Function of Activation Domains in Gene-Specific Transcription Factors

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Despite major advances in characterizing components of eukaryotic transcription machinery, the mechanisms of the recruitment of these components to gene promoters are poorly understood. Activation domains (ADs) of gene-specific transcription factors are critical for these recruitment steps. Amazingly, there are little requirements for the sequences and structure of ADs, and they are easily interchangeable with preservation of functionality not only between different gene specific activators but even between activators belonging to different eukaryotic phyla. The excess of hydrophobic and acidic amino acid residues in natural and synthetic ADs suggests that their interacting targets may be hydrophobic and basic. The nucleosomal histones are the most abundant proteins in the nucleus with such properties. The PI hypothesizes that histones are among the targets of some ADs and that distortion of promoter nucleosomes by ADs triggers a chain of chromatin remodeling events involving different coactivators. In this project, yeast HSF will be used as a model system. The chromatin remodeling mediated by HSF ADs at heat shock promoters will be tested in yeast strains bearing different AD deletions employing Chromatin ImmunoPrecipitation (ChIP) technique. The involvement of known histone-modifying and nucleosome-remodeling activities will be tested in the strains where these activities will be inactivated. The above hypothesis of AD-nucleosome interactions will be addressed in vivo by an attempt to create a synthetic activator with AD substituted by known histone-binding protein and in vitro by DNase I footprinting experiments using a reconstituted nucleosomal template and recombinant HSF. This project potentially can have an impact on the dynamically developing area of gene transcription regulation by characterizing the general chromatin remodeling mechanisms taking place in any eukaryotic cell. Understanding of general mechanisms of natural and especially synthetic ADs function potentially will have an impact on pharmaceutical directions related to the design of synthetic activators and repressors targeting disease-related genes.