Chromatin remodeling by the CHD7 protein is impaired by mutations that cause human developmental disorders

Abstract

Mutations in the CHD7 gene cause human developmental disorders including CHARGE syndrome. Genetic studies in model organisms have further established CHD7 as a central regulator of vertebrate development. Functional analysis of the CHD7 protein has been hampered by its large size. We used a dual-tag system to purify intact recombinant CHD7 protein and found that it is an ATP-dependent nucleosome remodeling factor. Biochemical analyses indicate that CHD7 has characteristics distinct from SWI/SNF- and ISWI-type remodelers. Further investigations show that CHD7 patient mutations have consequences that range from subtle to complete inactivation of remodeling activity, and that mutations leading to protein truncations upstream of amino acid 1899 of CHD7 are likely to cause a hypomorphic phenotype for remodeling. We propose that nucleosome remodeling is a key function for CHD7 during developmental processes and provide a molecular basis for predicting the impact of disease mutations on that function.

Fig. 1.

CHD7 is an ATP-dependent nucleosome remodeling factor. (A) Schematic representation of CHD7. For domain terminology see text. (B) Proteins used in assays were separated by SDS-PAGE and stained with Coomassie blue. (C) Analysis of nucleosome remodeling activities by restriction enzyme accessibility (REA) assay. The assay measured the ability of the remodeling factors to expose an MfeI restriction site in the nucleosome at +28-bp (see methods for details). Control (buffer only), remodeling factors (hSWI/SNF ∼75 nM, CHD7 10 nM, CHD7 K999R 10 nM) were incubated with radiolabeled nucleosomes (20 nM) with a 50-bp DNA overhang, in the presence or absence of ATP (2 mM) as indicated. (D) Nucleosome mobilization assay. Increasing concentrations of enzymes (hSWI/SNF: ∼6 nM, ∼12 nM, ∼25 nM, SNF2H: 42 nM, 85 nM, 170 nM; CHD7 and CHD7 K999R: 0.25 nM, 0.5 nM, 1 nM) were incubated with a radiolabeled end-positioned +50-bp nucleosome (20 nM) in the absence or presence of ATP, as indicated. Migration of the end- or centrally positioned nucleosome (filled circle) is indicated on the left.

Fig. 2.

CHD7 presents biochemical properties that are distinct from SWI/SNF- and ISWI-type enzymes. (A) REA assay. The assay measured the ability of the remodeling factors (control: buffer only, hSWI/SNF: 8 nM, SNF2H: 32 nM, CHD7: 1 nM, as indicated) to expose an MfeI restriction site in radiolabeled nucleosomes (4 nM) with a 30-bp, 40-bp, or 50-bp DNA overhang, as indicated. Aliquots of the reactions were quenched at 2.5 min, 5 min, 10 min, and 20 min, and cut/uncut DNAs were separated by PAGE. (B) Quantification of gels in (A). Error bars reflect SEM and were derived from 3 independent experiments. (C) REA assay. These experiments were performed as in A using radiolabeled +50-bp nucleosomes only and nucleosomes were assembled using either untreated (intact) or partially trypsinized (tail-less) histones from HeLa cells, as indicated. (D) Quantification of gels in C. Error bars reflect SEM and were derived from three independent experiments.

Fig. 3.

Mutations leading to C-terminal protein truncations upstream of the amino acid residue 1899 produce proteins with very low or no remodeling abilities. (A) Schematic representation of WT CHD7 and deletion mutants. Numbers refer to amino acid residues. (B) Deletion mutant protein preparations were separated by SDS-PAGE and stained with Coomassie blue. (C) ATPase assay. Ten nM of WT CHD7 or deletion mutants were incubated in the presence of buffer only (white bars), “+50-bp” DNA (60 nM; gray bars) or +50-bp nucleosomes (60 nM; black bars) and 20 μM ATP/ 60 μM Mg²⁺ supplemented with γ-[³²P]ATP. Reactions were quantified using TLC plates at various time points; the 24’ time point is displayed as a bar graph. Error bars reflect SEM derived from at least two independent experiments. (D) Analysis of WT CHD7 and deletion mutants in REA assays. REA reactions: Control (buffer only) or remodeling factors: WT CHD7 (10 nM), deletion mutants (10 and 100 nM as indicated on top of the gel lanes) were incubated with radiolabeled +50-bp nucleosomes (20 nM). (E) Quantification of gels shown in D. Error bars reflect SEM and were derived from at least two independent experiments.

Fig. 4.

CHD7 point mutations reported in CHARGE syndrome and IHH patients variably affect the nucleosome remodeling activity of CHD7. (A) CHD7 point mutant protein preparations were separated by SDS-PAGE and stained with Coomassie blue. (B) ATPase assay. Ten nM of WT or mutant CHD7 proteins were incubated and processed as in Fig. 3C. (C) Analysis of WT CHD7 and point mutants in REA assays. Control (buffer only) or WT CHD7 and point mutants (10 nM) were incubated at with radiolabeled +50-bp nucleosomes (1 nM), in the presence of ATP (2 mM). Samples and data were processed as in Fig. 3D. (D) Quantification of gels shown in C. Error bars reflect SEM and were derived from at least two independent experiments.