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. 2001 Jun 15;29(12):2517-21.
doi: 10.1093/nar/29.12.2517.

Mi2, an auto-antigen for dermatomyositis, is an ATP-dependent nucleosome remodeling factor

H B Wang  1 , Y Zhang
Affiliations

Mi2, an auto-antigen for dermatomyositis, is an ATP-dependent nucleosome remodeling factor

H B Wang et al. Nucleic Acids Res. .

Abstract

Dynamic changes in chromatin structure play an important role in transcription regulation. Recent studies have revealed two mechanisms that alter chromatin structure. One involves ATP-dependent chromatin remodeling, and the other involves acetylation of the core histone tails. We have previously purified and characterized a multi-subunit protein complex, NuRD, which possesses both nucleosome remodeling and histone deacetylase activities. Despite extensive biochemical characterization of the complex, little is known about the functions of its individual components. In this study, we focused on Mi2, a component of the NuRD complex. We found that, similar to the native NuRD complex, recombinant Mi2 is a DNA-dependent, nucleosome-stimulated ATPase. Kinetic analysis of the ATP hydrolysis reaction indicated that the differential stimulation of the Mi2 ATPase by DNA and nucleosomes were primarily due to their differential effects on the turnover number of the reaction. Furthermore, we demonstrated that recombinant Mi2 is an efficient nucleosome remodeling factor when compared to that of the native NuRD complex. Our results define the biochemical function of Mi2 and set the stage for understanding the mechanism of nucleosome remodeling in a defined reconstituted system.

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Figures

Figure 1
Figure 1
Mi2 is a DNA-dependent, nucleosome-stimulated ATPase. (A) Silver-stained SDS–PAGE containing the NuRD complex and recombinant Mi2. The identities of the proteins are indicated. (B) A comparison of the ATPase activity stimulated by DNA, histones or nucleosomes of equal amounts of Mi2 alone (rMi2) or in a complex (NuRD). (C) Quantification of the results shown in (B). Error bars represent the average deviation of two independent experiments.
Figure 2
Figure 2
Kinetic analysis of the ATP hydrolysis reaction catalyzed by NuRD and rMi2. (A and C) Plots of the ATP hydrolysis velocity, number of ATP molecules hydrolyzed by each molecule of rMi2 (C) or each NuRD (A) complex in 1 min, as a function of the ATP concentration. The plot for reactions in the presence of DNA or nucleosomes is indicated. (B and D) Double-reciprocal plots for NuRD (B) and rMi2 (D) kinetic analysis. The regression equations are shown in the Figures.
Figure 3
Figure 3
Mi2 is an ATP-dependent nucleosome remodeling factor. Mononucleosome disruption assay comparing the efficiency of rMi2 (lanes 2–7) with the NuRD complex (lanes 8–10). End-labeled mononucleosomes with a concentration of 20 nM were incubated with increasing concentrations of rMi2 as indicated, followed by DNase I digestion. The presence or absence of ATP in the reactions is indicated. The concentration of NuRD in the reactions is also indicated.
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