Studies on nuclear factor 1 binding to nucleosomal DNA

Sammanfattning: The organisation of DNA into chromatin will influence on transcriptional regulation.The first event in transcriptional activation is the binding of transcription factorsto specific binding sites in the regulatory regions of the gene. To understand transcriptionalregulation, we need to understand how transcription factor binding is influencedby nucleosomes. We have shown that the ability of nuclear factor I (NF-1) to interact with a nucleosomalNF-1 site is not affected by the translational or rotational position of the bindingsite. NF-I binds 100 to 300 times less strongly to nucleosomal binding sitesthanit does to binding to sites in free DNA. This result was shown by experiments whichinserted the NF-I binding site from the MMTV LTR into blocks of an artificial nucleosomepositioning sequence. Binding of recombinant NF-I to reconstituted nucleosomes wasfollowed using DNase I footprinting and DMS methylation protection. Introductionof homopolymeric stretches of (dA:dT) bp, called A-tracts, flanking the NF-1 bindingsite could increase or decrease the binding. We propose that the A-tracts will, dueto their rigidity and straightness, change the DNA curvature and thereby change thenucleosome 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- I binding did notdepend on the rotational position of the NF-1 binding site. NF-1 did not affect theSWI/SNF nucleosome remodeling. The nucleosome remodeling by SWI/SNF was increased,however, by the glucocorticoid receptor (GR) and a nucleosome containing a GR bindingsite. For the first time it has been shown that nucleosome remodeling affected bySWI/SNF can be enhanced by a transcription factor binding to DNA. This is consistentwith a model where transcription factors interact with chromatin and target the SWI/SNFnucleosome remodeling to discrete regions of the chromosome. We have also shown that a triple helix forming oligonucleotide (TFO) can preventnucleosome formation overlapping the triple helix region. The TFO functioned as anucleosome barrier and could be used as a tool to reposition nucleosomes and to investigatethe role of nucleosome position in transcriptional regulation. Keywords: nuclear factor 1, nucleosome, glucocorticoid receptor, triplex DNA,the SWI/SNF complex, A-tracts, MMTV LTR ISBN: 91-628-2633-6