Abstract
The NuRD (nucleosome remodeling and deacetylase) complex serves as a crucial epigenetic regulator of cell differentiation, proliferation, and hematopoietic development by coupling the deacetylation and demethylation of histones, nucleosome mobilization, and the recruitment of transcription factors. The core nucleosome remodeling function of the mammalian NuRD complex is executed by the helicase-domain-containing ATPase CHD4 (Mi-2β) subunit, which also contains N-terminal plant homeodomain (PHD) and chromo domains. The mode of regulation of chromatin remodeling by CHD4 is not well understood, nor is the role of its PHD and chromo domains. Here, we use small-angle X-ray scattering, nucleosome binding ATPase and remodeling assays, limited proteolysis, cross-linking, and tandem mass spectrometry to propose a three-dimensional structural model describing the overall shape and domain interactions of CHD4 and discuss the relevance of these for regulating the remodeling of chromatin by the NuRD complex.
Highlights
The nucleosome is the basic element of chromatin and is composed of approximately 147 bp of DNA tightly wrapped around a histone octamer comprising two copies of each of the core histones H2A, H2B, H3, and H4
Recombinant human CHD4 proteins containing either an N-terminal hexahistidine or a C-terminal decahistidine tag were expressed in Sf-9 insect cells and were purified on a nickel affinity column followed by size-exclusion chromatography (Fig. 1 and Supplementary Fig. 1)
Lane 1, total lysate; lanes 2–4, the elutions from nickel beads using buffer supplemented with 10, 30, and 300 mM imidazole, respectively; lane 5, Tobacco etch virus (TEV)-cleaved protein; lane 6, flow-through following reapplication of TEV-cleaved protein to a second nickel resin column; lanes 7–14, fractions from a peak with an elution volume corresponding to the predicted molecular mass of CC-AH-D, eluted from HiLoad Superdex 200 16/60 Prep Grade filtration column
Summary
The nucleosome is the basic element of chromatin and is composed of approximately 147 bp of DNA tightly wrapped around a histone octamer comprising two copies of each of the core histones H2A, H2B, H3, and H4. The following protein constructs were expressed and purified in this way (Fig. 1a): PP-CC-AH-D, PP-CC-AH, PP-CC, PP, CC-AH-D, and AH [where P = PHD domain, C = chromo domain, AH = ATPase-helicase domain, and D = domain of unknown function (DUF) 1].
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