Drug-Responsive Regulation and Comparison of Engineered Transcription Factors Containing Different Activation Domains

Abstract

The pursuit of effective tools for probing biological systems has led to a growing interest in approaches that offer both rapid, reversible, and accurate methods. Specifically, the ability to perturb the function of specific proteins with precision is highly sought after, as it contributes significantly to our capacity to unravel the intricacies of complex biological processes. Effective methods to regulate gene expression or protein function within mammalian hosts are essential for understanding basic biological mechanisms (Banaszynski 2008). Examples of these methods include generating animal knockouts, siRNA, the Cre-loxP system, and small molecule activators or inhibitors. However, these methods are limited because they may not be reversible, are non-specific, or—in the case of animal knockouts—the gene being knockout out is essential for development thus confounding the study of the protein’s function. Another method involves synthetic transcription factors paired with destabilizing-domain fusion proteins that target a gene of interest. The destabilizing-domain are stabilized in the presence of a ligand, thus providing the ability to control and regulate gene expression. This technique can provide the regulatability, specificity, and ease of use in understanding complex biological mechanisms. One component of synthetic transcription factors is the design of activation domains utilized. Activation domains promote recruitment of chromatin modifiers, which cause chromatin decondensation and accumulation of histone marks, thus promoting transcription (Martella 2021). The variability of transcription efficacy is based on the properties of different activation domains. In this study, three different domains are investigated: VPR, VP64, and p65, with the goal of determining which one provides the highest dynamic range in a lentiviral-transduced mammalian cell system using a synthetic transcription factor fused to a drug-dependent destabilizing protein domain.