Facilitated, "cooperative" binding of GAILA-AH to nucleosomal DNA occurred in response to inhibition from the core histone amino termini. The binding of GAL4-AH (which contains the DNA-binding and dimerization domains of GALA) to (5,15,34,43,64) and is affected in vivo by the stability of nucleosomes located over core promoter sequences (50) and mutations in the N termini of histone H4 (17).Before upstream activators can act on core promoters, they must first gain access to their respective upstream binding elements (reviewed in reference 1). Studies thus far implicate at least three criteria which govern the ability of factors to access their binding sites on nucleosomes. The first is an inherent difference in the ability of factors to bind nucleosomal DNA, perhaps dictated by their particular DNA-binding motifs. Those found to bind at least in some instances include TFIIIA, the glucocorticoid receptor, and GAL4 derivatives, while those unable to bind in similar circumstances include nuclear factor 1 and the human heat shock factor (2,31,42,44,45,52,62). Second, nucleosome positioning has been implicated in determining factor access.

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.

Facilitated, "cooperative" binding of GAL4-AH to nucleosomal DNA occurred in response to inhibition from the core histone amino termini. The binding of GAL4-AH (which contains the DNA-binding and dimerization domains of GAL4) to nucleosome cores containing multiple binding sites initiated at the end of a nucleosome core and proceeded in a cooperative manner until all sites were occupied. However, following tryptic removal of the core histone amino termini, GAL4-AH binding appeared to be noncooperative, similar to binding naked DNA. Binding of GAL4-AH to nucleosomes bearing a single GAL4 site at different positions indicated that inhibition of GAL4 binding was largely mediated by the histone amino termini and primarily occurred at sites well within the core and not near the end. When the histone amino termini were intact, binding of GAL4-AH to sites near the center of a nucleosome core was greatly enhanced by the presence of additional GAL4 dimers bound to more-accessible positions. These data illustrate that the binding of a factor to more-accessible sites, near the end of a nucleosome, allows facilitated binding of additional factors to the center of the nucleosome, thereby overcoming repression from the core histone amino termini. This mechanism may contribute to the binding of multiple factors to complex promoter and enhancer elements in cellular chromatin.

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