key sequences of BRE2 by site-directed mutagenesis have not yielded consistent results, perhaps indicating the presence of overlapping or redundant elements that will require alternative approaches to unravel. There are two adjacent octamer sequences present in the Xvent-2 promoter, neither of which matches exactly the single site of the gata2 promoter. POU transcription factors bind DNA via their unique POU domain, consisting of two sub-domains: a POU-type homeodomain (POUH) and a POU- speciﬁc domain (POUS), tethered to each other by a linker of variable length and sequence (61). In the case of Oct-1, both these domains are indispensable for DNA binding to the octamer consensus sequence 50-ATGCAAAT-30 (62). The Oct-1 binding site in the gata2 BRE1, 50-ATGTAAAG, retains the four (underlined) essential bases, and overall is identical at six of the eight nucleotides. Furthermore, super- shift experiments using an antibody speciﬁc to Oct-1 suggest that Oct-1 is the protein in Xenopus nuclear extracts that binds to the gata2 BRE1. Mutations that abolish this binding also block BMP-4 mediated induction of the gata2 promoter. How- ever, at this point it seems feasible that Oct-25 could also bind to the gata2 BRE1 (and also that Oct-1 might be able to interact at the Xvent-2 promoter). Until speciﬁcity is further documented, it seems reasonable to consider that POU-domain octamer binding proteins represent functional components that mediate BMP signaling. Based on the studies of the vestigial and Xvent-2 promoters, and our data showing that Smad1 can substitute for BMP4, while Smad6 blocks the BRE response, it seems likely that Oct-1 functions with Smad1/4 and perhaps Vent homeodomain proteins for activation of the gata2 promoter. Our attempts to demonstrate that Smad1/4 or Vent2 bind directly to sequences in the proximal BRE2 element have so far yielded negative results, but this may be due to relatively low binding afﬁnities or the requirement of additional co-factors. Although the cooperating elements in the vestigial locus were located within 25 bp of each other (60), it is conceivable that proteins bound to the gata2 elements could interact over larger distances, presumably facilitated by DNA bending, since the Oct-1 POU domain can bend DNA in vitro (63). The 20 bp region deﬁned as the BRE2 has sequences that might be capable of binding Smad (50-AGAC) and/or Vent (50- TAAT) proteins (Figure 7C). The core sequence 50-TAAT-30 has been previously shown to be absolutely required for bind- ing and activity of Xvent-2 (64,65). Xvent-2 is an excellent candidate as a co-modulator of the gata2 response to BMP signaling. First, it is a direct target of BMP-4 (66). Second, zebraﬁsh embryos in which the Vent-related homeobox genes vox and vent are inactivated by mutation have reduced expres- sion of gata2 at mid-gastrula stages (67). Third, Xvent-2 can associate with Smad1 to regulate the transcription of down- stream target genes (68). Fourth, Xvent-2 and gata2 co-regulate the expression of Xvent-1, placing both these genes in the same ventralizing pathway (33). Collectively, these data are consistent with the hypothesis that gata2 expres- sion is induced by a combination of Smad1/4, Oct-1 and Xvent-2 (and perhaps other co-factors), followed by coopera- tion of these transcription factors to activate other proteins important for ventral fate and the speciﬁcation of ventrally derived tissues. Nucleic Acids Research, 2005, Vol. 33, No. 13 4365 ACKNOWLEDGEMENTS The full-length zebrafish gata2 promoter was provided by Shuo Lin (UCLA) and the initial set of deletions was generated based on the sequences analyzed previously in his laboratory in transgenic animals. We are also grateful to J. Thomsen (SUNY, Stony Brook), J. Christian (Oregon Health Sciences University), M. Mullins (University of Pennsylvania) and C. Wright (Vanderbilt University) for providing expression constructs. These studies were supported by grants to T.E. from the National Institutes of Health (HL056182) and the Irma T. Hirschl Trust. Funding to pay the Open Access publication charges for this article was provided by the National Institutes of Health. Conflict of interest statement. None declared. REFERENCES 1. Shi,Y. and Massague,J. (2003) Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell, 113, 685–700. 2. Wrana,J.L. and Attisano,L. (2000) The Smad pathway. 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