OAZ uses distinct DNA- and protein-binding zinc fingers in separate BMP-Smad and Olf signaling pathways.

We have identified the 30-zinc finger protein OAZ as a DNA-binding factor that associates with Smads in response to BMP2, forming a complex that transcriptionally activates the homeobox regulator of Xenopus mesoderm and neural development, Xvent-2. OAZ contains a BMP signaling module formed by two clusters of fingers that bind Smads and the Xvent-2 BMP response element, respectively. Previously implicated as a transcriptional partner of Olf-1/EBF in olfactory epithelium and lymphocyte development in the rat, OAZ fulfills this role through clusters of fingers that are separate from the BMP signaling module. The mutually exclusive use of OAZ by the BMP-Smad and Olf pathways illustrates the dual role of a multi-zinc finger protein in signal transduction during development.

Negative regulation of axis formation and Wnt signaling in Xenopus embryos by the F-box/WD40 protein beta TrCP.

Screening a maternal Xenopus expression library for activities that synergize with low levels of injected beta-catenin, we have isolated a clone encoding the C-terminal end of x-beta TrCP-2, a highly conserved protein belonging to the F-box/WD40 family of ubiquitin-ligase specificity factors. We show that x-beta TrCP-2 expression reduces dorsal axis formation in Xenopus embryos. A dominant negative mutant lacking the F-box triggers the opposite effect, inducing secondary axes and activating the expression of Wnt responsive genes in ectodermal explants. In light of the existence of beta TrCP transcripts associated with the vegetal cortex, we propose that beta TrCP plays a fundamental role in the establishment of the dorsal determinants during cortical rotation in Xenopus.

A molecular basis for Smad specificity.

Bone morphogenetic proteins (BMPs) and activins are members of the TGFbeta superfamily of growth factors, a crucial group of regulators of induction and patterning of embryonic germ layers in metazoa. In early Xenopus embryos, activin, Vgl, and nodal are potent inducers of dorsal mesoderm, whereas BMPs can ventralize mesoderm, repress neural fate, and induce blood differentiation. These characteristic responses rely on ligand-specific signaling pathways, encompassing transmembrane kinase receptors and signal transducers belonging to the Smad family. The overexpression in Xenopus embryos of BMP-activated Smad1 and of activin/Vg1/ nodal-activated Smad2 is sufficient to specifically recapitulate ligand responses. In a search for determinants of a Smad specificity code, we have identified two small regions within the conserved carboxyl-domain that are necessary and sufficient for specific Smad action. Swapping both residue clusters (C1 and C2) between Smadl and Smad2 completely switches Smad effects in vivo. Thus, Smadl with swapped Smad2 clusters responds specifically to BMP but elicits an activin response, while a Smad2 protein containing the Smadl clusters is activated by activin and elicits a BMP response. Furthermore, association between Smads and FAST-1, a mediator of mesoderm induction by activin, is dependent upon the presence of the Smad2 C1 sequence. Finally, the Smadl-specific antagonist Smad6 can inhibit a Smad2 molecule harboring Smadl C1 and C2 sequences. Thus, the C1 and C2 regions of Smads specify the association between Smads and pathway-specific partners, such as FAST-1 and Smad6, and account for activin- and BMP- specific responses in vertebrate embryogenesis.

Smad6 inhibits BMP/Smad1 signaling by specifically competing with the Smad4 tumor suppressor.

Bone morphogenetic protein (BMP) receptors signal by phosphorylating Smad1, which then associates with Smad4; this complex moves into the nucleus and activates transcription. Here we report the existence of a natural inhibitor of this process, Smad6, a longer version of the previously reported JV15-1. In Xenopus embryos and in mammalian cells, Smad6 specifically blocks signaling by the BMP/Smad1 pathway. Smad6 inhibits BMP/Smad1 signaling without interfering with receptor-mediated phosphorylation of Smad1. Smad6 specifically competes with Smad4 for binding to receptor-activated Smad1, yielding an apparently inactive Smad1-Smad6 complex. Therefore, Smad6 selectively antagonizes BMP-activated Smad1 by acting as a Smad4 decoy.

Concentration-dependent patterning of the Xenopus ectoderm by BMP4 and its signal transducer Smad1.

Morphogens are thought to establish pattern in early embryos by specifying several cell fates along a gradient of concentration; a well-studied example is the Drosophila protein decapentaplegic (DPP) acting in the wing disc. Recent work has established that bone morphogenetic protein 4 (BMP4), the vertebrate homologue of DPP, controls the fundamental choice between neural and epidermal fates in the vertebrate ectoderm, under the control of antagonists secreted by the organizer region of the mesoderm. We now show that BMP4 can act as a morphogen, evoking distinct responses in Xenopus ectodermal cells at high and low concentrations, in a pattern consistent with the positions of the corresponding cell types in the embryo. Moreover, this complex cellular response to extracellular BMP4 concentration does not require subsequent cell-cell communication and is thus direct, as required of a classical morphogen. We also show that the same series of cell types--epidermis, cement gland and neural tissue--can be produced by progressively inhibiting endogenous BMP signaling with specific antagonists, including the organizer factor noggin. Finally, expression of increasing doses of the signal transduction molecule Smad1 accurately reproduces the response to BMP4 protein. Since Smads have been shown to act in the nucleus, this finding implies a direct translation of extracellular morphogen concentration into transcription factor activity. We propose that a graded distribution of BMP activity controls the specification of several cell types in the gastrula ectoderm and that this extracellular gradient acts by establishing an intracellular and then nuclear gradient of Smad activity.

Mutations increasing autoinhibition inactivate tumour suppressors Smad2 and Smad4.

Smad2 and Smad4 are related tumour-suppressor proteins, which, when stimulated by the growth factor TGF-beta, form a complex to inhibit growth. The effector function of Smad2 and Smad4 is located in the conserved carboxy-terminal domain (C domain) of these proteins and is inhibited by the presence of their amino-terminal domains (N domain). This inhibitory function of the N domain is shown here to involve an interaction with the C domain that prevents the association of Smad2 with Smad4. This inhibitory function is increased in tumour-derived forms of Smad2 and 4 that carry a missense mutation in a conserved N domain arginine residue. The mutant N domains have an increased affinity for their respective C domains, inhibit the Smad2-Smad4 interaction, and prevent TGF beta-induced Smad2-Smad4 association and signalling. Whereas mutations in the C domain disrupt the effector function of the Smad proteins, N-domain arginine mutations inhibit SMAD signalling through a gain of autoinhibitory function. Gain of autoinhibitory function is a new mechanism for inactivating tumour suppressors.

Partnership between DPC4 and SMAD proteins in TGF-beta signalling pathways.

The TGF-beta/activin/BMP superfamily of growth factors signals through heteromeric receptor complexes of type I and type II serine/threonine kinase receptors. The signal originated by TGF-beta-like molecules appears to be transduced by a set of evolutionarily conserved proteins known as SMADs, which upon activation directly translocate to the nucleus where they may activate transcription. Five SMAD proteins have so far been characterized in vertebrates. These factors are related to the mediator of decapentaplegic (dpp) signalling, mothers against dpp (Mad), in Drosophila and to the Sma genes from Caenorhabditis elegans. Smad1 and Smad2 have been shown to mimic the effects of BMP and activin, respectively, both in Xenopus and in mammalian cells, whereas Smad3 (a close homologue of Smad2) and the related protein DPC4, a tumour-suppressor gene product, mediate TGF-beta actions. We report here that DPC4 is essential for the function of Smad1 and Smad2 in pathways that signal mesoderm induction and patterning in Xenopus embryos, as well as antimitogenic and transcriptional responses in breast epithelial cells. DPC4 associates with Smad1 in response to BMP and with Smad2 in response to activin or TGF-beta. DPC4 is therefore a regulated partner of SMADs that function in different signalling pathways of the TGF-beta family.

Overlapping transcription by RNA polymerases II and III of the Xenopus TFIIIA gene in somatic cells.

Xenopus TFIIIA gene expression is developmentally regulated. The per cell level of steady-state TFIIIA mRNA present in oocytes is drastically reduced (10(6)-fold) in adult somatic cells, suggesting regulation at the transcriptional level. This is supported by the presence of TFIIIA mRNAs with different 5'-ends in oocytes and in somatic cells, which suggests the presence of distinct cell-/stage-specific promoters, i.e. a strong oocyte-specific and a weak somatic-specific promoter. Here, by mapping the 5'-ends of TFIIIA RNAs found in Xenopus somatic cells, we document the activity not only of an upstream somatic-specific promoter, but also of a down-stream "oocyte-specific" promoter that gives rise to a minor population of somatic TFIIIA mRNAs initiated from the same transcription start site utilized in oocytes. This result explains the presence of an oocyte-type form of TFIIIA protein in somatic cells. Furthermore, by characterizing transcription from the Xenopus TFIIIA promoter in somatic cell nuclear extracts and comparing the in vitro transcription start sites with the 5'-ends of endogenous somatic TFIIIA transcripts in poly(A)+ and poly(A)- RNA fractions, we show that RNA polymerase III initiates at position -70 of the "oocyte-specific" core promoter and transcribes through the promoter and into the TFIIIA coding sequences in somatic cells. These results suggest a possible polymerase III transcription-mediated down-regulation of the oocyte-specific TFIIIA promoter in Xenopus somatic cells.

Cloning and characterization of an evolutionarily divergent DNA-binding subunit of mammalian TFIIIC.

Transcription factor IIIC (TFIIIC) is required for the assembly of a preinitiation complex on 5S RNA, tRNA, and adenovirus VA RNA genes and contains two separable components, TFIIIC1 and TFIIIC2. TFIIIC2 binds to the 3' end of the internal control region of the VAI RNA gene and contains five polypeptides ranging in size from 63 to 220 kDa; the largest of these directly contacts DNA. Here we describe the cloning of cDNAs encoding all (rat) or part (human) of the 220-kDa subunit (TFIIIC alpha). Surprisingly, TFIIIC alpha has no homology to any of the yeast TFIIIC subunits already cloned, suggesting a significant degree of evolutionary divergence for RNA polymerase III factors. Antibodies raised against the N terminus of recombinant human TFIIIC alpha specifically inhibit binding of natural TFIIIC to DNA. Furthermore, immunodepletion assays indicate that TFIIIC alpha is absolutely required for RNA polymerase III transcription of 5S RNA, tRNA, and VAI RNA genes but not for the 7SK RNA and U6 small nuclear RNA genes. Transcription from the tRNA and VAI RNA genes in TFIIIC-depleted nuclear extracts can be restored by addition of purified TFIIIC. In contrast, restoration of 5S RNA gene transcription requires readdition of both TFIIIC and TFIIIA, indicating a promoter-independent interaction between these factors. Immunoprecipitation experiments demonstrate a tight association of all five polypeptides previously identified in the TFIIIC2 fraction, confirming the multisubunit structure of the human factor.

HrpF, a human sequence-specific DNA-binding protein homologous to XrpFI, a Xenopus laevis oocyte transcription factor.

The identification in HeLa nuclei of a novel DNA-binding protein, designated HrpF, is presented. This factor recognizes and binds a sequence of the Xenopus laevis L14 ribosomal protein (r-p) gene promoter bound by the Xenopus r-p transcription factor I (XrpFI). We show here that XrpFI and HrpF share a conserved DNA-binding domain. We also present evidences suggesting that the two factors perform similar functions in the cell. We discuss the hypothesis that closely related factors might be involved in the control of rp-gene transcription in vertebrates.