sequence (data not shown). Although these are negative results, they indicate that Smad proteins might not function directly by binding sequences in this region. Therefore, we considered other potential binding sites, and identiﬁed a strong match with the consensus binding site (50-ATGCAAAT) for the POU homeodomain transcription factor Oct-1 (50–52). The related sequence 50-ATGTAAAG is truncated precisely at the junction of the 68 bp deletion. Interestingly, Oct-1 binding is maximal if the ﬂanking sequences are A/T rich (51), which is the case in the gata2 sequence. Mutation of this putative Oct-1 binding site in the gata2 promoter nearly eliminated induction of the promoter by BMP-4, equivalent to truncation of the promoter at 751 (Figure 5B, mutation #7). It is important to point out that the mutation does not alter the basal activity of the promoter, but only the activation by BMP. Thus, the Oct-1 site mediates BMP responsiveness in this system. In gel mobility-shift assays, nuclear extracts derived from stage 13 Xenopus embryos generate a single predominant complex with a probe that is centered on the putative Oct-1 binding site (arrow in Figure 6A). This complex was not detected using probes from the original 68 bp BRE, since the octamer site is truncated on those sequences. This binding activity is competed in the presence of excess unlabeled probe corresponding to the wild-type sequence, but not with an Figure 6. The octamer site of BRE1 binds Oct-1 in Xenopus nuclear lysates. (A) Gel mobility shift assays were performed using a labeled probe containing the BRE1 sequences including the putative octamer binding site. Lane 0 represents probe alone and the position of the free probe is indicated (P). In lanes marked with a ‘+’, a specific complex forms using nuclear extracts derived from stage 13 embryos, as indicated by the arrow in the second lane. The complex is competed specifically by the addition in the reaction of excess (10·, 25· or 100·) unlabeled probe DNA (self) or DNA containing the octamer consensus (octamer), but not by DNA containing the same mutation of the octamer that blocks the induction by BMP4 (mutant). (B) Similar gel mobility-shift assays were performed. In this case, the lanes include probe alone (0), nuclear extract (1), nuclear extract and antibody to Xenopus Oct-1 (2) or a control isotype- matched antibody (3). The free probe is indicated (P) as are the positions of a non-specific complex (NS), the specific complex (SP) and the complex that is super-shifted by addition of the Oct-1 antibody (SS). Nucleic Acids Research, 2005, Vol. 33, No. 13 4363 unlabeled oligomer probe corresponding to the mutated sequence that fails to support a BMP response. Furthermore, this complex is fully competed by an oligomer probe contain- ing an Oct-1 consensus binding site (52). Finally, the complex that forms with the wild-type probe derived from the gata2 BRE is super-shifted speciﬁcally by addition of a monoclonal antibody raised against the C-terminus of the Xenopus Oct-1 protein (Figure 6B). Incubation with control isotype-matched antibodies does not alter migration of the complex. Thus, Oct- 1 binds to the gata2 BRE (designated BRE1), and a mutation that abolishes Oct-1 binding abrogates the ability of BMP-4 to induce the gata2 promoter. We tested if BMP signaling modiﬁes the interaction of Oct-1 with the BRE1 site. For this purpose, fertilized eggs were injected with BMP-4 RNA or control RNA. Nuclear extracts were subsequently prepared from the embryos at stage 13 and compared for binding activities. Although extracts from BMP-4 injected embryos appeared to have a modest relative decrease in Oct-1 binding activity, this difference is essentially the same difference found for a distinct complex formed with a control oligomer containing a CCAAT binding site (data not shown). Therefore, the amount and activity of normalized Oct-1 complex formation is not obviously altered by BMP-4 signaling. The Oct-1 site is not sufficient for mediating the BMP response, but cooperates with a downstream element Since BMP signaling does not appear to directly alter the Oct-1 interaction with the BRE1, we next considered whether Oct-1 cooperates with one or more additional complexes, which themselves might be direct targets of BMP signaling. Note also that the deletion of the BRE1 or mutation of the Oct-1 site does not eliminate entirely the BMP response, typi- cally reducing the effect from 5- to 2-fold, indicating that additional proximal sequences might contribute to activity. We ﬁrst tested whether the BRE1 was sufﬁcient to function autonomously upstream of a heterologous promoter. Because the Oct-1 binding site spanned the 30 junction of the 68 bp region, a larger region of the gata2 promoter including the Oct-1 site and ﬂanking sequences (134 bp, from 819 to 685) was tested for the ability to mediate BMP responsive- ness when placed as a single copy or in triplicate upstream of the SV40 promoter. These sequences were unable to mediate a BMP response with the SV40 promoter, indicating that the Oct-1 dependent BRE1 is necessary but not sufﬁcient for mediating induction (Figure 7A). When a larger region including sequences downstream of the BRE was tested (377 bp, from 819 to 443), BMP-4 induction was conferred to the SV40 promoter. This result is consistent with the presence of one or more cooper- ating cis-elements located within the more proximal sequences. Therefore, progressive 30 deletions were tested to deﬁne the 30 boundary of this activity. As shown in Figure 7A, sequences between positions 666 and 646 (BRE2) are required for conferring a BMP-4 response onto the SV40 promoter. To conﬁrm that this sequence cooperates with the original BRE1 deﬁned by the 50 deletion series (the Oct-1 site), the upstream sequences were removed from the context of the BRE2. Deletion of 68 bp from the larger 377 bp