). DNA was resolved on a 10% polyacrylamide gel and visualized with SYBR Gold. Representative of n = 2 independent experiments. (B) Native gel nucleosome remodeling assay with HELLS–CBP and MBP–CDCA7e. Center-positioned mononucleosomes (same as in A) were incubated with the indicated remodeling proteins. Reactions were stopped, resolved on a 5% polyacrylamide gel, and visualized with SYBR Gold. Sliding results in end-positioned nucleosomes, which migrate faster. Representative of n = 2 independent experiments.
	Fig. 5. CDCA7 ICF mutants are defective in chromatin recruitment of the HELLS–CDCA7 complex. (A) Alignment of CDCA7 zinc finger domains from the indicated species. The 4CXXC motif is underlined, and residues with identified ICF mutations (*) are highlighted in red. (B) Coomassie-stained gel of a pulldown of DNA beads incubated with MBP–CDCA7e harboring the indicated ICF mutations. Uncoupled beads (Bottom) were used to control for nonspecific binding. Representative of n = 2 independent experiments. (C) Western blot analyses of proteins copurified with chromatin beads recovered from interphase extracts mock depleted or depleted of CDCA7e. Beads coated with 19 × 601 naked DNA were chromatinized in interphase extract for 90 min before addition of 1 μM recombinant MBP–CDCA7e harboring the indicated ICF mutations. Following an additional 60-min incubation, chromatin beads were recovered. Representative of n = 2 independent experiments. (D) Restriction enzyme accessibility nucleosome remodeling assay with HELLS–CBP and MBP–CDCA7e with the indicated ICF mutations. The 601-positioned mononucleosomes with a 34 and 15 bp flanking DNA on the 5′ and 3′ end, respectively, incubated with the indicated remodeling proteins and MspI endonuclease. Productive nucleosome sliding exposes an MspI site, resulting in cleaved DNA (arrow). DNA was resolved on a 10% polyacrylamide gel and visualized with SYBR Gold.
	Fig. 6. Functional mapping of proteins related to ICF syndrome. ZBTB24 stimulates transcription of CDCA7. CDCA7 associates with chromatin, recruits HELLS, and remodels nucleosomes to allow DNMT3b-mediated DNA methylation. CDCA7 ICF mutations inhibit chromatin binding and recruitment of HELLS to chromatin.
	Fig. S1.{panel A, B, C] Proteomic profiling of chromatin regulation by the cell cycle, CPC, and H3K9me3 to identify how protein complexes are regulated. (A–E) Abundance of HP1-gamma-SUV39H1 (A), ATRX–DAXX (B), SSRP1–SPT16 (C), FANCI–D2 (D), and Astrin–SKAP (E) identified by LC-MS/MS on the indicated chromatin beads. Abundance (arbitrary units) was calculated by integrating LC-MS signals for each peptide, as described in Methods. (F) Abundance of the indicated protein complexes on M phase and interphase chromatin identified by LC-MS/MS. (G–K) Dendrogram of the mitosis-specific branch (F), general branch (H and J), M phase enriched branch (I), or HELLS–CDCA7 branch (K) of hierarchical clustering shown in Fig. 2A. A heatmap of each protein’s normalized profile is displayed, with proteins enriched on a given chromatin sample colored yellow. Note the condensin subunits cluster within the larger mitosis-specific cluster.
	Fig. S1. [panels D E F] Proteomic profiling of chromatin regulation by the cell cycle, CPC, and H3K9me3 to identify how protein complexes are regulated. (A–E) Abundance of HP1-gamma-SUV39H1 (A), ATRX–DAXX (B), SSRP1–SPT16 (C), FANCI–D2 (D), and Astrin–SKAP (E) identified by LC-MS/MS on the indicated chromatin beads. Abundance (arbitrary units) was calculated by integrating LC-MS signals for each peptide, as described in Methods. (F) Abundance of the indicated protein complexes on M phase and interphase chromatin identified by LC-MS/MS. (G–K) Dendrogram of the mitosis-specific branch (F), general branch (H and J), M phase enriched branch (I), or HELLS–CDCA7 branch (K) of hierarchical clustering shown in Fig. 2A. A heatmap of each protein’s normalized profile is displayed, with proteins enriched on a given chromatin sample colored yellow. Note the condensin subunits cluster within the larger mitosis-specific cluster.
	Fig. S1. [panels G-K] Proteomic profiling of chromatin regulation by the cell cycle, CPC, and H3K9me3 to identify how protein complexes are regulated. (A–E) Abundance of HP1-gamma-SUV39H1 (A), ATRX–DAXX (B), SSRP1–SPT16 (C), FANCI–D2 (D), and Astrin–SKAP (E) identified by LC-MS/MS on the indicated chromatin beads. Abundance (arbitrary units) was calculated by integrating LC-MS signals for each peptide, as described in Methods. (F) Abundance of the indicated protein complexes on M phase and interphase chromatin identified by LC-MS/MS. (G–K) Dendrogram of the mitosis-specific branch (F), general branch (H and J), M phase enriched branch (I), or HELLS–CDCA7 branch (K) of hierarchical clustering shown in Fig. 2A. A heatmap of each protein’s normalized profile is displayed, with proteins enriched on a given chromatin sample colored yellow. Note the condensin subunits cluster within the larger mitosis-specific cluster.
	Fig. S2. HELLS and CDCA7e are stoichiometrically equivalent on chromatin beads. (A) Quantification of Western blot bands of CDCA7e and HELLS in Xenopus egg extract. The amount loaded of each protein was calculated from their concentration in extract (34). (B) Western blot quantification of the amount of HELLS and CDCA7e on nucleosome beads recovered from interphase extract. Each connected pair of dots is an independent incubation of chromatin beads in extract. Wilcoxon matched pairs test was used to determine significance. (C) Western blots used to quantify A and B
	Fig. S3. [panel A B C] Characterization of HELLS and CDCA7 interaction and chromatin binding. (A) Coomassie-stained gel of purified MBP–CDCA7e and HELLS–CBP used in this study. (B) Coomassie-stained gel of a pulldown of 19 × 601 DNA beads incubated with MBP, MBP–CDCA7e, or HELLS–CBP. Uncoupled beads were used to control for nonspecific binding. (C) Western blot analysis of HELLS coimmunoprecipitation with CDCA7 paralogs. MYC–CDCA7 paralogs were expressed from mRNA in interphase Xenopus egg extract. Immunoprecipitation was performed using beads coupled with anti-HELLS antibodies or control IgG. Representative of n = 2 independent experiments. (D) Coomassie-stained gel of a pulldown of mononucleosomes with 5 bp 5′ linker or the naked DNA equivalent incubated with MBP–CDCA7e. Uncoupled beads were used to control for nonspecific binding.
	Fig. S3. [panel D] Characterization of HELLS and CDCA7 interaction and chromatin binding. (A) Coomassie-stained gel of purified MBP–CDCA7e and HELLS–CBP used in this study. (B) Coomassie-stained gel of a pulldown of 19 × 601 DNA beads incubated with MBP, MBP–CDCA7e, or HELLS–CBP. Uncoupled beads were used to control for nonspecific binding. (C) Western blot analysis of HELLS coimmunoprecipitation with CDCA7 paralogs. MYC–CDCA7 paralogs were expressed from mRNA in interphase Xenopus egg extract. Immunoprecipitation was performed using beads coupled with anti-HELLS antibodies or control IgG. Representative of n = 2 independent experiments. (D) Coomassie-stained gel of a pulldown of mononucleosomes with 5 bp 5′ linker or the naked DNA equivalent incubated with MBP–CDCA7e. Uncoupled beads were used to control for nonspecific binding.
	Fig. S4. Human HELLS chromatin binding is reduced upon CDCA7 depletion. (A) Western blot of chromatin from HeLa cells after control (siCNT) and CDCA7 (siCDCA7) RNAi, blotted as indicated. Numbers indicate signal intensity normalized to the loading and to lane 1 (red). (B) Whole cell extract of control (siCNT) and CDCA7 (siCDCA7) RNAi, and 130% (1.3), 50% (0.5), and 25% (0.25) of the control sample, blotted as indicated. (C) Western blot of chromatin fraction from HeLa cells treated with nocodazole (Noc) alone, or in combination with ZM 447439 (ZM) for 24 h, and blotted as indicated.
	Fig. S5. Characterization of HELLS–CDCA7e ATPase and remodeling activity. (A) Entire gel from Fig. 4B. (B) Native gel nucleosome remodeling assay. Endpositioned (Left) or center-positioned mononucleosomes (Right) were incubated with the indicated remodeling proteins. Reactions were stopped, resolved on a 5% polyacrylamide gel, and visualized with SYBR Gold. Representative of n = 2 independent experiments. (C) Restriction enzyme accessibility nucleosome remodeling assay. The 601-positioned mononucleosomes with a HaeIII site 11 bp into the nucleosome with 60 bp flanking DNA on the 3′ end were incubated with the indicated remodeling proteins and HaeIII. Productive nucleosome sliding exposes the HaeIII site, resulting in cleaved DNA (arrow). DNA was resolved on a 10% polyacrylamide gel and visualized with SYBR Gold. (D and E) Quantification of ATPase activity. The indicated proteins were incubated with gamma33P ATP for the indicated times at 37 °C (D) or 16 °C (E) for the indicated time. Reaction was separated by TLC, exposed to a PhosphorStorage screen, and the fraction of hydrolyzed ATP was quantified. In E, mean and SD from n = 3 distinct replicates are displayed.
	Fig. S6. Characterization of CDCA7e ICF mutations. (A) Coomassie staining (Top) and autography (Bottom) of HELLS–CDCA7e ICF mutant immunoprecipitation. Recombinant MBP–CDCA7e harboring the indicated ICF mutations was immunoprecipitated from reticulocyte lysate expressing 35S-labeled HELLS–GFP or GFP alone. Representative of n = 2 independent experiments. (B) Quantification of A. Mean and range from two independent experiments are plotted. (C) Western blot analyses of proteins copurified with chromatin beads recovered from interphase extracts mock depleted or depleted of CDCA7e. Beads coated with 19 × 601 naked DNA were chromatinized in interphase extract for 90 min before addition of 1 μM recombinant MBP–CDCA7e harboring the indicated ICF mutation. Following an additional 60-min incubation, chromatin beads were recovered. (D and E) Restriction enzyme accessibility nucleosome remodeling assay. The 601-positioned mononucleosomes (15 nM) with a 34 and 15 bp flanking DNA on the 5′ and 3′ end, respectively, incubated with the indicated remodeling proteins at their indicated concentration and MspI endonuclease. Productive nucleosome sliding exposes an MspI site, resulting in cleaved DNA (arrow). DNA was resolved on a 10% polyacrylamide gel and visualized with SYBR Gold. Representative of n = 2 (D) or n = 1 (E) independent experiments.
	Fig. S7. The specificity of CMA314 evaluated by ELISA. A serial dilution series of CMA314 hybridoma culture supernatant was incubated with plates coated with different peptides. CMA314 reacted with peptides containing H3S10ph. Details of peptides can be found in ref. 64.

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