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Characterization and functional analysis of the 5′-flanking promoter region of the mouse Tcf3 gene

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Abstract

Tcf3 is a nuclear mediator of canonical Wnt signaling which functions in many systems as a repressor of target gene transcription. In this study, we have cloned and characterized a 6.7 kb fragment of the 5′-flanking promoter region of the mouse Tcf3 gene. In silico analysis of the promoter sequence identified the existence of GC boxes and CpG islands, but failed to identify any TATA box. In addition, the promoter sequence contained putative binding sites for multiple transcription factors, including a few known to regulate the function of mTcf3. Of those, we confirmed functional binding sites for NFκB and Oct1 using a luciferase assay and ChIP. In vitro analysis revealed five potential transcription start sites which resulted in a 298 base pair 5′-untranslated region upstream of the mTcf3 translation start site ATG. Using a luciferase assay, we analyzed the activity of the cloned promoter fragment in embryonically derived neural stem cells. The luciferase activity of a 3.5 kb core promoter fragment (−3243/+211) showed up to 40-fold increased activity compared to the empty vector. Addition of sequences 5′ to the 3.5 kb core promoter fragment resulted in a 20-fold drop in luciferase activity, indicating the presence of further upstream repressive elements. In vivo analysis of a 4.5 kb promoter fragment (−4303/+211) driving, the expression of EGFP in mouse embryos highly resembled endogenous expression of mTcf3.

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Acknowledgment

This research was funded by the Norwegian Research Council (Grant no. 174938/130). The authors thank Monika Gelazauskaite for excellent technical help with cloning.

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Authors and Affiliations

  1. Unit for Cell Signaling, Department of Microbiology, Oslo University Hospital, Rikshospitalet, Forskningsparken, Gaustadalléen 21, 0349, Oslo, Norway

    Nina Solberg, Ondrej Machon & Stefan Krauss

  2. SFI-CAST Innovation Center, Gaustadalléen 21, 0349, Oslo, Norway

    Nina Solberg, Ondrej Machon & Stefan Krauss

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  1. Nina Solberg
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  2. Ondrej Machon
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  3. Stefan Krauss
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Corresponding author

Correspondence to Nina Solberg.

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Supplementary Fig. 1

Identification of transcriptional start sites using 5′RACE. (a) Outer and inner PCR on mouse embryo RNA and mouse thymus RNA. Outer and inner positive control for 5′RACE on mouse embryo RNA is shown with gene specific primers to the right of the second ladder. Positive and negative control for 5′RACE on mouse thymus RNA is seen to the right. The inner Race PCR products were further cloned into the pCR-Blunt vector. (b) Ten colonies from mouse embryo RNA derived clones were randomly picked and analyzed with EcoRI restriction enzyme before sequencing. Clone ten was discarded due to the appearance of two bands. (c) Representative M13 fwd/rev PCR product analysis of 35 clones derived from mouse thymus RNA. Together these clones revealed five transcription start sites which are indicated with bold letters in Fig. 2. (TIFF 4318 kb)

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Solberg, N., Machon, O. & Krauss, S. Characterization and functional analysis of the 5′-flanking promoter region of the mouse Tcf3 gene. Mol Cell Biochem 360, 289–299 (2012). https://doi.org/10.1007/s11010-011-1068-y

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  • DOI: https://doi.org/10.1007/s11010-011-1068-y

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