AP-1 proteins constitute one of the largest families of transcription factors in eukaryotic cells. These factors regulate a wide range of cellular processes including proliferation, apoptosis, development, and stress responses. In this project, we will elucidate regulatory aspects of DNA-protein interactions with yeast members of the evolutionary conserved AP-1 protein family. The yeast AP-1 family (yAP) consists of eight proteins (yAP1 to yAP8), which regulate gene expression in response to various stress conditions. The yAP1 protein controls expression of genes encoding antioxidant and protective functions, whereas yAP8 controls expression of metal detoxification genes. These proteins are regulated in distinct manners. In the absence of stress, yAP1 is maintained in the cytoplasm, whilst during stress, the protein quickly accumulates in the nucleus where it binds DNA. In contrast, yAP8 is constitutively bound to DNA both in the absence and presence of stress. yAP1 and yAP8 require distinct binding sites on DNA to induce gene expression. Both proteins bind to DNA as dimers and possess a characteristic bZIP (basic leucine-zipper) domain: the basic domain binds to DNA and the leucine-zipper domain is involved in dimer formation. Despite extensive research, little is known about the DNA binding properties of AP-1 proteins and the mechanisms by which they activate transcription.
In this project, we will use molecular modelling, combined with cellular and biochemical studies, to advance our understanding of the DNA binding properties of yAP1 and yAP8 and to decipher how they achieve gene-regulatory specificity. Using molecular modelling as an in silico microscope, we will explore the mechanics and energetics of protein-DNA interactions. Using newly in-house developed structural constraints, as a complement to all-atom molecular dynamics software, we are able to control structural arrangements of the protein-DNA complexes as well as DNA bending and superhelical state. These structural constraints provide a unique possibility to monitor protein-DNA interactions in conditions closely resembling those in vivo where DNA is highly topologically constrained by nucleosome packaging.
Our preliminary experimental data suggests that yAP8 triggers chromatin remodelling to efficiently induce gene expression. Our data also indicates that while histones occupancy decreases during gene activation, histone acetylation status remains unmodified. Our preliminary molecular modelling data indicates that yAP8 protein has two equipotential conformations with respect to its DNA binding domain. In the same time, our calculations showed that the DNA binding site has a quite shallow free energy profile with respect to supercoiling transitions, suggesting higher plasticity of this very sequence. Our hypothesis is that yAP8 can induce a significant conformational change in DNA that triggers a cascade of structural modifications leading to release of the target gene-encoding DNA and subsequent efficient gene expression.
We firmly believe that the experiments proposed here will elucidate the intricate biological mechanisms of gene-activation and transcriptional regulation mediated by AP-1 proteins in eukaryotic cells.