New mode of DNA binding of multi-zinc finger transcription factors: deltaEF1 family members bind with two hands to two target sites.
SIP1, a Smad-interacting protein, and deltaEF1, a transcriptional repressor involved in skeletal and T-cell development, belong to the same family of DNA binding proteins. SIP1 and deltaEF1 contain two separated clusters of zinc fingers, one N-terminal and one C-terminal. These clusters show high sequence homology and are highly conserved between SIP1 and deltaEF1. Each zinc finger cluster binds independently to a 5'-CACCT sequence. However, high-affinity binding sites for full-length SIP1 and deltaEF1 in the promoter regions of candidate target genes like Xenopus Xbra2, and human alpha4-integrin and E-cadherin, are bipartite elements composed of one CACCT and one CACCTG sequence, the orientation and spacing of which can vary. Using transgenic Xenopus embryos, we demonstrate that the integrity of these two sequences is necessary for correct spatial expression of a Xbra2 promoter-driven reporter gene. Both zinc finger clusters must be intact for the high-affinity binding of SIP1 to DNA and for its optimal repressor activity. Our results show that SIP1 binds as monomer and contacts one target sequence with the first zinc finger cluster, and the other with the second cluster. Our work redefines the optimal binding site and, consequently, candidate target genes for vertebrate members of the deltaEF1 family.
Remacle
J E
JE
Department of Cell Growth, Flanders Interuniversity Institute for Biotechnology (VIB), Laboratory of Molecular Biology (CELGEN), University of Leuven, Herestraat 49, B-3000 Leuven, Belgium.
Kraft
H
H
Lerchner
W
W
Wuytens
G
G
Collart
C
C
Verschueren
K
K
Smith
J C
JC
Huylebroeck
D
D
D000818:Animals; D030801:Animals, Genetically Modified; D015703:Antigens, CD / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D015820:Cadherins / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D015342:DNA Probes / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020832:EF Hand Motifs / Q000235:genetics; D005260:Female; D015870:Gene Expression; D017930:Genes, Reporter; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D039441:Integrin alpha4; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020825:T-Box Domain Proteins / Q000235:genetics; D014157:Transcription Factors; D029867:Xenopus Proteins; D014982:Xenopus laevis; D000074165:Zinc Finger E-box Binding Homeobox 2; D000071799:Zinc Finger E-box-Binding Homeobox 1; D016335:Zinc Fingers / Q000235:genetics
D015703:Antigens, CD; D015820:Cadherins; D015342:DNA Probes; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; D012097:Repressor Proteins; D020825:T-Box Domain Proteins; C116765:TBXT protein, Xenopus; D014157:Transcription Factors; D029867:Xenopus Proteins; C486307:ZEB1 protein, human; C492364:ZEB2 protein, Xenopus; C492363:ZEB2 protein, human; D000074165:Zinc Finger E-box Binding Homeobox 2; D000071799:Zinc Finger E-box-Binding Homeobox 1; D039441:Integrin alpha4; D004247:DNA