Controlling gene expression in plants using synthetic zinc finger transcription factors.
Synthetic 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.
Stege
Justin T
JT
Department of Molecular Biology and The Skaggs Institute for Chemical Biology, Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Guan
Xuen
X
Ho
Thao
T
Beachy
Roger N
RN
Barbas
Carlos F
CF
D001665:Binding Sites / Q000235:genetics; D002478:Cells, Cultured; D018506:Gene Expression Regulation, Plant / Q000235:genetics* / Q000502:physiology; D017930:Genes, Reporter / Q000235:genetics; D017405:Herpes Simplex Virus Protein Vmw65 / Q000235:genetics; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D010944:Plants / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection; D003313:Zea mays / Q000235:genetics; D016335:Zinc Fingers / Q000235:genetics* / Q000502:physiology
D017405:Herpes Simplex Virus Protein Vmw65; D012367:RNA, Viral; D014157:Transcription Factors; D008156:Luciferases