Axin2/Conductin/Axil and its ortholog Axin are negative regulators of the Wnt signaling pathway, which promote the phosphorylation and degradation of -catenin. While Axin is expressed ubiquitously, Axin2 mRNA was seen in a restricted pattern during mouse embryogenesis and organogenesis. Because many sites of Axin2 expression overlapped with those of several Wnt genes, we tested whether Axin2 was induced by Wnt signaling. Endogenous Axin2 mRNA and protein expression could be rapidly induced by activation of the Wnt pathway, and Axin2 reporter constructs, containing a 5.6-kb DNA fragment including the promoter and first intron, were also induced. This genomic region contains eight Tcf/LEF consensus binding sites, five of which are located within longer, highly conserved noncoding sequences. The mutation or deletion of these Tcf/LEF sites greatly diminished induction by -catenin, and mutation of the Tcf/LEF site T2 abolished protein binding in an electrophoretic mobility shift assay. These results strongly suggest that Axin2 is a direct target of the Wnt pathway, mediated through Tcf/LEF factors. The 5.6-kb genomic sequence was sufficient to direct the tissue-specific expression of d2EGFP in transgenic embryos, consistent with a role for the Tcf/LEF sites and surrounding conserved sequences in the in vivo expression pattern of Axin2. Our results suggest that Axin2 participates in a negative feedback loop, which could serve to limit the duration or intensity of a Wnt-initiated signal.
TCF and SOX proteins belong to the high mobility group box transcription factor family. Whereas TCFs, the transcriptional effectors of the Wnt pathway, have been widely implicated in the development, homeostasis and disease of the intestine epithelium, little is known about the function of the SOX proteins in this tissue. Here, we identified SOX9 in a SOX expression screening in the mouse fetal intestine. We report that the SOX9 protein is expressed in the intestinal epithelium in a pattern characteristic of Wnt targets. We provide in vitro and in vivo evidence that a bipartite β-catenin/TCF4 transcription factor, the effector of the Wnt signaling pathway, is required for SOX9 expression in epithelial cells. Finally, in colon epithelium-derived cells, SOX9 transcriptionally represses the CDX2 and MUC2 genes, normally expressed in the mature villus cells of the intestinal epithelium, and may therefore contribute to the Wnt-dependent maintenance of a progenitor cell phenotype.
Requirements for intron recognition during pre-mRNA splicing in plants differ from those in vertebrates and yeast. Plant introns contain neither conserved branch points nor distinct 3 splice site-proximal polypyrimidine tracts characteristic of the yeast and vertebrate introns, respectively. However, they are strongly enriched in U residues throughout the intron, property essential for splicing. To understand the roles of different sequence elements in splicing, we are characterizing proteins involved in intron recognition in plants. Accurate and orderly splicing of nuclear pre-mRNAs requires that exon and intron sequences are effectively distinguished from each other and that matching 5Ј and 3Ј splice sites (5Јss and 3Јss) 1 are selected with precision and juxtaposed before the catalytic steps. Several short sequence elements in pre-mRNA contribute to intron recognition and splice site selection. The 5Јss and 3Јss represent the most universally conserved and functionally important elements. The 5Јss, containing the nearly invariant GU dinucleotide, is recognized through base pairing by the U1 small nuclear ribonucleoprotein (snRNP) early in spliceosome assembly and by other snRNPs and factors at later stages of the reaction. The precise function of the conserved AG at the 3Ј intron border is less well understood (reviewed in Refs. 1-4). Although the role of splice sites appears to be similar in different eukaryotes, the relative contribution of other signals differs significantly between different organisms. In vertebrates and insects, the characteristic and functionally important feature of introns is a polypyrimidine tract, which is located between the branch point and the 3Јss. The pyrimidine tract is recognized by the heterodimeric protein U2AF (U2 snRNP auxiliary factor) early in spliceosome assembly, and this interaction is essential for positioning the U2 snRNP at the branch site, the sequence of which is highly degenerate in metazoa (5-7). Another factor, mBBP/SF1, interacting directly with the branch point region, may also assist U2 snRNP to establish a base pairing interaction with the branch site (8 -10). The distinguishing feature of pre-mRNA introns in the yeast Saccharomyces cerevisiae is a highly conserved branch site sequence, UACUAAC (reviewed in Refs. 2 and 11). Early in spliceosome assembly, UACUAAC is specifically recognized by the branch point-bridging protein BBP, the yeast ortholog of mBBP/SF1 (8 -10). Most introns in S. cerevisiae lack 3Јss-proximal polypyrimidine tracts, and specific sequences positioned 3Ј of the branch site do not contribute to the first step of splicing despite the fact that a factor, Mud2p, sharing some structural and functional similarity with the large subunit of the metazoan U2AF, is present in yeast (8,12). Some yeast introns contain uridine stretches upstream of the 3Јss, but these sequences function during the second step of splicing (13,14).Requirements for intron recognition in higher plants differ from those in yeast and vertebrates. Plant introns contain n...
We have isolated, via differential hybridization screening of a floral cDNA library from sunflower, a cDNA clone that hybridizes to a 1100 nucleotide-long mRNA found exclusively in mature pollen grains. The cDNA encodes a 219 amino acid-long polypeptide containing two potential zinc fingers alternating with two basic domains. A similar organization is found in the erythroid-specific transcription factor Eryf1 from chicken and its murine homolog GF-1. The C-terminus of the protein contains a sixfold repeat of the pentapeptide sequence (S,T,A)(E,D)TQN. These features suggest that the SF3 protein is a transcription factor required for the expression of late pollen genes. The SF3 gene is a member of a multicopy gene family. A genomic copy of the gene has been isolated and sequenced; it is split by four short, AT-rich introns.
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