title: Heritable endogenous gene regulation in plants with designed polydactyl zinc finger transcription factors.
abstract: Zinc finger transcription factors (TFs(ZF)) were designed and applied to transgene and endogenous gene regulation in stably transformed plants. The target of the TFs(ZF) 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 TF(ZF) consisting of the ZFP fused to the tetrameric repeat of herpes simplex VP16's minimal activation domain. Histochemical staining of beta-glucuronidase (GUS) activity in transgenic AP3GUS reporter plants expressing GUS under control of the AP3 promoter was increased dramatically in petals when the AP3-specific TF(ZF) activator was cointroduced. TF(ZF)-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 AP3GUS-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 TF(ZF). 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.
authors.affiliation: Torrey Mesa Research Institute, San Diego, CA 92121, USA. xueni.guan@syngenta.com
authors.forename: Xuen
authors.initials: X
authors.lastname: Guan
authors.forename: Justin
authors.initials: J
authors.lastname: Stege
authors.forename: Myoung
authors.initials: M
authors.lastname: Kim
authors.forename: Zina
authors.initials: Z
authors.lastname: Dahmani
authors.forename: Nancy
authors.initials: N
authors.lastname: Fan
authors.forename: Peter
authors.initials: P
authors.lastname: Heifetz
authors.forename: Carlos F
authors.initials: CF
authors.lastname: Barbas
authors.forename: Steven P
authors.initials: SP
authors.lastname: Briggs
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D029681:Arabidopsis Proteins / Q000235:genetics*; D018506:Gene Expression Regulation, Plant; D005966:Glucuronidase / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics*; D006801:Humans; D026161:MADS Domain Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D010641:Phenotype; D018515:Plant Leaves / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics*; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016335:Zinc Fingers
chemical_list: C101342:APETALA 3 protein, Arabidopsis; D029681:Arabidopsis Proteins; D018398:Homeodomain Proteins; D026161:MADS Domain Proteins; D010940:Plant Proteins; D012333:RNA, Messenger; D005966:Glucuronidase