The ATP-dependent chromatin remodeler SWI/SNF regulates transcription and DNA recombination by making DNA more accessible. The objective of this study is to find the mechanism used by SWI/SNF to move and disassemble nucleosomes. Nucleosome remodeling will be examined at two levels: changes in histone- DNA interactions and the corresponding changes in the interactions of SWI/SNF with histones and DNA. Ultimately the goal is to correlate these two different sets of interactions with each other in a temporal manner to provide an unprecedented view of the process of nucleosome remodeling. We propose to apply the relatively new technology of an expanded genetic code to site-specifically incorporate a photoreactive amino acid analog for examining protein-protein and protein-DNA interactions both in vivo and in vitro. Domains in the catalytic subunit of SWI/SNF other than the ATPase domain will be systematically studied with the basic premise that these domains make critical contributions to mobilizing nucleosomes in coordination and cooperation with the ATPase domain. Several domains have already been identified that are necessary for SWI/SNF remodeling. Single molecule magnetic tweezer and DNA unwinding optical trap type experiments will be done to determine if these domains are important for DNA translocation and nucleosome remodeling. The effect of histone H3 acetylation on SWI/SNF remodeling will be examined to determine at which stage in remodeling it modulates the activity of SWI/SNF and RSC. SWI/SNF has many important regulatory roles such as in stem cell self renewal, cellular differentiation, chromatin maintenance and stability, and oncogenesis. While the yeast system has provided us with important insights as to how this enzyme functions and the corresponding models, there still remain many questions as to how SWI/SNF regulates chromatin structure. In this proposal we continue to take advantage of the yeast system to examine these questions at a biophysical, biochemical, and molecular genetic level.
. SWI/SNF is involved in regulation of transcription and DNA repair. Often mutations in SWI/SNF are associated with the onset of cancer and several of the proteins in SWI/SNF are from known tumor suppressor genes. We are investigating the mechanism of SWI/SNF that is used to regulate the epigenome and how it regulates chromatin structure and function.
|Bartholomew, Blaine (2014) Regulating the chromatin landscape: structural and mechanistic perspectives. Annu Rev Biochem 83:671-96|
|Hota, Swetansu K; Bartholomew, Blaine (2011) Diversity of operation in ATP-dependent chromatin remodelers. Biochim Biophys Acta 1809:476-87|
|Gangaraju, Vamsi K; Bartholomew, Blaine (2007) Mechanisms of ATP dependent chromatin remodeling. Mutat Res 618:3-17|