4358 Nucleic Acids Research, 2005, Vol. 33, No. 13 all, known BMP response elements (BREs) are also Smad binding elements (SBEs), including those in the promoters for the genes encoding Vestigial (17), xVent2B (16,18), Smad6 (19), Id1 (20,21), Dlx3 (22) and Hex (23). A BMP- 4 syn-expression motif was identiﬁed (TGGCGCC) as a conserved BRE (7), and SBEs have in some cases (17,24) been associated with GC-rich elements (GCCGnCGC) or as other deﬁned short (GTCT or GCAT) motifs (18,25). However, there is no single consensus sequence that can readily predict a functional Smad1 binding site. Given that Smads have weak afﬁnity for DNA, it is necessary to deﬁne the DNA-binding cofactor(s) to understand how Smads are targeted to any particular BRE. One of the best characterized BMP-regulated pathways relates to the induction of ventral/posterior cell fate during Xenopus development. The homeobox transcription factor Xvent2 was the ﬁrst target gene of BMP signaling to be inves- tigated mechanistically, and several elements that contribute to BMP responsiveness have been deﬁned, including an SBE/ OAZ BRE, GCAT motifs, Vent2 auto-regulatory binding sites, and most recently, binding sites for the POU-domain protein Oct-25 (26). Another gene important to the ventral pathway is gata2, encoding a zinc ﬁnger transcription factor that is expressed throughout ventral ectoderm and ventral and lateral mesoderm (27–29). The gata2 gene is induced by BMP-4 (30,31) and forced expression of an engrailed-Gata2 fusion protein is sufﬁcient to dorsalize Xenopus embryos (32), pro- viding strong evidence that Gata factor activity is essential for ventral cell fate. Gata2 and xVent2 cooperate to activate the xVent1 gene, placing these two transcription factors within a common ventral network (32,33). Here, we investigate the mechanism by which the gata2 gene is activated by BMP signaling. We establish a reporter assay to deﬁne sequences of the gata2 promoter that mediate induction by BMP-4 during embryonic development. We ﬁnd that an octamer binding site, interacting with Oct-1, is necessary for induction, but only functions in the context of a separate more proximal DNA sequence. The results provide an independent example of a POU-homeodomain protein mediating induction by BMP signaling, and implicate a common network controlling regulatory genes of the ventral pathway. MATERIALS AND METHODS Identification of the zebrafish gata2 transcriptional start site RNA was isolated from zebraﬁsh embryos (75% epiboly) with Tri-reagent (MRC, Inc.). Superscript Reverse Transcriptase (BRL) was used to generate cDNA, followed by treatment with RNAse. Adapters were ligated to the cDNA ends using the Invitrogen 50 RACE kit as per the manufacturer’s instruc- tions. A 30 gene-speciﬁc reverse primer, corresponding to position +92 (relative to the translation start site) of the gata2 transcript, was used to amplify cDNA by PCR. The primer sequence was 50-GTAAGCCCGTGATGATGTGAC- TCT (TE872). A single PCR product was identiﬁed in this analysis and subcloned in pCR-2.1 TOPO (Invitrogen). Sequence analysis conﬁrmed that the clone included the previously characterized gata2 cDNA, with additional 50- untranslated region. The start site and exon/intron boundaries were determined by comparing this new cDNA sequence with the genomic sequence. Reporter plasmids and deletion constructs A genomic region including 7.1 kb of sequence upstream of the initiation methionine (6.5 kb upstream of transcriptional start site) was provided by Shuo Lin (UCLA), and transferred to the Kpn1 site of the pGL3basic luciferase reporter plasmid (Promega). The deletion to 1070 was facilitated by a unique MluI restriction site at that position. The other 50 deletion constructs were generated by PCR, incorporating in the upstream primer a KpnI site and using a reverse primer cor- responding to position +58 of the gata2 promoter. PCR prod- ucts were cloned into the pCR-2.1 TOPO vector (Invitrogen). Subsequent cloning steps took advantage of an internal BssH2 site located at position 97. Therefore, each PCR-derived clone was digested with KpnI/BssH2 and the fragment used to substitute the full-length gata2 promoter sequence in the context of the pGL3basic luciferase reporter. PCR primers used to generate the progressive deletions were (KpnI sites underlined): 890 Kpn I forward, 50-AAAGGTACCGCCT- GCTGCTTGTTTTGCC (TE851); 819 KpnI forward, 50-AAAGGTACCGGCTTTATTTGGACCTGC (TE876); 751 KpnI forward, 50-AAA GGTACCAAGTGACGTTT- GATCGCT (TE878); 728 KpnI forward, 50-AAAGG- TACCCGGCATACAAAGACAGTG (TE852); +58 reverse, 50-CCCGATTGTTAAAGTCTCCC (TE837). To transfer a ‘minimal’ BRE region upstream of the SV40 promoter, a ClaI site at position 443 was used with promoter constructs truncated at 819 or 751. Plasmids were digested with KpnI/ClaI, transferred ﬁrst into pBluescript KS digested with KpnI/ClaI, re-excised with KpnI and SmaI, and ﬁnally transferred into similarly digested SV40 pGL3promoter luciferase plasmid (Promega). This resulted in reporter plas- mids that transfer from 819 to 443 (377 bp) or 751 to 443 (308 bp) of the gata2 promoter, respectively. Additional minimal promoter constructs (which transfer 233, 184 or 134 bp) were generated by PCR, ﬁrst cloning products into pCR-2.1 TOPO, followed by re-excision with KpnI and SacI, and transfer into the KpnI/SacI sites of the SV40 pGL3promoter luciferase plasmid (Promega). This resulted in the transfer of regions 819 to 586 (233 bp), 819 to 635 (184 bp), or 819 to 685 (134 bp), respectively. The PCR primers used for this purpose were (restriction sites underlined): 819 KpnI forward, 50-AAAGGTACCGGCTT- TATTTGGACCTGC (TE 876); 685 SacI reverse, 50-AAA- GAGCTCGTCACCACATTCTTCTTG (TE 1127); 635 SacI reverse 50-AAAGAGCTC CTCTAAATTGGGGGCTA- TTG (TE1161); 586 SacI reverse, 50-AAAGAGCTCCTCAA CTCCTAGCACCTC (TE1128). For site-directed mutagenesis, the GeneEditor in vitro mutagenesis system (Invitrogen) was used with the follow- ing oligonucleotides (mutations underlined): mutation #1, 50-TCGATAGGTACCACTTTTATTTGGACC (TE1009); mutation #2, 50-ACCGGCTTTATTGCTACCTGCCCATGC (TE1012); mutation #3, 50-GGCTTTATTTGGGAGTGC- CCATGCGAC (TE1013); mutation #4, 50-TGGACCTG- CCCAAAGGACCTGTCGGCAC (TE1016); mutation #5, 50-CACCTCCAAGAGACTAGTTCGCTATTAATATGTAA- AGTG (TE1134); mutation #6, 50-AGAGACGGGCTCGC-