Core histones isolated from normal and butyrate‐treated HeLa cells have been reconstituted into nucleosome cores in order to analyze the role of histone acetylation in enhancing transcription factor binding to recognition sites in nucleosomal DNA. Moderate stimulation of nucleosome binding was observed for the basic helix‐loop‐helix factor USF and the Zn cluster DNA binding domain factor GAL4‐AH using heterogeneously acetylated histones. However, by coupling novel immunoblotting techniques to a gel retardation assay, we observed that nucleosome cores containing the most highly acetylated forms of histone H4 have the highest affinity for these two transcription factors. Western analysis of gel‐purified USF‐nucleosome and GAL4‐AH‐nucleosome complexes demonstrated the predominant presence of acetylated histone H4 relative to acetylated histone H3. Immunoprecipitation of USF‐nucleosome complexes with anti‐USF antibodies also demonstrated that these complexes were enriched preferentially in acetylated histone H4. These data show that USF and GAL4‐AH preferentially interact with nucleosome cores containing highly acetylated histone H4. Acetylation of histone H4 thus appears to play a primary role in the structural changes that mediate enhanced binding of transcription factors to their recognition sites within nucleosomes.

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.

In order to investigate the interrelated roles of nucleosome cores and histone H1 in transcription repression, we have employed a purified system to analyze the function of H1 in the repression of transcription factor binding to nucleosomes. H1 binding to nucleosome cores resulted in the repression of USF binding to nucleosomes. By contrast, H1 only slightly inhibited the binding of GAL4‐AH, indicating that H1 differentially represses the binding of factors with different DNA‐binding domains. H1‐mediated repression of factor binding was dependent on the core histone amino‐terminal tails. Removal of these domains alleviated H1‐mediated repression and increased acetylation of these domains partly alleviated repression by H1. H1 binding assays suggest a less stable interaction of histone H1 with the core particle in the absence of the amino termini.

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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