Nucleosome positioning : impact on transcription factors access

Sammanfattning: In the eukaryotic cell nucleus, DNA is organized into nucleosomes. Some of the regulatory regions are integrated into sequence-specifically positioned nucleosomes. What impact nucleosome positioning has on the accessibility of the regulatory sequences, and how transcription factors gain access to their binding sites within nucleosomes, are fundamental to the understanding of the eukaryotic gene expression. These issues were addressed in a purified system by binding two different transcription factors to various nucleosomes displaying their cognatesites in a defined rotational and translational position. Glucocorticoid receptor (GR) is able to recognize a glucocorticoid response element (GRE) that is located at the nucleosome dyad and faces away from the histone octamer. The binding affinity is only 2-fold lower than that of a free GRE. The contacts of GR with this nucleosomal GRE are identical to that with the free GRE as revealed by dimethylsulfate methylation analysis. Conversely, a GRE at a similar translational position but facing towards the histone octamer is inaccessible for GR recognition. However, the influence of rotational positioning can be compromised by translational positioning of the GRE in a nucleosome. GRis capable of binding to an inward-facing GRE near the nucleosome boundary albeit with a 6-fold decreased affinity relative to an outward-facing GRE. The interaction of the GR homodimer with the GRE is via the two consecutive major grooves that face toward the histone octamer, and the formation of the ternary complex is accompanied by a local alteration of histone-DNA contacts. The helical path of the DNA on the histone octamer is anisotropic and this is reflected in the decreased GR binding to an outward-facing GRE located 20-bp from the nucleosome dyad. However, the reduced GR affinity can be restored by introducing a 5-bp A/T segment immediately after the GRE at the major groove compression. The introduction of an A/T segment at a major groove compression is detrimental to the nucleosomal formation, which alleviates the constraints imposed by the histone-DNA interaction at this translational position. The affinity of GR to two GREs organized in one nucleosome depends on the spatial proximity of the two GREs within the nucleosome rather than the linear distance along the DNA axis. The facilitated binding of GR is observed for the two GREs juxtapositioned by one turn of nucleosomal DNA (80-bp). This is in sharp contrast to the cooperative GR binding previously reported for free GRE. In contrast to GR, NF1 could not bind to its recognition sequences within a nucleosome regardless of the rotational and translational position. Taken together, these data demonstrate that both rotational and translational positioning of the regulatory sequences within nucleosomes modulate transcription factor access. However, the effect of translational positioning can be overridden by the sequences which flank the regulatory element. Furthermore, the ability of a transcription factor to recognize its cognate element in a nucleosome is an inherent property that differs among different transcription factors.