Chromatin remodeling as a fundamental regulator of gene activity
We are interested in the biochemical principles that govern chromatin structure and gene expression. Chromatin is the native complex of DNA and histone proteins in nucleosome arrays, associated with nonhistone proteins and RNA. Chromatin has an important role in the regulation of gene expression in the life of a cell. Because nucleosomes impede access to the genome, they must undergo dynamic alterations catalyzed by ATP-dependent chromatin remodeling enzymes, which change nucleosome composition or nucleosome positioning near regulatory DNA sequences.
We are currently studying the multi-component, 1-megadalton, SWR1 chromatin remodeling complex conserved from yeast to humans. SWR1 catalyzes exchange of the histone variant H2A.Z for nucleosomal histone H2A. H2A.Z marks nucleosomes next to nucleosome-deficient promoter and enhancer elements, modulating recruitment and activity of the RNA polymerase machinery. To elucidate the molecular mechanism of histone H2A.Z replacement, we employ budding yeast as a primary model system, for ease of genetic and biochemical manipulation.
How is SWR1 recruited to engage with promoter nucleosomes? How do the 14 components of this macromolecular machine use ATP hydrolysis to carry out the histone H2A.Z exchange reaction? What type of reaction intermediates are created, and what are their lifetimes? What is the atomic structure of the SWR1 complex and how does it change in the process of histone exchange? How do H2A.Z-nucleosomes influence the initiation of transcription? To answer these questions, we take a multi-disciplinary approach, using the tools of biochemistry, cell and molecular biology, combined with structural biology and single-molecule biophysics, in collaboration with colleagues at Hopkins and beyond.
We are additionally interested in the unusual biochemistry of variant nucleosomes at chromosome centromeres. Unlike the need for nucleosome remodeling at gene control elements, nucleosome stability at centromeres is obligatory for kinetochore assembly and chromosome segregation during cell division. Yeast centromeres assemble nucleosomes harboring a histone H3 variant called Cse4/ CENP-A. Both Cse4 histone residues and centromere DNA sequences are important for recruiting Mif2/CENP-C, a key kinetochore protein, and thus both histones and DNA have a role in centromere specification. We are exploring the molecular basis of these interactions.