DMi-2 Chromatin Binding and Remodeling Activities Are Regulated by dCK2 Phosphorylation

Karim Bouazoune,Alexander Brehm

doi:10.1074/jbc.m507084200

Karim Bouazoune, Alexander Brehm

Open Access

https://doi.org/10.1074/jbc.m507084200

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Abstract

A plethora of ATP-dependent chromatin-remodeling enzymes have been identified during the last decade. Many have been shown to play pivotal roles in the organization and expression of eukaryotic genomes. It is clear that their activities need to be tightly regulated to ensure their coordinated action. However, little is known about how ATP-dependent remodelers are regulated at the molecular level. Here, we have investigated the ATP-dependent chromatin remodeling enzyme Mi-2 of Drosophila melanogaster. Radioactive labeling of S2 cells reveals that dMi-2 is a phosphoprotein in vivo. dMi-2 phosphorylation is constitutive, and we identify dCK2 as a major dMi-2 kinase in cell extracts. dCK2 binds to and phosphorylates a dMi-2 N-terminal region. Dephosphorylation of recombinant dMi-2 increases its affinity for the nucleosome substrate, nucleosome-stimulated ATPase, and ATP-dependent nucleosome mobilization activities. Our results reveal a potential mechanism for regulation of the dMi-2 enzyme and point toward CK2 phosphorylation as a common feature of CHD family ATPases.

Highlights

CHD proteins harbor a pair of chromodomains, which have been implicated in binding of nucleosomal DNA and in the recognition of the methylated lysine 4 mark on histone H3 [4, 5]
NuRD complexes combine two enzymatic activities both of which are directed toward chromatin: a nucleosome remodeling ATPase activity, supplied by the Mi-2 subunit, and a type I histone deacetylase activity
Preparation of Whole Cell Extracts—Drosophila Schneider line 2 (S2) cells were lysed in lysis buffer (LyBu) 200 (20 mM Hepes, pH 7.6, 10 mM ␤-glycerophosphate, 0.1% Nonidet P-40, 10% glycerol, and protease inhibitors) containing 200 mM KCl by subjecting the cells to two freezethaw cycles followed by sonication

Summary

Introduction

CHD proteins harbor a pair of chromodomains, which have been implicated in binding of nucleosomal DNA and in the recognition of the methylated lysine 4 mark on histone H3 [4, 5]. NuRD complexes regulate cell fate determination during lymphoid and erythroid differentiation (19 –22) These cell type-specific functions of Mi-2 ATPases contrast sharply with their ubiquitous expression patterns [13]. It has been suggested that specificity is achieved by interaction with cell type-specific transcriptional repressors that direct NuRD complexes to particular chromosomal sites. The dMi-2 “corepressor” appears to localize to most sites of active transcription on polytene chromosomes [29] These results suggest that chromatin association alone is not sufficient for the formation of repressive chromatin structure. Rather, these studies emphasize the need for regulation of NuRD activity at steps subsequent to chromatin binding

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