No abstract

In this study, we examined the effect of acetylation of the NH 2 tails of core histones on their binding to nucleosomal DNA in the absence or presence of bound transcription factors. To do this, we used a novel UV laser-induced protein-DNA cross-linking technique, combined with immunochemical and molecular biology approaches. Nucleosomes containing one or five GAL4 binding sites were reconstituted with hypoacetylated or hyperacetylated core histones. Within these reconstituted particles, UV laser-induced histone-DNA crosslinking was found to occur only via the nonstructured histone tails and thus presented a unique tool for studying histone tail interactions with nucleosomal DNA. Importantly, these studies demonstrated that the NH 2 tails were not released from nucleosomal DNA upon histone acetylation, although some weakening of their interactions was observed at elevated ionic strengths. Moreover, the binding of up to five GAL4-AH dimers to nucleosomes occupying the central 90 bp occurred without displacement of the histone NH 2 tails from DNA. GAL4-AH binding perturbed the interaction of each histone tail with nucleosomal DNA to different degrees. However, in all cases, greater than 50% of the interactions between the histone tails and DNA was retained upon GAL4-AH binding, even if the tails were highly acetylated. These data illustrate an interaction of acetylated or nonacetylated histone tails with DNA that persists in the presence of simultaneously bound transcription factors.DNA in the cell nucleus exists in the form of chromatin. Chromatin structure is quite complex, and several different levels of chromatin packaging have to be perturbed in order for transcription factors to gain access to their binding sites within regulatory DNA sequences (12). In this study, we examined the binding of transcription factors to the first level of chromatin organization, the nucleosome. The nucleosome is the basic chromatin subunit; it consists of a DNA fragment about 180 to 200 bp long wrapped around an octamer of core histones, two each of H2B, H2A, H3, and H4. As demonstrated earlier, the histone octamer represents a tripartite assembly with an overall shape of a cylindrical wedge and a centrally located tetramer, (H3-H4) 2 , flanked by two H2A-H2B dimers (6). The surface of the histone octamer has 12 periodically located, binary structural motifs that permit the docking of DNA on the octamer; discovery of this structure yielded a model for the nucleosome in which the NH 2 termini emerge at alternating sides of the DNA (7). This model is in excellent agreement with the recently resolved crystal structure of the nucleosome core particle at 2.8 Å (45). The histone tails are located external to the core particle and are subject to acetylation, a posttranslational modification which is believed to be involved in transcriptional regulation (29,30,43). Until the late 1980s, only indirect circumstantial evidence existed to support the originally proposed hypothesis of Allfrey et al. (3) that acetylation remodels chromatin st...