Determining chromatin structure genome wide in yeast (564.2)

Abstract

The linear DNA in a human cell is compacted over a million fold, so much that, when compacted, 60,000 feet of DNA can fit on the tip of a needle. The first stage of compaction, nucleosome formation, is already known to impact gene regulation. The further compaction of nucleosomes into a chromatin fiber has also been shown to limit accessibility of DNA to DNA binding proteins. Despite the nucleosome crystal structure and decades of biochemical assays, the structure of this fiber is still a matter of debate. There are two competing structures proposed for the chromatin fiber: a two start and a one start model. A major difference between these models is the path of the linker DNA. Computational studies of chromatin structure suggest that details such as the length of linker DNA between nucleosomes can cause changes in the type of structure adopted by the fiber. Experiments using different linker lengths and different solution conditions support this claim and provide evidence in support of each model of the chromatin fiber. No study, however, has unambiguously accounted for the in vivo variability of linker lengths found in natural chromatin. We are developing a protocol that will allow us to assay the structure of chromatin in vivo using yeast DNA to account for the true variability of in vivo nucleosome spacing. We have shown our assay is sensitive to the structure of chromatin and are assaying how that structure differs at different genomic locations. We anticipate that the overall structure of the fiber at each genomic location is important for regulation. Our future aim is to explicitly test this prediction by first using our models to correlate changes in the fiber structure to differences in transcriptional regulation.