Native promoter reporters validate transcriptional targets.
The transcriptional activator beta-catenin is the key mediator of the canonical Wnt signaling pathway. However, beta-catenin does not itself bind DNA, but functions via interaction with T-cell factor (TCF)/ lymphoid-enhancing factor (LEF) transcription factors. These proteins contain a high-mobility group (HMG) box that binds DNA in a sequence-specific manner. Thus, in the case of active Wnt signaling, beta-catenin activates, in cooperation with proteins of the TCF/LEF family, the expression of a wide variety of genes. To date, the list of established Wnt targets is far from complete. The establishment of plasmids harbouring reporter genes under control of the native promoter sequences provides a tool to validate novel putative Wnt targets by directly quantifying the beta-catenin-dependent activation of each specific gene. In this chapter, we describe how to generate such reporter plasmids using the MMP7 promoter as an example.
Schmalhofer
Otto
O
Department of Surgery, University of Freiburg, Germany.
Spaderna
Simone
S
Brabletz
Thomas
T
D001483:Base Sequence; D030561:Databases, Nucleic Acid; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D020783:Matrix Metalloproteinase 7 / Q000235:genetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D015203:Reproducibility of Results; D015398:Signal Transduction / Q000502:physiology; D051820:TCF Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D051153:Wnt Proteins / Q000235:genetics / Q000378:metabolism; D051176:beta Catenin / Q000235:genetics / Q000378:metabolism
D051820:TCF Transcription Factors; D051153:Wnt Proteins; D051176:beta Catenin; D020783:Matrix Metalloproteinase 7