SpyCatcher-Associated, Tale-Transmitted Epigenetic Regulation (SCATTER)

We named our project SCATTER in honor of Scatte, the ancient Germanic goddess of life.

Just kidding: we only needed a pronounceable acronym and SCATTER fit the bill. (If you happen to find any deeper meaning in the abbreviation, please let us know.)

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 will transform reporter genes into specific loci in the yeast genome and create TALE-SpyTag constructs that can bind the promoter regions of the reporter genes. A fusion protein of a histone deacetylase and a SpyCatcher domain will be used as the TALEs' counterpart. Upon expressing both halves of the complex, we expect to observe a downregulation of the reporter gene.