ABSTRACT
Among Xenopus Lef/Tcfs, XTcf-4 has an outstanding role. In early development it is located exclusively in the midbrain where it is essential for midbrain and isthmus development. In order to identify transcription factors responsible for the restriction of XTcf-4 expression we isolated a 3.8kb fragment of the XTcf-4 promoter. We found that this promoter fragment is sufficient to mimic endogenous XTcf-4 expression in the midbrain. Characterization of putative binding sites for en2 and pax2/5 revealed that en2, but not pax2/5 directly represses XTcf-4 promoter activity. Gain-of-function experiments in Xenopus embryos confirmed this en2-mediated repression. Loss-of-function experiments demonstrate that both en2 and pax2/5 are essential for endogenous XTcf-4 expression. The primary effect of pax2/5 depletion thereby appears to be a reduced en2 expression at neurula stages. Because en2 can compensate for the depletion of pax2/5, we assume a hierarchical regulation of gene expression in the midbrain/isthmus region with pax2/5 acting upstream of en2. Furthermore, since the XTcf-4 expression domain does not overlap with the expression domains of the isthmus marker genes en2 and pax2/5, we conclude that the knock-down of en2 and pax2/5 results in a downregulation of a paracrine growth factor regulating XTcf-4 expression. We found that the growth factor for this non-cell-autonomous effect of en2 and pax2/5 is wnt-1 acting on the -1437 Lef/Tcf binding site on the XTcf-4 promoter. We provide evidence that the main nuclear wnt transducer for the autoregulation of XTcf-4 is XTcf-1.
Subject(s)
PAX2 Transcription Factor/metabolism , PAX5 Transcription Factor/metabolism , TCF Transcription Factors/metabolism , Transcription Factors/metabolism , Xenopus Proteins/metabolism , Xenopus/embryology , Animals , Animals, Genetically Modified , Binding Sites/genetics , Body Patterning/genetics , Brain/metabolism , Embryo, Nonmammalian/embryology , Genes, Reporter , Green Fluorescent Proteins/metabolism , In Situ Hybridization , Promoter Regions, Genetic , RNA, Messenger/metabolism , Transfection , Xenopus/genetics , Xenopus/metabolismABSTRACT
The restricted expression of XTcf-4 in the anterior midbrain is regulated via an active wnt/beta-catenin pathway (Kunz et al.,2004, Dev Biol 273:390-401). The molecular mechanism of this autoregulatory loop, however, remained elusive. Here we show that the activity of a 1,775 bp promoter fragment containing a consensus Lef/Tcf binding site at position -1,437 to -1,428 is upregulated by activating transcription factors of the Lef/Tcf family. Furthermore, chromatin immunoprecipitation revealed that endogenous beta-catenin is bound to the Lef/Tcf site on the promoter. Thus, regulation of XTcf-4 by canonical wnt-signaling is directly controlled by binding to and activating a consensus Lef/Tcf binding site within its own promoter.
