Regulation of gene expression in Arabidopsis thaliana by artificial zinc finger chimeras

Abstract

The artificial regulation of endogenous gene expression in plants is limited to only a few approaches. Here, we describe the use of artificial zinc finger chimeras to regulate the expression of a known reporter construct. The artificial zinc finger chimera TFIIIAZif is a fusion protein consisting of the four zinc fingers of TFIIIA linked through a spacer region to the three zinc fingers of Zif268. This artificial zinc finger chimera is able to bind specifically to a target DNA sequence (ZBS, zinc finger binding site) of 27 base pairs (bp). TFIIIAZif was fused to a transactivation domain from the herpes simplex virus VP16 or its tetramer VP64 to give ZF-VP16 or ZF-VP64, respectively. In transient expression assays, these two transcription activators were able to activate a target reporter gene (luc and GFP) expressed from a minimal -46 35S promoter linked to four copies of ZBS. The activation was confirmed in transgenic plants using an inducible XVE system [Zuo et al. (2000) Plant J. 24: 265] to express ZF-VP16 or ZF-VP64. Furthermore, to test the specificity of ZF-VP64 we have compared reporter gene expression from a wild type (1×ZBS) and a mutant (1×ZBSmu) binding site in transgenic plants. The 1×ZBS was used to express green fluorescent protein (GFP) whereas the 1×ZBSmu was used to express red fluorescent protein (RFP). Upon induction of ZF-VP64 we found a much higher expression of GFP (about 33-fold) as compared to RFP expression. These results suggest that artificial zinc finger chimeras can be used to target specific DNA sequences and to regulate gene expression in plants.