case, the activity of the reporter was measured in luciferase assays following injection into the two presumptive dorsal blastomeres of a four-cell embryo, and normalized to Renilla luciferase activity from co-injected Renilla luciferase RNA. A B Figure 2. The gata2 promoter directs reporter gene activity that is induced by BMP4 and dependent on Smad signaling. (A) The luciferase reporter was injected at the four-cell stage into blastomeres that contribute preferentially to ventral or dorsal regions of the embryo, as indicated. The reporter is more active in the ventral-posterior derivatives, but this is abrogated by co-injection of RNA encoding the inhibitory Smad6 (the error bar for this sample is present, but it is so small that it is not evident). The results indicate that the reporter responds as predicted to the endogenous Smad-dependent BMP signaling path- way. (B) The gata2 promoter is induced 5-fold by co-injection of RNA encoding BMP4. This activation is also blocked by co-expression of Smad6. Nucleic Acids Research, 2005, Vol. 33, No. 13 4361 As shown in Figure 3, deletion of sequences from 6520 to 819 has no effect on the levels of induction caused by BMP-4. However, removing a 68 bp region located between 819 and 751 (relative to the start of transcription) results in a signiﬁcant loss of induction, indicating that this region mediates a response to BMP-4 and might contain one or more BREs. BMP-dependent Smads can activate the zgata2 promoter via the 68 bp BRE We showed that the BMP response on the gata2 promoter is sensitive to Smad6 (the inhibitory Smad), so it is expected that BMP-dependent R-Smads should be at least part of the mecha- nism that mediates the induction between 819 and 751, although this could be direct or indirect. We therefore tested whether injection of RNA encoding BMP regulated Smads 1, 5 or 8 could function through the gata2 BRE to induce reporter activity. The reporter either contained the BRE ( 819) or lacked the BRE ( 751), as illustrated in the diagram of Figure 3. As shown in Figure 4, injection of mRNA encoding Xenopus Smad1 (xSmad1) or zebraﬁsh Smad5 (zSmad5) was sufﬁcient to activate the promoter containing the 68 bp BRE ( 819). Injection of Xenopus Smad8 (xSmad8) failed to acti- vate the same reporter, indicating that not all BMP regulated Smads can mediate this effect and that there are functional distinctions between Smads 1, 5 and 8 [shown also in other studies (34,49)]. Importantly, Smad1 failed to activate a reporter construct deleted of the 68 bp BRE ( 751), indicating that this region is required for both BMP-4 signaling and Smad1 activity to induce the gata2 promoter. The experiment does not distinguish whether Smads function directly by bind- ing this promoter, but only that their activity is mediated by the 68 bp sequence. An Oct-1 binding site is a necessary element within the 68 bp zGATA2 BRE We ﬁrst considered whether sequences similar to previously deﬁned SBEs exist within the 68 bp gata2 BRE identiﬁed by the deletion analysis. Alignment of the region to the syntenic region of the Fugu rubripes gata2 gene showed that the Figure 3. Deletion analysis maps a BRE to a 68 bp region upstream of the gata2 promoter. A progressive series of 50 deletions was used to measure the relative activity of the promoter when injected with RNA encoding BMP4, compared to the same reporter when injected with non-coding control RNA, as described in Materials and Methods. For each construct, the site of truncation is indicated, the +1 indicates the transcriptional start site, and the box represents the firefly luciferase reporter. The fold difference (induced/uninduced) is plotted and shows a significant drop when sequences are deleted between 819 and 751. This 68 bp sequence is designated BRE1.