Zinc finger transcription factors (TFsZF) were designed and applied to transgene and endogenous gene regulation in stably transformed plants. The target of the TFsZF is the Arabidopsis gene APETALA3 (AP3), which encodes a transcription factor that determines floral organ identity. A zinc finger protein (ZFP) was designed to specifically bind to a region upstream of AP3. AP3 transcription was induced by transformation of leaf protoplasts with a transformation vector that expressed a TFZF consisting of the ZFP fused to the tetrameric repeat of herpes simplex VP16's minimal activation domain. Histochemical staining of ␤-glucuronidase (GUS) activity in transgenic AP3::GUS reporter plants expressing GUS under control of the AP3 promoter was increased dramatically in petals when the AP3-specific TFZF activator was cointroduced. TFZF-amplified GUS expression signals were also evident in sepal tissues of these double-transgenic plants. Floral phenotype changes indicative of endogenous AP3 factor coactivation were also observed. The same AP3-specific ZFP AP3 was also fused to a human transcriptional repression domain, the mSIN3 interaction domain, and introduced into either AP3::GUS-expressing plants or wild-type Arabidopsis plants. Dramatic repression of endogenous AP3 expression in floral tissue resulted when a constitutive promoter was used to drive the expression of this TFZF. These plants were also sterile. When a floral tissue-specific promoter from APETALA1 (AP1) gene was used, floral phenotype changes were also observed, but in contrast the plants were fertile. Our results demonstrate that artificial transcriptional factors based on synthetic zinc finger proteins are capable of stable and specific regulation of endogenous genes through multiple generations in multicellular organisms.floral development ͉ APETALA3 ͉ APETALA1 I n nature, eukaryotic nuclear genes are tightly regulated at both the transcriptional and translational levels. Much of this control is achieved through DNA-binding transcription factors. The manipulation of plant traits in agricultural biotechnology would be greatly facilitated if preselected endogenous genes could be turned on or off in a controlled and selective manner. A conceptual approach to such manipulation is the engineered expression of specific native transcription factors that have evolved to control particular genes. Advances in whole-genome sequencing of Arabidopsis (1) and more recently rice (2), combined with informatics-based analysis have allowed the identification of numerous putative plant transcription factors (2, 3). However, the identification and characterization of the molecular targets of these transcription factors is still at a very early stage, and consequently it is not yet possible to use them broadly as gene-specific tools for controlled regulation of endogenous gene expression. Rational design of artificial transcription factors that target specific DNA sequences with non-native nucleotide binding domains fused to transcriptional activation or repression domain...

SummarySynthetic zinc finger proteins can be fused to transcriptional regulatory domains to create artificial transcription factors that modulate the expression of a specific target gene. Recent studies have demonstrated that synthetic zinc finger domains can be constructed to bind DNA sequences with a high degree of specificity. To devise a general strategy for controlling plant gene expression with artificial transcription factors, a rapid transient assay was developed to test the regulatory activity of synthetic zinc finger transcription factors (effectors) on target plasmids (reporters) in plant cells. Effective activation was demonstrated with zinc finger proteins fused to a derivative of the VP16 activation domain. The mSin3 interaction domain (SID) of the human MAD1 protein provided moderate repression of target reporters. Unlike many naturally occurring transcription factors, these synthetic effectors exhibit a strong dependence on binding site position. Reporter genes that are stably integrated into plant cells responded similarly to transiently transfected reporter plasmids, verifying that this assay accurately reflects the behavior of these transcription factors on an endogenous target within the context of chromosomal DNA. These results provide evidence that synthetic zinc finger proteins can be used to manipulate the expression of endogenous genes in plants.