Interaction of Smad complexes with tripartite DNA-binding sites.
The Smad family of transcription factors function as effectors of transforming growth factor-beta signaling pathways. Smads form heteromultimers capable of contacting DNA through the amino-terminal MH1 domain. The MH1 domains of Smad3 and Smad4 have been shown to bind to the sequence 5'-GTCT-3'. Here we show that Smad3 and Smad4 complexes can contact three abutting GTCT sequences and that arrays of such sites elevate reporter expression relative to arrays of binding sites containing only two GTCTs. Smad3/4 complexes bound synergistically to probes containing two of the four possible arrangements of three GTCT sequences and showed a correlated ability to synergistically activate transcription through these sites. Purified Smad3 and Smad4 were both able to contact three abutting GTCT sequences and reporter experiments indicated that either protein could mediate contact with all three GTCTs. In contrast, the Smad4 MH1 domain was essential for reporter activation in combination with Smad1. Together, these results show that Smad complexes are flexible in their ability to interact with abutting GTCT triplets. In contrast, Smads have high affinity for only one orientation of abutting GTCT pairs. Functional Smad-binding sites within several native response elements contain degenerate GTCT triplets, suggesting that trimeric Smad-DNA interaction may be relevant in vivo.
Johnson
K
K
Laboratory of Genetics, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Kirkpatrick
H
H
Comer
A
A
Hoffmann
F M
FM
Laughon
A
A
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004330:Drosophila; D011485:Protein Binding; D011487:Protein Conformation; D051900:Smad3 Protein; D015534:Trans-Activators / Q000378:metabolism*
D004268:DNA-Binding Proteins; D051900:Smad3 Protein; D015534:Trans-Activators; D004247:DNA