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A transcriptional response to Wnt protein in human embryonic carcinoma cells.

Willert J, Epping M, Pollack JR, Brown PO, Nusse R - BMC Dev. Biol. (2002)

Bottom Line: The promoters of nearly all the target genes we identified have putative TCF binding sites, and we show that the TCF binding site is required for induction of Follistatin.Several of the target genes have a cooperative response to a combination of Wnt and BMP.Wnt signaling activates genes that promote stem cell fate and inhibit cellular differentiation and regulates a remarkable number of genes involved in its own signaling system.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Developmental Biology, Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305 USA. jennifer.willert@cox.net

ABSTRACT

Background: Wnt signaling is implicated in many developmental decisions, including stem cell control, as well as in cancer. There are relatively few target genes known of the Wnt pathway.

Results: We have identified target genes of Wnt signaling using microarray technology and human embryonic carcinoma cells stimulated with active Wnt protein. The ~50 genes upregulated early after Wnt addition include the previously known Wnt targets Cyclin D1, MYC, ID2 and betaTRCP. The newly identified targets, which include MSX1, MSX2, Nucleophosmin, Follistatin, TLE/Groucho, Ubc4/5E2, CBP/P300, Frizzled and REST/NRSF, have important implications for understanding the roles of Wnts in development and cancer. The protein synthesis inhibitor cycloheximide blocks induction by Wnt, consistent with a requirement for newly synthesized beta-catenin protein prior to target gene activation. The promoters of nearly all the target genes we identified have putative TCF binding sites, and we show that the TCF binding site is required for induction of Follistatin. Several of the target genes have a cooperative response to a combination of Wnt and BMP.

Conclusions: Wnt signaling activates genes that promote stem cell fate and inhibit cellular differentiation and regulates a remarkable number of genes involved in its own signaling system.

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Related in: MedlinePlus

Response of a reporter containing the Follistatin promoter to Wnt-3A protein in NCCIT cells [37]. The Follistatin promoter [36] contains one putative TCF binding site (CTTTGAT). This promoter linked to luciferase was transfected in NCCIT cells, which were then exposed to Wnt-3A CM and CCM for 8 hours. This assay showed a significant increase in activity when cells were stimulated directly with Wnt-3A CM or co-transfected with activated β-catenin (not shown). This effect was abrogated by co-transfection of dominant-negative TCF-4 or axin (not shown). When the TCF site within the Follistatin promoter was mutated into CATCGAT, the Wnt-3A response was abolished.
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Figure 5: Response of a reporter containing the Follistatin promoter to Wnt-3A protein in NCCIT cells [37]. The Follistatin promoter [36] contains one putative TCF binding site (CTTTGAT). This promoter linked to luciferase was transfected in NCCIT cells, which were then exposed to Wnt-3A CM and CCM for 8 hours. This assay showed a significant increase in activity when cells were stimulated directly with Wnt-3A CM or co-transfected with activated β-catenin (not shown). This effect was abrogated by co-transfection of dominant-negative TCF-4 or axin (not shown). When the TCF site within the Follistatin promoter was mutated into CATCGAT, the Wnt-3A response was abolished.

Mentions: In the Wnt target gene promoters that are functionally mapped (MSX1, MSX2, ID2, ID3, Follistatin, REST/NRSF, Versican) we invariably found TCF binding sites. Moreover, we found that a luciferase reporter gene, placed under the control of the Follistatin promoter, was activated by Wnt-3A protein added to transiently transfected NCCIT cells (Figure 5). Mutating the single TCF binding site on the Follistatin promoter eliminated the response. These data suggest that Follistatin is activated directly through the Wnt signal transduction pathway and not by another pathway. Because the kinetics of Follistatin activation by Wnt are similar to that of the other target genes (Figure 2A), we suggest that there are no "earlier" targets, i.e. all identified targets are direct.


A transcriptional response to Wnt protein in human embryonic carcinoma cells.

Willert J, Epping M, Pollack JR, Brown PO, Nusse R - BMC Dev. Biol. (2002)

Response of a reporter containing the Follistatin promoter to Wnt-3A protein in NCCIT cells [37]. The Follistatin promoter [36] contains one putative TCF binding site (CTTTGAT). This promoter linked to luciferase was transfected in NCCIT cells, which were then exposed to Wnt-3A CM and CCM for 8 hours. This assay showed a significant increase in activity when cells were stimulated directly with Wnt-3A CM or co-transfected with activated β-catenin (not shown). This effect was abrogated by co-transfection of dominant-negative TCF-4 or axin (not shown). When the TCF site within the Follistatin promoter was mutated into CATCGAT, the Wnt-3A response was abolished.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC117803&req=5

Figure 5: Response of a reporter containing the Follistatin promoter to Wnt-3A protein in NCCIT cells [37]. The Follistatin promoter [36] contains one putative TCF binding site (CTTTGAT). This promoter linked to luciferase was transfected in NCCIT cells, which were then exposed to Wnt-3A CM and CCM for 8 hours. This assay showed a significant increase in activity when cells were stimulated directly with Wnt-3A CM or co-transfected with activated β-catenin (not shown). This effect was abrogated by co-transfection of dominant-negative TCF-4 or axin (not shown). When the TCF site within the Follistatin promoter was mutated into CATCGAT, the Wnt-3A response was abolished.
Mentions: In the Wnt target gene promoters that are functionally mapped (MSX1, MSX2, ID2, ID3, Follistatin, REST/NRSF, Versican) we invariably found TCF binding sites. Moreover, we found that a luciferase reporter gene, placed under the control of the Follistatin promoter, was activated by Wnt-3A protein added to transiently transfected NCCIT cells (Figure 5). Mutating the single TCF binding site on the Follistatin promoter eliminated the response. These data suggest that Follistatin is activated directly through the Wnt signal transduction pathway and not by another pathway. Because the kinetics of Follistatin activation by Wnt are similar to that of the other target genes (Figure 2A), we suggest that there are no "earlier" targets, i.e. all identified targets are direct.

Bottom Line: The promoters of nearly all the target genes we identified have putative TCF binding sites, and we show that the TCF binding site is required for induction of Follistatin.Several of the target genes have a cooperative response to a combination of Wnt and BMP.Wnt signaling activates genes that promote stem cell fate and inhibit cellular differentiation and regulates a remarkable number of genes involved in its own signaling system.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Developmental Biology, Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305 USA. jennifer.willert@cox.net

ABSTRACT

Background: Wnt signaling is implicated in many developmental decisions, including stem cell control, as well as in cancer. There are relatively few target genes known of the Wnt pathway.

Results: We have identified target genes of Wnt signaling using microarray technology and human embryonic carcinoma cells stimulated with active Wnt protein. The ~50 genes upregulated early after Wnt addition include the previously known Wnt targets Cyclin D1, MYC, ID2 and betaTRCP. The newly identified targets, which include MSX1, MSX2, Nucleophosmin, Follistatin, TLE/Groucho, Ubc4/5E2, CBP/P300, Frizzled and REST/NRSF, have important implications for understanding the roles of Wnts in development and cancer. The protein synthesis inhibitor cycloheximide blocks induction by Wnt, consistent with a requirement for newly synthesized beta-catenin protein prior to target gene activation. The promoters of nearly all the target genes we identified have putative TCF binding sites, and we show that the TCF binding site is required for induction of Follistatin. Several of the target genes have a cooperative response to a combination of Wnt and BMP.

Conclusions: Wnt signaling activates genes that promote stem cell fate and inhibit cellular differentiation and regulates a remarkable number of genes involved in its own signaling system.

Show MeSH
Related in: MedlinePlus