4364 Nucleic Acids Research, 2005, Vol. 33, No. 13 A B C Figure 7. The BRE1 cooperates with a distinct BRE2 element located at 100 bp proximal to the Oct-1 binding site. (A) The SV40 minimal promoter was used to test if the BRE activity could be transferred to a heterologous promoter. The SV40 promoter itself is not induced by BMP4. Shown below this construct are six additional reporters containing various regions of the gata2 upstream region as indicated on the map below, placed upstream of the SV40 promoter. Sequences from 819 to 685 containing just the BRE1 (construct #1, 134 bp) does not function as a BRE, whereas the entire region from 819 to 443 (construct #2, 377 bp) is sufficient to mediate 3-fold activation of the SV40 promoter by BMP-4. Subsequent constructs delineated a 20 bp sequence (between 666 and 646) that is necessary for BRE activity (designated BRE2). Dark grey boxes indicate gata2 genomic sequences, black boxes indicate the SV40 promoter, and the light grey box represents the luciferase reporter gene. Black boxes on the map below represent the BRE sequences. (B) BRE2 is not active in the absence of BRE1. This is shown by transferring sequences from 751 to 443 upstream of the SV40 promoter, which fails to support BMP-mediated induction. Therefore, neither BRE1 nor BRE2 has activity on its own, but the two regions cooperate to mediate the response. Boxes represent sequences as in (A). (C) Sequences around the BRE2, defined so far by the 20 bp region between 646 and 666. Potential Smad (AGAC) or Vent-2 (ATTA) binding sites are indicated, although we have so far been unable to confirm that they are functional, either for binding or activity (data not shown). region (308 bp, from 751 to 443) removes the Oct-1 site and eliminates induction of the SV40 promoter by BMP-4 (Figure 7B). We conclude that BMP induction requires both the distal Oct-1 site (BRE1) and sequences within or ﬂanking a 20 bp region of a proximal element (BRE2). DISCUSSION Gata2 is a transcription factor expressed in multiple tissues including those of the hematopoietic (31,53–55) and nervous systems (56–58). The gene is likely to be regulated by distinct pathways in diverse tissues, but the early activation in ventral and lateral embryonic mesoderm and ectoderm is fully dependent on the BMP signaling pathway. The gata2 gene is therefore a component of the BMP syn-expression network. Our results show that BMP signals activate the gata2 promoter, dependent on the participation of R-Smad proteins, and requiring a binding site for the POU homeodomain transcription factor Oct-1, which functions in conjunction with a more proximal regulatory region. It is important to point out that the gata2 gene is expressed broadly throughout the BMP syn-expression domain, at the stages of development we are investigating. The Oct-1 binding site is therefore presumably involved in mediating this broad activation of the gata2 promoter, and our experiments do not address whether it continues to be important for the regulatory mechanisms that later restrict expression of the gata2 gene to deﬁned cell lineages, e.g. in the hematopoietic system. Although originally considered a ubiquitous transcription factor (50), Xenopus Oct-1 is expressed during embryogenesis in a restricted pattern (59) in regions of both ectoderm and mesoderm coincident with gata2 expression. Furthermore, mis-expression of Oct-1 in developing Xenopus embryos causes axis defects (59), suggesting that Oct-1 might be a component of early dorsal–ventral patterning. Axis defects are similarly caused by expression of a dominant- negative Gata2 isoform (32). There is precedence for media- tion of the BMP pathway by POU domain proteins, provided ﬁrst by studies of the Dpp-regulated vestigial gene in Drosophila (60). A sequence from the vestigial locus, known as the vgQ enhancer, is directly regulated by Dpp signaling through binding of Mad to this element (17). However, over-expression of the POU domain protein Drifter (DFR) expands the domain of LacZ expression in transgenic ﬂies that carry a vgQ-LacZ reporter (60). Subsequent promoter deletion analysis identiﬁed a DFR binding element in close proximity to the already characterized Mad binding element, consistent with the idea that full Dpp-mediated transcriptional activity requires the cooperation of both DFR and Mad proteins at the vestigial promoter (60). Ectopic expression of Dpp or a constitutively activated form of the Dpp receptor Thick Veins fails to activate DFR expression, implying that DFR itself is not a direct target of the Dpp signaling pathway (60). Recently, Cao et al. (26) found using a one-hybrid assay that Oct-25 binds to and stimulates the activity of the Xvent-2B promoter, and that Oct-25 can also bind to components of the BMP signaling pathway, including Smad1, Smad4 and Xvent-2. Interestingly, Oct-25 expression is not sufﬁcient for activation of the Xvent-2B promoter, but requires the cis-elements that interact with Smad1/4 and OAZ. This is analogous to the situation we describe with the gata2 pro- moter, in that Oct-1 binding is not sufﬁcient but requires in addition the more proximal BRE2 sequences, which we pro- pose are direct or indirect targets for Smads and/or other activators. Our preliminary attempts at further deﬁning the