Functional selectivity of recombinant mammalian SWI/SNF subunits

Abstract

The selective expression of genes that are packaged into repressive chromatin structures is a fundamental process that controls gene regulation during development. Genetic and biochemical studies have defined several elegant mechanisms that relieve nucleosomal repression and increase accessibility of DNA for protein interactions that establish appropriate patterns of gene expression (for review, see Kadonaga 1998; Kingston and Narlikar 1999). These mechanisms often involve large enzymatic complexes that structurally disrupt or modify histone–DNA contacts within nucleosomes to facilitate transcription factor binding.

One important category of such complexes is the SWI/SNF family of ATP-dependent chromatin-remodeling proteins (for review, see Muchardt and Yaniv 1999). SWI/SNF is a 2 Mda, multisubunit, DNA-dependent ATPase that has been shown genetically to regulate subsets of inducible genes in yeast (for review, see Winston and Carlson 1992) and biochemically to facilitate the interaction of a variety of transcription factors with nucleosomal DNA (Utley et al. 1997; for review, see Workman and Kingston 1998). Mammalian SWI/SNF complexes consist of ∼15 subunits and fall into two broad classes, depending on whether they contain hBRM or BRG1 as the ATPase. BRG1-associated factors (BAFs) tightly bind to either ATPase subunit to form distinct SWI/SNF complexes. These complexes are subject to cell cycle control by changes in phosphorylation (Muchardt et al. 1996; Sif et al. 1998) and can be quite biochemically diverse, which suggests that they may have specialized cellular functions (Wang et al. 1996).

Current studies support the view that SWI/SNF causes the partial unwrapping of DNA from the nucleosome without actual loss of histones, in contrast to the histone acetyl transferase p300 (Ito et al. 2000), and can promote both octamer sliding and transfer to neighboring DNA (for review, see Peterson and Workman 2000). Interestingly, human SWI/SNF (hSWI/SNF) can convert nucleosomal structure from a base state to a remodeled structure in a reversible manner (Schnitzler et al. 1998) and the recombinant ATPase subunit, hBRM or BRG1, is sufficient to disrupt histone–DNA contacts (Phelan et al. 1999).

Although considerable information is available concerning the mechanism by which SWI/SNF alters nucleosomal structure by ATP hydrolysis, little is known about how this complex is targeted to specific promoters to generate transcriptionally active genes. In this regard, SWI/SNF has been found to associate with diverse regulators of gene activation and cell proliferation. These include the glucocorticoid receptors (GRs) and estrogen receptors (Yoshinaga et al. 1992; Muchardt and Yaniv 1993; Ichinose et al. 1997; Ostlund-Farrants et al. 1997; Fryer and Archer 1998), the retinoblastoma tumor suppressor protein, Rb (Dunaief et al. 1994), and cyclin E (Shanahan et al. 1999). The c-MYC proto-oncogene and papillomavirus E1 protein can each associate and function with SWI/SNF, further supporting the notion that this complex participates in cell growth control (Cheng et al. 1999; Lee, D. et al. 1999; for review, see Muchardt and Yaniv 1999). Moreover, activation of the mammalian hsp70 gene in response to certain signaling pathways is also dependent on SWI/SNF components (de La Serna et al. 2000). Mammalian SWI/SNF has functional interactions with tissue-restricted activators such as EKLF (Armstrong et al. 1998) and C/EBPβ (Kowenz-Leutz and Leutz 1999) and cooperates with these proteins to regulate expression of β-globin and myeloid genes, respectively. Involvement of SWI/SNF in the developmental regulation of the human β-globin locus has been demonstrated recently in vivo (Lee, C.H. et al. 1999; O'Neill et al. 1999). Taken together, these studies clearly show that SWI/SNF has a critical role in a wide variety of transcriptional programs and that the specificity of chromatin remodeling must be a highly regulated process.

We have previously shown that a mammalian SWI/SNF complex (E-RC1) regulates transcription of chromatin-assembled human β-globin genes in combination with the erythroid factor EKLF in vitro (Armstrong et al. 1998). SWI/SNF facilitates the targeted interaction of EKLF to its binding site at −90 within the β-globin promoter, resulting in the generation of a DNase hypersensitive region, which is indicative of structurally remodeled chromatin. In contrast, SWI/SNF is unable to activate expression from chromatin-assembled HIV-1 promoters by the E-box binding protein, TFE3, which indicates that remodeling and activation by SWI/SNF is transcription factor selective in vitro. We have examined the basis for this apparent functional selectivity and the role of specific SWI/SNF subunits in factor-directed nucleosomal targeting and gene activation. Here we show that mammalian SWI/SNF cooperates with several proteins containing zinc finger DNA-binding domains (DBDs) to disrupt chromatin structure and to activate transcription. Targeted remodeling by SWI/SNF results from direct interaction with the zinc finger DBDs but not with the activation domains (ADs) of these factors. In contrast, SWI/SNF does not interact or function with two non–zinc finger proteins, TFE3 and NF-κB. We have examined the role of individual SWI/SNF subunits in this process and found that a minimal recombinant complex composed of two proteins is sufficient for factor-directed chromatin disruption and transcription. Thus functional selectivity by mammalian SWI/SNF occurs by direct interactions between specific protein domains and individual SWI/SNF subunits to achieve targeted chromatin remodeling.

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