Dynamic Chromatin Invasion and Remodeling by the Transcription Factor Rap1

Pioneer transcription factors (PTF) are a subset of transcription factors with nucleosome-binding properties allowing them to bind specific sequences in condensed chromatin. Although the biological functions of PTFs including target genes and epigenetic changes have been well characterized in vivo, their mechanism of action remains unknown. Here, we investigate dynamic chromatin binding of Rap1 (an essential Yeast PTF) in highly defined in-vitro single-molecule experiments. Rap1 is an essential regulatory protein whose DNA-binding sites are found in three types of chromosomal elements: promoters, silencers and telomeres. Moreover, its DNA targets are well characterized on a genome wide scale. Further, Rap1 binding sites have been found to co-localize with fragile, i.e. unstable and partially accessible nucleosomes. Due to its role as a PTF, Rap1 might be able to directly interact with compact chromatin and open chromatin structure in the process. Alternatively, it might capture transiently open states, stabilizing fragile nucleosomes. To discriminate between these possibilities, we will investigate dynamic chromatin binding of Rap1 in highly defined in vitro single-molecule experiments. Our experiments include single-molecule FRET and single-molecule TIRF to probe the search and binding kinetics of Rap1 in different environments (i.e free DNA, mono-nucleosomes and chromatin) but also study the effect of Rap1 binding on chromatin. For this, we have designed a library of nucleosome positioning sequences containing Rap1 binding sites based on a well characterized promoter region found in vivo (Ribosomal Protein L30 promoter). Subsequently, these modular DNA sequences can be fluorescently labelled and inserted into large arrays of DNA allowing too later form defined chromatin fibres. We have so far shown that Rap1 is indeed capable of binding nucleosomal DNA and that its binding is specific and modulated by the local chromatin environment. Furthermore, the sequence composition and position of its recognition site within the nucleosome and chromatin structure alters binding kinetics. Importantly, we also find that Rap1 opens local chromatin structure upon binding, thereby potentially providing access to the gene expression machinery.