Team:FAU Erlangen/Description
Project Description
Synthetic epigenetics is an emerging field in synthetic biology. The modification of histones allows to artificially alter the structure of chromatin and thus increase or decrease the expression of target genes. Fusing a DNA-binding TAL domain to a histone modifying enzyme, such as a demethylase, has been proven to influence gene expression. However, in order to use this in cancer therapy, for example, it will be necessary to regulate the expression of several oncogenes simultaneously. The usage of fusion proteins for this task would be impractical, as a new fusion protein would be required for every single gene to be regulated.
Our approach differs from the existing methods by utilizing the established SpyTag/SpyCatcher system to express the DNA binding domain and the histone modifying domain separately, which would then covalently bind to each other in the nucleus. This yields a single molecule that possesses both the capability to bind specific DNA sequences and to modify the gene loci at their binding sites, thus regulating gene expression. The distinct advantage of our approach consists in shorter sequences containing the information necessary to express the proteins required to facilitate the simultaneous regulation of several genes. In the future, we aim to compose a system of exchangeable elements enabling us to freely decide the sequence to bind and the epigenomic modification to induce in any eukaryotic cell.
During the course of our experiments, we transformed the reporter gene eYFP into the trp locus in the yeast genome and created TALE-SpyTag constructs that can bind the promoter region of the reporter gene. A fusion protein of a histone deacetylase and a SpyCatcher domain were used as the TALEs' counterpart. Upon expressing both halves of the complex, we observed a decreased YFP signal.