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Abstract
A major challenge in chromatin biology is to understand the mechanisms by which chromatin is remodeled into active or inactive states as required during development and cell differentiation. One complex implicated in these processes is the nucleosome remodeling and histone deacetylase (NuRD) complex, which contains both histone deacetylase and nucleosome remodeling activities and has been implicated in the silencing of subsets of genes involved in various stages of cellular development. Chromodomain-helicase-DNA-binding protein 4 (CHD4) is a core component of the NuRD complex and contains a nucleosome remodeling ATPase domain along with two chromodomains and two plant homeodomain (PHD) fingers. We have previously demonstrated that the second PHD finger of CHD4 binds peptides corresponding to the N terminus of histone H3 methylated at Lys(9). Here, we determine the solution structure of PHD2 in complex with H3K9me3, revealing the molecular basis of histone recognition, including a cation-π recognition mechanism for methylated Lys(9). Additionally, we demonstrate that the first PHD finger also exhibits binding to the N terminus of H3, and we establish the histone-binding surface of this domain. This is the first instance where histone binding ability has been demonstrated for two separate PHD modules within the one protein. These findings suggest that CHD4 could bind to two H3 N-terminal tails on the same nucleosome or on two separate nucleosomes simultaneously, presenting exciting implications for the mechanism by which CHD4 and the NuRD complex could direct chromatin remodeling.
Highlights
Is translated to chromatin in the form of specific post-translational modification (PTM)4 patterns on histone tails
Chromodomain-helicase-DNA-binding protein 4 (CHD4)-PHD1 Recognizes Histone H3—We have previously shown that PHD2 of CHD4 is capable of recognizing H3 unmodified at Lys4 and methylated at Lys9
Conservation of the Histone Binding Ability of plant homeodomain (PHD) in CHD4-related Proteins—The nucleosome remodeling and histone deacetylase (NuRD) complex has been purified from human (64 – 66), amphibian [67], and insect cells [68], and proteins with homology to individual NuRD components, including CHD4, have been identified in nematodes and plants [3]
Summary
Sequence Analysis and Molecular Diagrams—Sequence analyses and alignments of DNA and proteins were carried out using ClustalW [33] and BioManager 3.0 (no longer available) followed by manual adjustment. Combinatorial On-bead Screening Assay—A 5000-member PTM-randomized combinatorial peptide library based on the first 10 residues of the histone H3 N terminus was incubated first with the GST-tagged version of CHD4-PHD1; second with a GST-specific primary antibody; third with a biotinylated secondary antibody, and with streptavidin-conjugated alkaline phosphatase, catalyzing the turnover of 5-bromo-4chloroindol-3-yl phosphate, which results in the formation of a turquoise precipitate on beads bearing sequences that bind to the target protein. 1H assignments of unlabeled PHD2-bound H3K9me peptide (ARTKQTARKme3STG synthesized by the Peptide Core Facility, University of Colorado Denver, or ARTKQTARKme3STGGY purchased from Peptide 2.0, Chantilly, VA) were obtained from 15N/13C double half-filteredNOESY, two-dimensional COSY and two-dimensional TOCSY spectra as well as a 13C/13C double half-filtered-NOESY acquired on a sample in D2O buffer. The 50 lowest energy structures were subjected to water refinement, and the 20 lowest energy water-refined structures analyzed by PROCHECK NMR [52]
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