The compaction of DNA in the eukaryotic chromosome presents an obstacle that must be unraveled to allow the processes of gene transcription, replication, recombination and repair. Our group studies the structure of chromatin and its role in gene expression using the evolutionarily conserved, heat shock-inducible gene hsp70 as a model system. We have previously discovered that heat shock promoter sequences are organized in an altered chromatin structure that is hypersensitive to cleavage by nucleases, and have shown that these regions of altered chromatin contain sequence-specific DNA binding proteins. More recently, the heat shock transcription factor HSF was purified and the Drosophila and human HSF genes were cloned. Our current studies are focused on the elucidation of the heat stress signaling pathway and the activation of HSF, on energy-dependent processes of chromatin remodeling for transcription, and on changes in chromatin structure during the cell division cycle.
HSF is activated by an monomer to trimer transition which appears to be dependent on a choice between intramolecular and intermolecular leucine zipper interactions. This activation step is independent of the absolute environmental temperature, and requires the participation of undefined cellular factors (Wu, 1995). A search for these factors and other upstream regulators of HSF is underway. Domains important for HSF localization and transactivation have been identified, and mutants for the HSF gene have been isolated in Drosophila. These mutants have a larval-lethal phenotype, indicating a requirement for HSF function during normal development.
We are undertaking a major effort to characterize a recently discovered ATP-dependent nucleosome remodeling factor that facilitates the binding of transcription factors to nucleosomes (Tsukiyama et al., 1994). This factor (NURF) has been purified to homogeneity from Drosophila embryo extracts by seven chromatographic steps, and is composed of at least four discrete subunits. The amino acid sequences of these polypeptides are being determined. Our goal is to clone the genes encoding the NURF subunits, determine the mechanism by which NURF perturbs nucleosome structure, and demonstrate its physiological role in chromosomal functions.
In cytological and molecular studies of chromatin structure in the cell cycle, the displacement of a number of transcription factors from the mitotic chromatin of HeLa cells was observed (Martinez et al., 1995). The mechanism underlying transcription factor displacement in the mitotic phase of the cell cycle is being studied. The correlation between transcription factor displacement and chromosome condensation in mitotic prophase suggests that the compaction of chromatin during mitosis may play a key role in this process.
Kim, S.J. et al. Protein Science 3:1040-1051 (1994).
Martinez, M. et al. Cell 83:29-38 (1995).
Tsukiyama, T. et al. Nature 367: 525-532 (1994).
Wu, C. Ann. Review Cell & Devel. Biol. 11: 441-469 (1995).