Wnt and its intracellular effector

Mammalian gene promoters for transcription by RNA polymerase II are typically organized in the following order: upstream sequence motif(s)/TATA box/initiation site. Here we report studies in which the order, orientation and DNA sequences of these three elements are varied to determine how these affect polarity of transcription. We have constructed promoters with an 'octamer' upstream sequence ATTTGCAT (or its complement ATGCAAAT) in combination with several different TATA boxes and initiation (cap) sites, and tested these promoters in transfection experiments with cultured cells. TATA boxes derived from the adenovirus major late promoter (TATAAAA), immunoglobulin kappa light chain (TTATATA) and heavy chain (TAAATATA) promoter functioned equally well or even better when inverted. Only the beta-globin TATA box (CATAAAA) was poorly active when inverted. In addition, a symmetrical TATA box (TATATATA) derived from a casein gene was very active. Our results suggest that the asymmetry of most TATA boxes (consensus TATAAAA) is not a primary determinant of the polarity of transcription. We also found that the initiation (cap) site, which usually consists of an adenine embedded in a pyrimidine-rich region (PyPyCAPyPyPyPyPy), was permissive towards sequence alterations; even a randomly composed sequence worked well. However, an inverted, hence purine-rich, cap site reduced transcript levels to 1/7th, as did an oligo G sequence. Irrespective of the presence of a cap site, the configuration: 'TATA box/octamer' yielded a strong leftward, rather than rightward transcription. From this, we conclude that the polarity of transcription is primarily determined by the linear order of an upstream sequence relative to a TATA box, rather than by the individual orientations of either of these two elements.

Many protein domains for transcriptional activation also function when fused to a heterologous DMA binding domain. In mammalian HeLa cells, we have previously characterized the activation domains of several transcription factors using GAL4 fusion proteins. Here we have tested their transcriptional activity in oocytes and developing embryos of the clawed toad Xenopus laevis. We find that the "acidic 11 C-terminal domain of the herpesvirus VP16 (= Vmw65) activator, which is active from yeast to man, is also very active in the two Xenopus systems. The constitutive nature of this viral domain may have evolved to be refractory to cellular defense mechanisms. By contrast, activation domains from cellular eukaryotic transcription factors (TFE3, ITF2, MTF-1) are differentially active in oocytes and early embryos. This indicates that their activity can be regulated by protein modification and/or availability of specific coactivators. We have also compared VP16 induced enhancement of transcription from remote and promoter-proximal positions. In both oocytes and late blastula embryos, activation from a promoter-proximal position was more than 50 fold, while only a moderate stimulation (3-8 fold) was observed from remote positions. This may mean that frog oocyte and early embryos are not yet fully geared for gene control by remote enhancers, i.e. respond predominantly to close-by regulatory sequences. The fact that cellular enhancers are naturally located at various distances from the responsive promoters may thus be exploited by multicellular organisms for differential gene control at early and late stages of development.

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In addition to measuring transcription in vitro (Gerber et al. 1992) or in transient transfection assays (e.g. Xu et al. 1991), an alternative method to study the function of transcription factors is to inject reporter DNA together with either nuclear extract or factor gene into Xenopus oocytes (Rungger et al. 1990; Theulaz et al. 1988). Here we introduce the GALA-derived assay, which was established in other systems, to study transcriptional activation in the frog oocyte. In our experiment, purified recombinant transactivator fusion protein GAL-VP16(40N) (Seipel et al. 1992; P.Rigoni and W.Schaffner, unpublished) was coinjected with a (3-globin reporter gene into the Xenopus oocytes nucleus (germinal vesicle). The reporter promoter contained five GALA binding sites upstream of an octamer motif. Xenopus oocytes were prepared as described previously (Rungger et al. 1990; Bertrand et al. 1991). Mixtures of 4 ng reporter gene plasmid, 1 ng reference gene and 1 ng purified protein G-AL-VP16(40N) in 10 nl of DNA injection buffer (89 mM NaCl, 1 mM KCl, 20 mM Hepes, pH 7) were injected into the nucleus (germinal vesicle). Following the injections, the oocytes were incubated for 15 to 20 hrs in OR2 solution (82.5 mM NaCl, 2.5 mM KCl, 1 mM Na2HPO4, 5 mM Hepes, 0.5 mM PVP with calcium (Wallace et al. 1973)) containing antibiotics (50 iU of