title: Tailor-made zinc-finger transcription factors activate FLO11 gene expression with phenotypic consequences in the yeast Saccharomyces cerevisiae.
abstract: 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.
authors: Department of Biomedical Sciences, Chung Shan Medical University, Taichung, Taiwan. jcs@csmu.edu.tw
authors: Jia-Ching
authors: JC
authors: Shieh
authors: Yu-Che
authors: YC
authors: Cheng
authors: Mao-Chang
authors: MC
authors: Su
authors: Michael
authors: M
authors: Moore
authors: Yen
authors: Y
authors: Choo
authors: Aaron
authors: A
authors: Klug
mesh_terms: D001483:Base Sequence; D002448:Cell Adhesion; D015966:Gene Expression Regulation, Fungal; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D019151:Peptide Library; D010641:Phenotype; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016335:Zinc Fingers
chemical_list: C110690:FLO11 protein, S cerevisiae; D008562:Membrane Glycoproteins; D019151:Peptide Library; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors
references: Mol Biol Cell. 1998 Jan;9(1):161-71
references: 9436998
references: Methods Enzymol. 1999;310:91-109
references: 10547784
references: J Mol Biol. 1999 Oct 22;293(2):215-8
references: 10529348
references: Proc Natl Acad Sci U S A. 2000 Feb 15;97(4):1495-500
references: 10660690
references: Curr Opin Struct Biol. 2000 Aug;10(4):411-6
references: 10981627
references: Proc Natl Acad Sci U S A. 2000 Oct 24;97(22):12158-63
references: 11027318
references: Proc Natl Acad Sci U S A. 2000 Dec 5;97(25):13619-24
references: 11095711
references: Science. 2001 Feb 2;291(5505):878-81
references: 11157168
references: Science. 2001 Feb 16;291(5507):1304-51
references: 11181995
references: Proc Natl Acad Sci U S A. 2001 Feb 13;98(4):1432-6
references: 11171968
references: Proc Natl Acad Sci U S A. 2001 Feb 13;98(4):1437-41
references: 11171969
references: Nature. 2001 Feb 15;409(6822):832-3
references: 11237001
references: Nature. 2001 Feb 15;409(6822):860-921
references: 11237011
references: Nat Biotechnol. 2001 Jul;19(7):656-60
references: 11433278
references: Nucleic Acids Res. 2001 Jul 1;29(13):2860-74
references: 11433033
references: J Biol Chem. 2001 Aug 3;276(31):29466-78
references: 11340073
references: Methods Enzymol. 2001;340:593-609
references: 11494872
references: Genes Dev. 2002 Jan 1;16(1):27-32
references: 11782442
references: Annu Rev Biochem. 2001;70:313-40
references: 11395410
references: Nat Biotechnol. 2002 Feb;20(2):135-41
references: 11821858
references: Microbiology. 2002 Apr;148(Pt 4):893-907
references: 11932437
references: Eukaryot Cell. 2002 Oct;1(5):673-86
references: 12455687
references: Proc Natl Acad Sci U S A. 2003 Feb 18;100(4):1621-6
references: 12574501
references: Proc Natl Acad Sci U S A. 2003 Feb 18;100(4):1615-20
references: 12574502
references: Nat Biotechnol. 2003 Mar;21(3):275-80
references: 12592413
references: Science. 2003 May 2;300(5620):763
references: 12730593
references: Science. 2003 May 2;300(5620):764
references: 12730594
references: Nat Rev Drug Discov. 2003 May;2(5):361-8
references: 12750739
references: Stem Cells. 2003;21(6):632-7
references: 14595122
references: J Virol. 2004 Feb;78(3):1301-13
references: 14722285
references: Cell. 2004 Feb 6;116(3):405-15
references: 15016375
references: Expert Opin Investig Drugs. 2004 Jul;13(7):829-39
references: 15212621
references: Circulation. 2004 Oct 19;110(16):2467-75
references: 15477407
references: Proc Natl Acad Sci U S A. 1976 Mar;73(3):804-8
references: 1062791
references: Cell. 1984 Dec;39(3 Pt 2):499-509
references: 6096007
references: FEBS Lett. 1985 Jul 8;186(2):271-4
references: 4007166
references: Nucleic Acids Res. 1987 Jul 10;15(13):5017-30
references: 3299263
references: Nature. 1988 Oct 6;335(6190):563-4
references: 3047590
references: Science. 1989 Aug 11;245(4918):635-7
references: 2503871
references: Curr Genet. 1989 Dec;16(5-6):339-46
references: 2692852
references: Science. 1991 May 10;252(5007):809-17
references: 2028256
references: EMBO J. 1992 Dec;11(13):4961-8
references: 1464321
references: Proc Natl Acad Sci U S A. 1994 Nov 8;91(23):11163-7
references: 7972027
references: Proc Natl Acad Sci U S A. 1994 Nov 8;91(23):11168-72
references: 7972028
references: Genes Dev. 1995 Sep 1;9(17):2117-30
references: 7657164
references: Proc Natl Acad Sci U S A. 1996 Aug 6;93(16):8419-24
references: 8710886
references: J Bacteriol. 1996 Dec;178(24):7144-51
references: 8955395
references: Science. 1997 Jan 31;275(5300):657-61
references: 9005850
references: Science. 1997 Feb 28;275(5304):1314-7
references: 9036858
references: Proc Natl Acad Sci U S A. 1997 May 27;94(11):5525-30
references: 9159105
references: Yeast. 1997 Jul;13(9):837-48
references: 9234672
references: EMBO J. 1985 Jun;4(6):1609-14
references: 4040853
references: Mol Cell Biol. 1985 Dec;5(12):3610-6
references: 3915782
references: Proc Natl Acad Sci U S A. 1998 Mar 17;95(6):2812-7
references: 9501172
references: Microbiol Mol Biol Rev. 1998 Jun;62(2):334-61
references: 9618445
references: Proc Natl Acad Sci U S A. 1998 Dec 8;95(25):14628-33
references: 9843940
references: Mol Microbiol. 1999 Jan;31(1):103-16
references: 9987114
references: EMBO J. 1999 Mar 1;18(5):1257-69
references: 10064592
references: Cell. 2004 Dec 29;119(7):981-90
references: 15620356
references: Proc Natl Acad Sci U S A. 2005 Aug 16;102(33):11716-21
references: 16081541
references: Nature. 2006 Jun 1;441(7093):656-9
references: 16738662
references: Proc Natl Acad Sci U S A. 2006 Jul 25;103(30):11228-33
references: 16844788
references: Proc Natl Acad Sci U S A. 2006 Sep 26;103(39):14423-8
references: 16983072
references: Biophys Chem. 2007 Jan;125(1):59-71
references: 16863676
references: Nucleic Acids Res. 2007;35(1):100-12
references: 17151075