Using Chromatin Conformation Capture (3C) to Investigate Length-Dependent Saccharomyces Cerevisiae Gene Looping with and without HMGB Proteins

Abstract

HMGB proteins are able to enhance the apparent flexibility of DNA, presumably through the creation of flexible kinks in the DNA. These sequence non-specific proteins may facilitate chromatin looping and dynamics by acting on the rigid segments of inter-nucleosomal linker DNA and might affect chromatin stability by directly interacting with nucleosomes. We are interested in understanding how loss of HMGB proteins in yeast affects the ability of chromatin to form loops over the length scale of full yeast genes (several thousand base pairs). We hypothesize that gene looping over this scale will be inhibited in a length-dependent manner in Saccharomyces cerevisiae strains disrupted for NHP6A/B, NHP10, or HMO1 genes encoding sequence non-specific HMGB proteins. We have engineered these gene disruptions in yeast and have adapted the conventional Chromatin Conformation Capture (3C) assay for quantitative PCR analysis to compare gene looping in wild-type versus mutant strains. Using this system with suitable gene loops, the yeast BLM10 gene that was recently shown to have a clear 3C looping signal in vivo as well as the yeast GAL10 and HEM3 genes, we report progress to date on testing the effects of HMGB deletion on gene looping. These data address the issue of how the intrinsic physical properties of DNA are managed in a biological context.