Heritable endogenous gene regulation in plants with designed polydactyl zinc finger transcription factors - PubMed (original) (raw)

Heritable endogenous gene regulation in plants with designed polydactyl zinc finger transcription factors

Xuen Guan et al. Proc Natl Acad Sci U S A. 2002.

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.

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Figures

Figure 1

Figure 1

(A) _ZFP_AP3 target sequence (boxed) and its position in the AP3 5′ UTR. Numbers indicate the distance from the ATG translation initiation codon. The arrowed boxes indicate the exon of AP3. (B) DNA recognition helix sequences of the _ZFP_AP3 protein. The underlined amino acids are the components of the new zinc fingers that provide specificity for the selected nucleotide sequences indicated in A. The recognition helices of fingers 1–6 (F1–F6) are underlined.

Figure 2

Figure 2

Activation of the silenced endogenous AP3 gene in Arabidopsis leaf cells. RT-PCR was used to detect AP3 expression in Arabidopsis leaf protoplasts transformed with GFP (control), ZFP m4 -VP64 (nonspecific activation control), and ZFP_AP3_-VP64 (_AP3_-specific activation). The last lane (−) is no template control amplification.

Figure 3

Figure 3

GUS staining flowers of background plant with AP3∷GUS only and double transgenic plant with both AP3∷GUS and activation construct AP1∷ZFPAP3-VP64//nos. (A). Predicted GUS staining patterns of AP3∷GUS and AP1∷GUS (based on personal communication with Martin Yanofsky, University of California, San Diego). (B) Flowers from background plant with AP3∷GUS only stained for GUS activity. GUS signal is detected only in the petal (p) and stamen (not visible here, see Fig. 6_A_), but not in the carpal (not visible) and sepal (se). Picture taken directly after staining procedure. (C) Flowers from double transgenic plant expressing AP3∷GUS and AP1∷ZFPAP3-VP64//nos simultaneously. GUS signal is increased in petal and is detected throughout the young flower primordia. (D) Mounted flowers from background plant with AP3∷GUS only stained for GUS activity. GUS signal is detected only in the petal (p) and stamen (not visible here, see Fig. 7_A_) and not in the carpal and sepal (se). (E) Mounted flowers from double transgenic plant expressing AP3∷GUS and AP1∷ZFPAP3-VP64//nos simultaneously. GUS signal is increased in petal (p), extended to sepal (se), and detected throughout the young flower primordia (arrow).

Figure 4

Figure 4

Floral phenotypic changes in double transgenic plant expressing AP3∷GUS and AP1∷ZFPAP3-VP64//nos simultaneously. A seven-petal flower is shown here. Two extra petals are fully converted (f), and the third one is partially converted (p).

Figure 5

Figure 5

Repression of endogenous AP3 expression by the constitutive repression construct UBQ3∷Sid-ZFPAP3//nos in transgenic plant ND0052–2e. (A) RT-PCR identification of transgene _ZFP_AP3 in transgenic event ND0052–2e and wild-type control plant. (B) RT-PCR evaluation of endogenous gene AP3 expression level in transgenic event ND0052–2e and wild-type plant. In plant ND0052–2e, the expression of AP3 is significantly repressed by the expression of repressor _Sid-ZFP_AP3 fusion protein. Quantitative PCR indicated 46-fold repression.

Figure 6

Figure 6

GUS staining flowers of background plant with AP3∷GUS only and double transgenic plant with both AP3∷GUS and repression construct AP1∷Sid-ZFPAP3//nos. (A) Flowers from background plant with AP3∷GUS only stained for GUS activity. GUS signal is detected only in the petal (p) and stamen (st) and not in the carpal and sepal (se). Picture taken directly after staining procedure. (B) Flowers from double transgenic plant expressing AP3∷GUS and AP1∷Sid-ZFPAP3//nos simultaneously. GUS signal disappeared from petals but is detectable in stamens. (C) Flowers from a different double transgenic plant expressing AP3∷GUS and AP1∷Sid-ZFPAP3//nos simultaneously. Low level GUS activity is detectable in stamens. In addition, the petals were absent in this flower.

Figure 7

Figure 7

Floral phenotypic changes in double transgenic plant expressing AP3∷GUS and AP1∷Sid-ZFPAP3//nos simultaneously. (A) Flowers from background plant with AP3∷GUS only. (B) Flowers from double transgenic plant expressing AP3∷GUS and AP1∷Sid-ZFPAP3//nos simultaneously. A three-petal flower is shown here. In this flower, an extra sepal is fully replaced by one petal (f), and another petal is partially replaced (p). (C) Flowers from a different double transgenic plant expressing AP3∷GUS and AP1∷Sid-ZFPAP3//nos simultaneously with one missing petal.

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