We will be assaying the ability of transcription factors to bind nucleosomal DNA and nucleosomes in the presence and absence of HMG-14 and -17. The transcription factor we will be using will be various deletion mutants of a bZIP protein, C/EBP. bZIP proteins form dimers via an alpha-helical, coiled-coil region (i.e. a leucine zippper), yielding a Y-shaped complex. The extended alpha-helical arms of the Y constitute the DNA contact surfaces, which can """"""""scissors-grip"""""""" symmetrical recognition sites within the major groove. This DNA-binding protein bends free DNA , but no information is available on their ability to approach and bind to nucleosomes core particles. In these experiments, a strong binding sequence appropriate for C/EBP is inserted into nucleosomal length DNA. We are designing a DNA sequence which will result in a positioned nucleosome and will test to see whether our design matches the theory. Within this positioned nucleosome, we will be inserting C/EBP recognition sequences at different positions on the nucleosomal dyad axis and near the region of the nucleosomal DNA where HMG-14 and -17 bind. We will measure the binding constants for these interactions both in the presence and absence of HMG-14 and -17. The results from this series of experiments will allow for the assignment of a function to a highly conserved and well-characterized set of proteins. An understanding of the ability of the HMG proteins to make chromatin """"""""transcriptionally competent"""""""" and their preferential localization in active chromatin regions will significantly enhance our ability to understand the importance of chromatin structure in the regulation of genes.
