4362 Nucleic Acids Research, 2005, Vol. 33, No. 13 Figure 4. BRE1 mediates activation by BMP4 or Smads. The inducible reporter ( 819) or the reporter lacking BRE1 ( 751), as diagrammed in Figure 3, was co-injected into Xenopus embryos with RNA encoding Smad1, BMP4, Smad5 or Smad8, as indicated. Smad1 activates the reporter equivalent to BMP4, and in both cases this is dependent on the presence of the BRE1 sequences. Smad5 is only slightly less active, while Smad8 fails to induce reporter activity. Figure 5. Mutation of an octamer element located at the 30 end of the BRE1 is sufficient to block BMP-induced activation of the promoter. (A) A series of specific mutations were introduced into the BRE1 by site-directed mutagenesis for potential Smad-binding sites (mutations #1–5) or for an A/T-rich sequence (mutation #6), or a putative octamer binding site (mutation #7). (B) When compared to the BMP-inducible reporter containing the intact BRE1 ( 819), none of the first six mutations had a significant effect on induction. In contrast, mutation of the putative octamer binding site (mutation #7) blocked BMP-mediated induction, similar to deletion of the BRE1 ( 751). sequence is 69% conserved (data not shown), but with no obvious clustering, and with no sequences that match perfectly a known SBE. However, we tested several potential candidate sequences by site-speciﬁc mutagenesis (Figure 5A). The sequence 50-TGGAGC is important for mediating BMP-4 activation of the xVent2 promoter by directing the assembly of a Smad1/Smad4/OAZ transcriptional complex (16). The zebraﬁsh gata2 BRE contains a related sequence: 50-TGGACC (Figure 5A, #2 and 3). Also, Drosophila Mad binds to the GC-rich sequence 50-GCCGnCGC (17), and a related sequence 50-GGGCTCGC is present in the zebraﬁsh gata2 promoter (Figure 5A, #5). Both these elements and several other sequences with some similarity to previously identiﬁed SBEs (e.g. GGCT, Figure 5A, #1), or A/T-rich sequences (Figure 5A, #6) were altered using site-directed mutagenesis. However, none of these mutations (#1–6) had any effect on the ability of BMP-4 to induce the promoter (Figure 5B). Gel mobility shift assays using the 68 bp BRE as a probe failed to reveal any speciﬁc protein complexes using whole cell or nuclear extracts from Xenopus embryos injected with xSmad1 or mSmad4 (data not shown). Neither GST–Smad1 nor GST–Smad4 fusion proteins were able to bind the 68 bp