Interaction of Smad complexes with tripartite DNA-binding sites

The Smad family of transcription factors function as effecters of transform ing growth factor-beta signaling pathways. Smads form heteromultimers capab le 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'. Her e we show that Smad3 and Smad4 complexes can contact three abutting GTCT se quences and that arrays of such sites elevate reporter expression relative to arrays of binding sites containing only two GTCTs. Smad3/4 complexes bou nd synergistically to probes containing two of the four possible arrangemen ts of three GTCT sequences and showed a correlated ability to synergistical ly activate transcription through these sites. Purified Smad3 and Smad4 wer e both able to contact three abutting GTCT sequences and reporter experimen ts indicated that either protein could mediate contact with all three GTCTs . In contrast, the Smad4 MH1 domain was essential for reporter activation i n combination with Smad1. Together, these results show that Smad complexes are flexible in their ability to interact with abutting GTCT triplets. In c ontrast, Smads have high affinity for only one orientation of abutting GTCT pairs. Functional Smad-binding sites within several native response elemen ts contain degenerate GTCT triplets, suggesting that trimeric Smad-DNA inte raction may be relevant in vivo.