Comprehensive Description of Specific Chromosomal Regions
We are working towards developing a set of tools that will allow us to analyze chromatin in unprecedented detail (for preliminary data see Tackett et al., 2005). Specifically, we would like to be able to take any portion of a given chromosome under specified cellular conditions and define where the nucleosomes are positioned, quantitate the site-specific modifications on the histones making up these nucleosomes, and define the position and makeup of every protein and protein complex that is resident on this particular piece of chromatin. These are transparently lofty goals that will stretch our analytical capabilities to their limits. In particular, our sensitivity needs will be very high because we expect that frequently there may be £ 1 protein molecule of interest per portion of chromatin. We can offset this difficulty, to some extent in yeast, by using large numbers of cells (~1012 cells). This strategy will in turn place high demands on our ability to differentiate specifically-binding from contaminant proteins. Thus, we plan to attack this site-specific analysis of chromatin complexes problem in a series of steps with increasing levels of difficulty. First, we will insert sections of chromosomes on plasmids, isolate these sections of chromatin, and analyze their macromolecular content. The advantage of using plasmids is that they are straightforward to manipulate genetically and that they can be expressed at fairly high copy number – giving us the ability to amplify the number of proteins of interest by at least a factor of 20. The second step will be to isolate any chosen chromatin region. Ultimately, we hope to elucidate the sets of proteins that associate with any given region of a chromosome, and how they are arranged upon the DNA sequence. Isolation of defined intact sections of chromatin should ultimately allow us to “walk across” a chromosome and determine the identity and organization of specifically bound proteins.