A Hot-Swappable Genetic Switch: Building an Inducible and Trackable Functional Assay for the Essential Gene MEDIATOR 21

Essential genes, estimated to be approximately 20% of
the Arabidopsis genome, are broadly expressed and
required for
reproductive success. They are difficult to study, as interfering
with their function leads to premature death. Transcription is one
of the essential functions of life, and the multiprotein Mediator
complex coordinates the regulation of gene expression at nearly every
eukaryotic promoter. In this study, we focused on a core mediator
component called MEDIATOR21 (MED21), which is required for activation
of transcription. Our previous work has also shown a role for MED21
in repression of gene expression through its interaction with a corepressor
protein. Here, we sought to differentiate the role MED21 plays in
activation versus repression using the model plant Arabidopsis. As mutations in MED21 lead to embryo-lethal phenotypes, we constructed
a set of synthetic switches using PhiC31 serine integrases to create
an “on-to-off” inducible loss-of-function MED21 in a
nonessential tissue. Our technology, which we call Integrase Erasers,
enabled med21 mutant plants to survive into adulthood
by ablating protein expression selectively in lateral root primordia,
allowing quantification and characterization of med21 mutant phenotypes in a postembryonic context. In addition, we engineered
chemical induction of the Integrase Eraser to ablate MED21 expression
in whole seedlings at a user-specified time point. Finally, we extended
this technology to build a hot-swappable Integrase Isoform Switch
where expression of the integrase toggled cells from expressing wild-type
MED21 to expressing MED21 sequence variants. Our analysis of the entire
set of new integrase-based tools demonstrates that this is a highly
efficient and robust approach to the study of essential genes.