324. The Degron Motif: A Protein Destruction Module with Potent Transcriptional Activation Properties for ZFP-Targeted Gene Regulation

Abstract

Engineered zinc finger protein transcription factors (ZFP TFs) can be designed to regulate any intended gene target, with single gene specificity within the genome. This mode of gene up-regulation outperforms that of the cDNA since (i) very low levels of expression of the ZFP TF activator are required for a dramatic effect on the gene target; and (ii) the ZFP TF also upregulates all of the splice variants, a fact which may, in many instances, be critical to therapeutic efficacy. To fully exploit the remarkably low steady-state levels of engineered ZFP TFs necessary for in vivo function we describe here a ZFP TF construct that has been modified to induce proteasome mediated self-destruction following transcriptional activation of the target gene. To do this, we have made use of the motif, a protein degradation module present in the most potent of natural transcription factors, including VP16. We show that incorporation of the degron motif into an engineered ZFP TF resulted in the proteasome-dependent degradation of ZFP TF, leading to an almost undetectable level of ZFP TF protein at steady state. Importantly, the low protein levels observed for these degron-containing ZFP TFs did not impair their ability to activate gene expression. Indeed, for the genes tested, equivalent or even increased activation of the target gene was obtained by the degron-constructs. Furthermore, we show that a degron containing ZFP TF may be combined with a regulatable switch such as the progesterone receptor ligand-binding domain, resulting in mifpristone-dependent up-regulation of the target gene. The degron motif thus represents a potent, self-limiting activation domain that permits maximal efficacy of the ZFP TF while simultaneously restricting ZFP TF protein levels. The specificity and potency of ZFP TFs can thus be controlled even within the context of gene therapy.