Architectural DNA binding by a high-mobility-group/kinesin-like subunit in mammalian SWI/SNF-related complexes

Abstract

The SWI/SNF complex in yeast and Drosophila is thought to facilitate transcriptional activation of specific genes by antagonizing chromatin-mediated transcriptional repression. The mechanism by which it is targeted to specific genes is poorly understood and may involve direct DNA binding and/or interactions with specific or general transcription factors. We have previously purified a mammalian complex by using antibodies against BRG1, a human homologue of SWI2/SNF2. This complex is likely functionally related to the yeast SWI/SNF complex because all five subunit identified so far (referred to as BAFs, for BRG1-associated factors) are homologues of the yeast SWI/SNF subunits. However, we now describe the cloning of the 57-kDa subunit (BAF57), which is present only in higher eukaryotes but not in yeast. BAF57 is shared by all mammalian complexes and contains a high-mobility-group (HMG) domain adjacent to a kinesin-like region. Both recombinant BAF57 and the whole complex bind four-way junction (4WJ) DNA, which is thought to mimic the topology of DNA as it enters or exits the nucleosome. Surprisingly, complexes with mutations in the HMG domain of BAF57 can still bind 4WJ DNA and mediate ATP-dependent nucleosome disruption. Our work describes the first DNA binding subunit for SWI/SNF-like complexes and suggest that the mechanism by which mammalian and Drosophila SWI/SNF-like complexes interact with chromatin may involve recognition of higher-order chromatin structure by two or more DNA binding domains.

Figure 1

BAF57 is a shared core subunit of different mammalian BAF complexes. The purified mammalian BAF complex was analyzed by SDS/PAGE (8%) and silver staining (a). The antibodies used for immunopurification are marked on the top of each lane. MW refers to the molecular weight markers. (b and c) The load, flow-through (FT), and eluted fractions from the indicated immunoaffinity columns were analyzed by Western blot analysis by using antibodies indicated at the side of each panel by an arrow. The presence of IgG and crosslinked IgG (∗) were marked.

Figure 2

BAF57 contains a single HMG domain and a coiled-coil region similar to kinesin. (a) The sequence of human and mouse BAF57 proteins are compared, with identical residues represented by dashed lines. The underlined sequences represent peptide sequences obtained from microsequencing tryptic peptides of human BAF57 protein. (b) The domain structures of BAF57 are diagrammed. (c) The HMG domain of BAF57 is aligned with other HMG domains. The arrows indicate amino acid residues that distinguish between specific and nonspecific HMG families. The asterisk indicates the conserved lysine that has been mutated in the experiment described in Fig. 5d. dEST represents an EST sequence (ID AA201846 at dbEST databank maintained at NCBI) that likely encodes the Drosophila homologue of BAF57 (70% identical, 90% similar within the HMG domain). (d) The predicted coiled-coil region of BAF57 is aligned with kinesin. The arrows indicate the hydrophobic residues of the heptad repeats.

Figure 3

BAF57 is widely expressed in different tissues and cell lines. (a) RNA from different mouse tissues were examined by quantitative RNase protection analysis (35). The tissue source for the RNA and the tRNA control are indicated on the top of each lane. The probe for actin mRNA is used to normalize the RNA loaded for each lane (Lower). The RNA products were separated on a 6% polyacrylamide-urea gel and autoradiographed by using x-ray films. (b) Nuclear extracts from several cell lines derived from different tissue sources were analyzed by Western blot analysis by using anti-BAF57 antibodies. The name of each cell line and its species is indicated on the top of each lane and their description can be found in ref. .

Figure 4

The BAF57 HMG domain binds 4WJ DNA similar to the HMG1 protein. The probes illustrated in a are used to examine the binding properties of the mammalian SWI/SNF complexes and recombinant BAF57. The diagrams illustrate the structures of the 4WJ DNA and the two regular duplex DNA that made up its “arms.” (b) The truncated recombinant BAF57 proteins purified from E. coli were analyzed on a Coomassie blue-stained SDS gel (12%). (c) The DNA binding activities of wild-type and K112I mutant BAF57 proteins were analyzed by gel-shift assays by using 4WJ DNA as the probe. The concentrations of proteins are 30, 100, 300, and 1,000 nM, respectively. Also shown in the figure are binding of three other HMG box-containing proteins: HMG1, LEF1, and mTF1. Their concentrations are 300 and 1000 nM, respectively. (d) The DNA binding activities of recombinant BAF57 proteins were analyzed by gel-shift assays by using two duplex “arms” of 4WJ DNA as probes. The protein concentration is 1,000 nM for all proteins. Note that the recombinant proteins may not be 100% active. The arrows indicate the DNA–protein complex formed.

Figure 5

Purification of BAF complex containing exogenously introduced BAF57 tagged with the HA epitope. (a) The subunit composition of the HA-tagged BAF57 complexes purified by 12CA5 mAb is shown on a silver-stained SDS gel (8%). The complex containing the wild-type (WT) or the deletion mutant (del) are marked on the top of each lane. Mock refers to the control experiment by using the parental cell line without the exogenous BAF57 gene as the source for 12CA5 immunopurification. BRG1 represents the complex purified with BRG1 antibody. Several contaminating proteins were marked by ∗. The arrows indicate the HA-tagged wild-type BAF57 (WT), the deletion mutant whose entire HMG domain was removed (del), and the endogenous BAF57. They showed the different mobilities on the SDS gel caused by the addition of the HA tag. (b) The load, flow-through (FT), and elute fractions of 12CA5 immunoaffinity column for purification of the complex containing the HA-tagged wild-type BAF57 were analyzed by Western blotting. The arrows indicate the presence of immunoreactivity to 12CA5 antibody (Upper) or BAF57 antibody (Lower).

Figure 6

The mammalian BAF complex binds 4WJ DNA, a property similar to the yeast complex and HMG proteins. The DNA binding activity of HA-tagged BAF57 complex is analyzed by gel-shift assays by using either 4WJ DNA (a) or its two duplex DNA “arms” (b), as indicated by the arrows. The concentration of the complex is 5 nM. The complex containing either the HA-tagged wild-type BAF57 (WT) or the HMG deletion mutant (del) are indicated on the top of each lane. Mock refers to the negative control as described in the legend to Fig. 5. The presence or absence of 1 mM ATP is also indicated.

Figure 7

The HMG domain of BAF57 is not essential for the in vitro nucleosome-disruption activity of the complex. The BAF complexes containing either the wild-type (WT) or the deletion mutant (del) of BAF57, as indicated on the top of the figure, were analyzed for the ATP-dependent nucleosome disruption activities. The naked DNA template and the nucleosomal template are shown on the top of the figure. The presence or absence of 1 mM ATP is also indicated.