Dna-binding properties of the recombinant high-mobilitygroup-

Article in press - uncorrected proof
Biol. Chem., Vol. 387, pp. 1469–1478, October/November 2006 • Copyright by Walter de Gruyter • Berlin • New York. DOI 10.1515/BC.2006.184

DNA-binding properties of the recombinant high-mobilitygroup-like AT-hook-containing region from human BRG1 protein

Mahavir Singh, Loyola D’Silva and Tad A. Holak*
Max Planck Institute for Biochemistry, D-82152Martinsried, Germany
* Corresponding author e-mail: holak@biochem.mpg.de

Abstract
The hBRG1 protein, a central ATPase of the human switching/sucrose non-fermenting (SWI/SNF) remodeling complex, has a catalytic ATPase domain, an AT-hook motif and a bromodomain. Bromodomains, found in many chromatin-associated proteins, recognize N-acetyl-lysine in histones and other proteins. The AT-hook motif,first described in the high-mobility group of nonhistone chromosomal proteins HMGA1/2, is a DNA-binding motif. The AT-hook binds to the AT-rich DNA sequences in the minor groove of B-DNA in a nonsequence specific manner. AT-hook motifs have been identified in many other DNA-binding proteins. In this study we cloned and purified a fragment of hBRG1 encompassing the AT-hook region and the bromodomain.Nuclear magnetic resonance (NMR) and circular dichroism (CD) analyses show that the recombinant domains are structured. The functionality of subdomains was checked by assessing their interactions with N-acetylated peptides from histones and with DNA. Isothermal titration calorimetric (ITC) analysis demonstrates that the primary micromolar interaction is through the AT-hook motif. The AT-hookregion binds to linear DNA by unwinding it. These properties resemble the characteristics of the HMGA1/2 proteins and their interaction with DNA. Keywords: AT-hook; AT-hook-DNA interaction; BRG1; bromodomain; chromatin remodeling; histone acetylation; NMR; SWI/SNF.

Introduction
The BRG1 protein is a central ATPase of the ATP dependent switching/sucrose non-fermenting (SWI/SNF) chromatin-remodelingcomplex. SWI/SNF is a 2-MDa multisubunit remodeling complex that is highly conserved among eukaryotes (Flaus and Owen-Hughes, 2001; Muchardt and Yaniv, 2001). The mammalian SWI/ SNF complex contains each of two ATPases BRM1 and BRG1, in a mutually exclusive manner, and a number of BAFs (BRG1 associated factors) (Wang et al., 1996; Sif et al., 2001). BRG1 and BRM1 proteins share a high degree ofsequence similarity ()80%). The sequence similarity extends beyond the ATPase domains and both

proteins have other domains, for example, the bromodomain and the HMGA1 (high-mobility-group 1)-like AThook region, and two other uncharacterized domains at their N-termini (Figure 1A). High sequence homology suggests common general functions for the two proteins; however, contrasting phenotypes wereobtained for BRG1 and BRM1 gene knockout mice. BRG1 heterozygous mice display soft tissue sarcoma with features of rhabdoid tumors, whereas homozygous inactivation results in early embryonic lethality (Klochendler-Yeivin et al., 2000; Bultman et al., 2000; Roberts et al., 2000; Guidi et al., 2001). In contrast, BRM1 gene inactivation in mice is not lethal, and only an increase in body weight wasobserved (Reyes et al., 1998). Moreover, BRG1 was also found to be mutated in multiple human cancer cell lines (Wong et al., 2000; Roberts and Orkin, 2004). These results suggest that BRG1 can compensate the loss of BRM1, but probably not vice versa. Bromodomain is a conserved acetyl-lysine-recognizing and -binding domain found in many chromatin-associated proteins and in almost all histoneacetyltransferases (HATs) (Jeanmougin et al., 1997). It plays a crucial role in chromatin remodeling and signal transduction (Zeng and Zhou, 2002; Yang, 2004). The acetylated histone recognition by bromodomain of BRG1 has been implicated in translating the histone code (Strahl and Allis, 2000; Kouzarides, 2002). In vivo in the SWI/SNF complex, the bromodomain of BRG1 was capable of interacting with the...