Research and Teaching Interest
DNA in eukaryotic organisms exists in a hierarchy of distinctive levels of nucleoprotein organization that are collectively called chromatin. The most basic unit of chromatin structure is the core nucleosome, a complex of core histones and 147 bp of DNA. In cells, nucleosomes reside in arrays, core nucleosomes spaced by variable lengths of less rigidly structured, linker DNA.
Functionally important processes like replication and transcription must occur in ways which involve this periodic compact structure of DNA. My research focuses on the relation between one of these necessary functional processes, transcription, and chromatin structure. Important questions are: where do nucleosomes locate themselves on particular gene sequences; what factors are responsible for the specific initiation of transcription in eukaryotic chromatin; what happens to nucleosomes during transcription.
Our research involves the in vitro studies of model systems. One such system is a dodecameric, head to tail repeat of the 5S rDNA gene of sea urchin, present in a multinucleosomal template. Each unit specifically positions a nucleosome and thus this dodecameric template allows us to reconstitute defined multinucleosomal arrays suitable for biophysical analysis. The other system is derived from a mammalian promoter, the MMTV (Mouse Mammary Tumor Virus) promoter. This promoter has been a major model for chromatin structure changes in association with transcription a activation in vivo and we are adapting the system for in vitro studies of this process.
Using these models, we are studying basic structural features of chromatin arrays: how histones load onto DNA, how they are released from DNA; the role of histone acetylation in these processes; how nucleosome remodelling factors work to alter the basic chromatin structure to permit processes like transcription, how transcription factors find their binding sites on chromatinized promoters..
Among techniques used are gel electrophoresis (including transfer techniques and probe hybridization); isolation and analysis of recombinant DNA; general nucleic acid methodology, protein characterization. The major structural approaches used are Atomic Force Microscopy and single molecule fluorescence techniques. |
