Studies on nuclear factor 1 binding to nucleosomal DNA

The organisation of DNA into chromatin will influence on transcriptional regulation. The first event in transcriptional activation is the binding of transcription factors to specific binding sites in the regulatory regions of the gene. To understand transcriptional regulation, we need to understand how transcription factor binding is influenced by nucleosomes.

We have shown that the ability of nuclear factor I (NF-1) to interact with a nucleosomal NF-1 site is not affected by the translational or rotational position of the binding site. NF-I binds 100 to 300 times less strongly to nucleosomal binding sites than it does to binding to sites in free DNA. This result was shown by experiments which inserted the NF-I binding site from the MMTV LTR into blocks of an artificial nucleosome positioning sequence. Binding of recombinant NF-I to reconstituted nucleosomes was followed using DNase I foot printing and DMS methylation protection. Introduction of homopolymeric stretches of (dA:dT) bp, called A-tracts, flanking the NF-1 binding site could increase or decrease the binding. We propose that the A-tracts will, due to their rigidity and straightness, change the DNA curvature and thereby change the nucleosome stability, which will have impact on NF-I binding.

NF-1 binding increases if the nucleosome is remodeled by the SWI/SNF complex, which we partially purified from rat liver. This increase in NF-1 binding did not depend on the rotational position of the NF-1 binding site. NF-1 did not affect the SWI/SNF nucleosome remodeling. The nucleosome remodeling by SWI/SNF was increased, however, by the glucocorticoid receptor (GR) and a nucleosome containing a GR binding site. For the first time it has been shown that nucleosome remodeling affected by SWI/SNF can be enhanced by a transcription factor binding to DNA. This is consistent with a model where transcription factors interact with chromatin and target the SWI/SNF nucleosome remodeling to discrete regions of the chromosome. We have also shown that a triple helix forming oligonucleotide (TFO) can prevent nucleosome formation overlapping the triple helix region. The TFO functioned as a nucleosome barrier and could be used as a tool to reposition nucleosomes and to investigate the role of nucleosome position in transcriptional regulation.