Nucleic Acids Research, 2005, Vol. 33, No. 13 4357–4367 doi:10.1093/nar/gki746 An Oct-1 binding site mediates activation of the gata2 promoter by BMP signaling Tal Oren, Ingrid Torregroza and Todd Evans* Albert Einstein College of Medicine, Bronx NY 10461, USA Received May 5, 2005; Revised July 5, 2005; Accepted July 13, 2005 ABSTRACT The gata2 gene encodes a transcription factor implicated in regulating early patterning of ectoderm and mesoderm, and later in numerous cell-specific gene expression programs. Activation of the gata2 gene during embryogenesis is dependent on the bone morphogenetic protein (BMP) signaling path- way, but the mechanism for how signaling controls gene activity has not been defined. We developed an assay in Xenopus embryos to analyze regulatory sequences of the zebrafish gata2 promoter that are necessary to mediate the response to BMP signaling during embryogenesis. We show that activation is Smad dependent, since it is blocked by expression of the inhibitory Smad6. Deletion analysis identified an octamer binding site that is necessary for BMP- mediated induction, and that interacts with the POU homeodomain protein Oct-1. However, this element is not sufficient to transfer a BMP response to a heterologous promoter, requiring an additional more proximal cooperating element. Based on recent studies with other BMP-dependent promoters (Drosophila vestigial and Xenopus Xvent-2), our studies of the gata2 gene suggest that POU-domain proteins comprise a common component of the BMP signaling pathway, cooperating with Smad proteins and other transcriptional activators. INTRODUCTION Bone morphogenetic proteins (BMPs) are members of the TGF-b superfamily of extracellular ligands, which signal by binding to heterodimeric serine/threonine kinase receptor complexes. Much progress has been made identifying the molecular components that regulate transduction of a BMP signal from the plasma membrane to the nucleus (1,2). Ligand binding activates the receptor complex, which relays the signal by recruiting downstream mediators. A commonly used subset of mediators are known collectively as Smads, which are categorized into three functional groups: (i) receptor regulated Smads (R-Smads) that are directly phosphorylated by the activated receptor; (ii) Smad4, which cooperates with R-Smads to form an active signaling complex; and (iii) inhibitory Smads (I-Smads), which serve as negative regu- lators of the pathway (3). Although there may be exceptions (4,5), generally R-Smads 1, 5 and 8 are phosphorylated by the BMP pathway, whereas R-Smads 2 and 3 function instead to transduce a TGF-b, nodal or activin signal. The R-Smad/ Smad4 complex interacts with speciﬁc nuclear transcription factors to activate gene transcription. There are also Smad- independent mechanisms to mediate BMP signaling, e.g. by p38 MAPK (6). Deﬁning the mechanisms by which BMP-induced Smads activate speciﬁc target genes is an important goal (7) given the wide range of developmental and physiological responses that are under the control of the pathway. BMP signaling patterns early germ layers to establish a dorsal/ventral mesoderm axis, the anterior/posterior endoderm character and the dis- tinction of neural/epidermal ectoderm (8–12). BMP signaling also regulates lineage and morphogenetic programs relevant to bone, cartilage, kidney, heart and reproductive organ devel- opment. Thus, a wide range of highly speciﬁc gene expression programs is coordinated by the action of this common signal- ing pathway, presumably by the presence or absence of inter- secting signaling pathways and speciﬁc nuclear co-factors. An R-Smad/Smad4 complex binds DNA weakly on its own, relying on interaction with other nuclear partners to achieve stable and functional binding (13). A paradigm of Smad– cofactor interaction was established for TGF-b/activin signal- ing by the identiﬁcation of winged helix proteins, such as FAST-1 as DNA-binding partners that interact with Smad2/ 3 at target promoters (14,15). With respect to the BMP path- way, an analogous example is the 30-zinc ﬁnger protein OAZ, shown to interact with BMP-induced Smads to activate the promoter for the homeobox gene Xvent2 (16). Most, but not *To whom correspondence should be addressed at Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Jack and Pearl Resnick Campus, 1300 Morris Park Avenue, Chanin Building, Room 501, Bronx NY 10461, USA. Tel: +1 718 430 3506; Fax: +1 718 430 8988; Email: tevans@aecom.yu.edu The Author 2005. Published by Oxford University Press. All rights reserved. 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