Jacques Côté

Purification and biochemical heterogeneity of the mammalian SWI-SNF complex.

We have purified distinct complexes of nine to 12 proteins [referred to as BRG1‐associated factors (BAFs)] from several mammalian cell lines using an antibody to the SWI2‐SNF2 homolog BRG1. Microsequencing revealed that the 47 kDa BAF is identical to INI1. Previously INI1 has been shown to interact with and activate human immunodeficiency virus integrase and to be homologous to the yeast SNF5 gene. A group of BAF47‐associated proteins were affinity purified with antibodies against INI1/BAF47 and were found to be identical to those co‐purified with BRG1, strongly indicating that this group of proteins associates tightly and is likely to be the mammalian equivalent of the yeast SWI‐SNF complex. Complexes containing BRG1 can disrupt nucleosomes and facilitate the binding of GAL4‐VP16 to a nucleosomal template similar to the yeast SWI‐SNF complex. Purification of the complex from several cell lines demonstrates that it is heterogeneous with respect to subunit composition. The two SWI‐SNF2 homologs, BRG1 and hbrm, were found in separate complexes. Certain cell lines completely lack BRG1 and hbrm, indicating that they are not essential for cell viability and that the mammalian SWI‐SNF complex may be tailored to the needs of a differentiated cell type.

Persistent Site-Specific Remodeling of a Nucleosome Array by Transient Action of the SWI/SNF Complex

The SWI/SNF complex participates in the restructuring of chromatin for transcription. The function of the yeast SWI/SNF complex in the remodeling of a nucleosome array has now been analyzed in vitro. Binding of the purified SWI/SNF complex to a nucleosome array disrupted multiple nucleosomes in an adenosine triphosphate-dependent reaction. However, removal of SWI/SNF left a deoxyribonuclease I-hypersensitive site specifically at a nucleosome that was bound by derivatives of the transcription factor Gal4p. Analysis of individual nucleosomes revealed that the SWI/SNF complex catalyzed eviction of histones from the Gal4-bound nucleosomes. Thus, the transient action of the SWI/SNF complex facilitated irreversible disruption of transcription factor-bound nucleosomes.

Stimulation of Transcription Factor Binding and Histone Displacement by Nucleosome Assembly Protein 1 and Nucleoplasmin Requires Disruption of the Histone Octamer

To investigate the mechanisms by which transcription factors invade nucleosomal DNA and replace histones at control elements, we have examined the response of the histone octamer to transcription factor binding in the presence of histone-binding proteins (i.e., nucleosome assembly factors). We found that yeast nucleosome assembly protein 1 (NAP-1) stimulated transcription factor binding and nucleosome displacement in a manner similar to that of nucleoplasmin. In addition, disruption of the histone octamer was required both for the stimulation of transcription factor binding to nucleosomal DNA and for transcription factor-induced nucleosome displacement mediated by nucleoplasmin or NAP-1. While NAP-1 and nucleoplasmin stimulated the binding of a fusion protein (GAL4-AH) to control nucleosome cores, this stimulation was lost upon covalent histone-histone cross-linking within the histone octamers. In addition, both NAP-1 and nucleoplasmin were able to mediate histone displacement upon the binding of five GAL4-AH dimers to control nucleosome cores; however, this activity was also forfeited when the histone octamers were cross-linked. These data indicate that octamer disruption is required for both stimulation of factor binding and factor-dependent histone displacement by nucleoplasmin and NAP-1. By contrast, transcription factor-induced histone transfer onto nonspecific competitor DNA did not require disruption of the histone octamer. Thus, histone displacement in this instance occurred by transfer of complete histone octamers, a mechanism distinct from that mediated by the histone-binding proteins nucleoplasmin and NAP-1.

[6] Basic analysis of transcription factor binding to nucleosomes

Publisher Summary This chapter discusses the basic preparations of reagents and their application to the analysis of transcription factor binding to nucleosomes. It provides protocols for the preparation of nucleosomes and histones, nucleosome reconstitution, and the analysis of transcription factor binding. The chapter also provides protocols that are applicable to any nucleosome-length DNA fragment that can be easily performed by any biochemist or molecular biologist with minimal protein experience. The only limitation to performing these experiments is the availability of sufficient quantities of the transcription factors of interest. The direct analysis of transcription factor binding to nucleosomes has revealed many general properties of factor-nucleosome interactions. Different factors are inhibited in binding to different degrees by the occupancy of their binding sites in nucleosomes. For example, the glucocorticoid receptor exhibits very efficient nucleosome binding abilities, whereas similar analyses indicate that the binding of NF1 and the human heat-shock factor are severely inhibited by nucleosomes. Other factors demonstrate intermediate levels of affinity for nucleosomal DNA.

[7] Experimental analysis of transcription factor-nucleosome interactions

Publisher Summary This chapter describes advanced techniques to analyze the interactions of an individual or group of factors with nucleosome cores. The methods presented in the chapter allow an investigation of the parameters governing the binding of a particular factor to nucleosomes, including nucleosome position effects and the role of the core histone amino termini. In addition, approaches to analyze the cooperativity of factor binding and the function of accessory protein complexes in factor binding are described in the chapter. It also provides protocols to directly assess the proteins present (factors and histones) in ternary complexes resulting from the binding of transcription factors to nucleosomes. These approaches can be applied to analyze the binding of an individual transcription factor. Alternatively, the methods can be used to address the function of multiple factors in binding and displacing nucleosomes reconstituted on complex enhancer or promoter elements. The histone-binding protein, nucleoplasmin, stimulates transcription factor binding to nucleosomes and can remove H2A/H2B dimers from factor/nucleosome ternary complexes.

Mechanisms and Consequences of Transcription Factor Binding to Nucleosomes

Publisher Summary Biochemical analysis of the proteins involved in transcription by RNA polymerase II has revealed two classes of transcription factors. These include the general initiation factors, which are required in addition to RNA polymerase II for accurate initiation, and regulatory factors, which bind upstream promoter and/or enhancer elements and activate transcription initiation. Biochemical studies of the interactions of regulatory transcription factors with nucleosome cores have illustrated several parameters that restrict the binding of individual transcription factors to nucleosomal DNA. These include a differential intrinsic affinity of different factors for their recognition sites on nucleosomes, the location of the binding sites within the nucleosome core, and inhibition from the core histone amino termini. However, nucleosome-mediated repression of factor binding can be overcome by the facilitated binding of multiple factors, relief of inhibition from the core histone amino termini (that is, by histone acetylation), and through the function of accessory proteins that stimulate factor binding. The binding of regulatory factors to nucleosomes results in the formation of factor/nucleosome ternary complexes that contain bound factors, core histones, and DNA.