Structural Basis and Functional Consequences of Alternative ATF2-Jun Heterodimer Orientations at the Interferon-Beta Enhancer

The selective activation of genes is essential for diverse biological processes such as growth, development, and responses to environmental cues. Unlike lower organisms that often use individual proteins to control gene activation, transcription regulation in higher organisms generally requires cooperation among multiple proteins. Cooperation can be achieved via interactions between DNA-binding proteins that bind to adjoining DNA sequences. Such interactions can stabilize DNA binding by these proteins. Many eukaryotic transcription factors form heterodimers that can bind to DNA in two opposite orientations. Because of the asymmetry of such heterodimers, cooperative DNA binding has been predicted, and in some cases observed, to require a specific orientation of heterodimer binding. Interferon regulatory factor 3 (IRF3) and a heterodimer containing activating transcription factor 2 and c-Jun (ATF2-Jun) bind cooperatively to the human interferon-beta enhancer, and opposite orientations of ATF2-Jun binding have been observed using different experimental approaches. High mobility group protein I (HMGI) binds to sequences overlapping the ATF2-Jun-IRF3 site within the interferon-beta enhancer and facilitates DNA-binding and synergistic transcriptional activation by components of the enhancer complex, yet its effects on ATF2-Jun-IRF3 complex formation have not been investigated. This thesis presents the identification of the structural determinants of ATF2-Jun heterodimer orientation at the interferon-beta enhancer in vitro as well as functional characterization in cells. Using gel-based fluorescence resonance energy transfer analysis, I found that ATF2-Jun binds to the interferon-beta enhancer in both orientations alone and in association with IRF3 and HMGI. Two symmetry-related sets of amino acid residues in ATF2 and Jun facilitated the opposite orientations of heterodimer interactions with IRF3 at the interferon-beta enhancer. Expression of ATF2 and Jun variants that bound the interferon-beta enhancer in opposite orientations together with IRF3 produced distinct levels of interferon-beta transcription in Sendai-virus infected Hela cells. Expression of these proteins resulted in different relative levels of transcription of different genes regulated by ATF2 and Jun. Collectively, this work illustrates a novel mode of cooperative DNA-binding by transcription factors and suggests that alternative nucleoprotein arrangements can influence transcriptional activity through distinct mechanisms at different genes.Ph.D.Biological ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/89855/1/vburns_1.pd