Tailor-made zinc-finger transcription factors activate FLO11 gene expression with phenotypic consequences in the yeast Saccharomyces cerevisiae.
Tailor-made zinc-finger transcription factors activate FLO11 gene expression with phenotypic consequences in the yeast Saccharomyces cerevisiae.
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DOI：
10.1371/journal.pone.0000746
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发表时间：
2007-08-15
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影响因子：
3.7
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中科院分区：
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Cys2His2锌指结构是真核生物DNA结合基序，能够区分不同的DNA序列，适合于工程化人工转录因子。在这项工作中，我们使用芽殖酵母酿酒酵母研究定制的锌指蛋白激活FLO11基因表达的能力，表型的后果。鉴定了两个三指肽，其识别来自FLO11基因的5′ UTR的位点，具有纳摩尔DNA结合亲和力。三指结构域及其组合的六指基序，识别一个18 bp的位点，融合到VP16或VP64的激活结构域。这些转录因子构建体保留了它们的DNA结合能力，其中六指转录因子的亲和力最高。然而，当在单倍体酵母细胞中表达时，只有一个三指重组转录因子能够有效地激活FLO11的表达。与野生型不同，具有这种转录激活的细胞显示侵入性生长和生物膜形成，而不需要任何葡萄糖消耗。VP16和VP64结构域似乎在FLO11表达的激活中同样有效，在表型改变中具有相当的效果。我们的结论是，定制的转录因子在细胞中的功能活性是不容易预测的体外DNA结合活性。
Cys2His2 zinc fingers are eukaryotic DNA-binding motifs, capable of distinguishing different DNA sequences, and are suitable for engineering artificial transcription factors. In this work, we used the budding yeast Saccharomyces cerevisiae to study the ability of tailor-made zinc finger proteins to activate the expression of the FLO11 gene, with phenotypic consequences. Two three-finger peptides were identified, recognizing sites from the 5′ UTR of the FLO11 gene with nanomolar DNA-binding affinity. The three-finger domains and their combined six-finger motif, recognizing an 18-bp site, were fused to the activation domain of VP16 or VP64. These transcription factor constructs retained their DNA-binding ability, with the six-finger ones being the highest in affinity. However, when expressed in haploid yeast cells, only one three-finger recombinant transcription factor was able to activate the expression of FLO11 efficiently. Unlike in the wild-type, cells with such transcriptional activation displayed invasive growth and biofilm formation, without any requirement for glucose depletion. The VP16 and VP64 domains appeared to act equally well in the activation of FLO11 expression, with comparable effects in phenotypic alteration. We conclude that the functional activity of tailor-made transcription factors in cells is not easily predicted by the in vitro DNA-binding activity.