325. Identification of Mechanistically Distinct Repression Domains at Endogenous Genes Using Engineered Zinc Finger Proteins

Abstract

Engineered zinc finger protein transcription factors (ZFP TFs) are capable of controlling the expression of targeted endogenous genes, and thus provide potential therapeutics for the treatment of disease. The functionality of ZFP TFs results from the modularity of the DNA binding and effector domains allowing such ZFP-fusion proteins to address a given domain to a specific chromosomal gene in vivo. This technology thus allows novel effector domains to be assayed for function directly at an endogenous gene. Since this system requires no insertion of binding sites or other foreign DNA into the region of interest, the efficacy of a given ZFP-domain fusion can be determined in the context of truly native chromatin. This is of particular significance to transcriptional regulation since promoter specific modification of histones plays a dynamic role in the process of gene control. We show here that a ZFP targeted to an endogenous reporter gene can be used to successfully identify new effector domains capable of repressing gene expression in vivo. Furthermore we show that the requirements for gene repression at an endogenous gene are not always recapitulated by more standard plasmid-based reporter assays. Finally we use this system to identify different effector domains that employ alternative repression pathways, and show that a domain from the TGF-beta inducible early gene is capable of driving gene repression in an HDAC and DNA methylation independent manner. The ability to screen for, and employ alternative effector domains with defined sensitivity toward known drugs opens the potential for the combinatorial use of such compounds with ZFP TF therapeutic interventions to both accentuate, or limit their function.