Role of nucleic acid binding in Sir3p-dependent interactions with chromatin fibers.

Recent studies of the mechanisms involved in the regulation of gene expression in eukaryotic organisms depict a highly complex process requiring a coordinated rearrangement of numerous molecules to mediate DNA accessibility. Silencing in Saccharomyces cerevisiae involves the Sir family of proteins. Sir3p, originally described as repressing key areas of the yeast genome through interactions with the tails of histones H3 and H4, appears to have additional roles in that process, including involvement with a DNA binding component. Our in vitro studies focused on the characterization of Sir3p-nucleic acid interactions and their biological functions in Sir3p-mediated silencing using binding assays, EM imaging, and theoretical modeling. Our results suggest that the initial Sir3p recruitment is partially DNA-driven, highly cooperative, and dependent on nucleosomal features other than histone tails. The initial step appears to be rapidly followed by the spreading of silencing using linker DNA as a track.

Characterization of chromatin samples in the presence of Drosophila embryo extract by quantitative agarose gel electrophoresis.

Recent studies have focused attention on chromatin as both a negative and positive regulator of specific nuclear events. The vast majority of this research has been centered on chromatin remodeling and histone post-translational modifications over the regulatory regions of specific genes. However, due the technical difficulties of such studies, the contribution of the higher-order structure of chromatin on the regulation of gene expression has not been as thoroughly investigated and the majority of the initial studies have used biophysical methods or microscopy. Until recent technical developments, the main hindrance for these biophysical investigations of chromatin has been an almost absolute requirement for large amounts of highly purified material. The development of an agarose gel electrophoresis method (quantitative agarose gel electrophoresis), initially designed for the analysis of the three-dimensional structure of purified and in vivo-assembled chromatin over a promoter region, has been expanded to include studies of chromatin in the presence of a Drosophila crude extract. The technique presented in the study reported here will help in paving the way for subsequent analyses of structural modifications of chromatin that are linked with the recruitment of various chromatin-associated factors present in the provided extract(s).

Chromatin remodeling complexes: ATP-dependent machines in action.

Since the initial characterization of chromatin remodeling as an ATP-dependent process, many studies have given us insight into how nucleosome-remodeling complexes can affect various nuclear functions. However, the multistep DNA-histone remodeling process has not been completely elucidated. Although new studies are published on a nearly weekly basis, the nature and roles of interactions of the individual SWI/SNF- and ISWI-based remodeling complexes and DNA, core histones, and other chromatin-associated proteins are not fully understood. In addition, the potential changes associated with ATP recruitment and its subsequent hydrolysis have not been fully characterized. This review explores possible mechanisms by which chromatin-remodeling complexes are recruited to specific loci, use ATP hydrolysis to achieve actual remodeling through disruption of DNA-histone interactions, and are released from their chromatin template. We propose possible roles for ATP hydrolysis in a chromatin-release/target-scanning process that offer an alternative to or complement the often overlooked function of delivering the energy required for sliding or dislodging specific subsets of core histones.