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