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, 38 (6), 1964-81

Alternative Splicing of Tcf7l2 Transcripts Generates Protein Variants With Differential Promoter-Binding and Transcriptional Activation Properties at Wnt/beta-catenin Targets

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Alternative Splicing of Tcf7l2 Transcripts Generates Protein Variants With Differential Promoter-Binding and Transcriptional Activation Properties at Wnt/beta-catenin Targets

Andreas Weise et al. Nucleic Acids Res.

Abstract

Alternative splicing can produce multiple protein products with variable domain composition from a single gene. The mouse Tcf7l2 gene is subject to alternative splicing. It encodes TCF4, a member of the T-cell factor (TCF) family of DNA-binding proteins and a nuclear interaction partner of beta-catenin which performs essential functions in Wnt growth factor signalling. Multiple TCF4 isoforms, potentially exhibiting cell-type-specific distribution and differing in gene regulatory properties, could strongly influence tissue-specific Wnt responses. Therefore, we have examined mouse Tcf7l2 splice variants in neonatal tissues, embryonic stem cells and neural progenitors. By polymerase chain reaction amplification, cloning and sequencing, we identify a large number of alternatively spliced transcripts and report a highly flexible combinatorial repertoire of alternative exons. Many, but not all of the variants exhibit a broad tissue distribution. Moreover, two functionally equivalent versions of the C-clamp, thought to represent an auxiliary DNA-binding domain, were identified. Depending upon promoter context and precise domain composition, TCF4 isoforms exhibit strikingly different transactivation potentials at natural Wnt/beta-catenin target promoters. However, differences in C-clamp-mediated DNA binding can only partially explain functional differences among TCF4 variants. Still, the cell-type-specific complement of TCF4 isoforms is likely to be a major determinant for the context-dependent transcriptional output of Wnt/beta-catenin signalling.

Figures

Figure 1.
Figure 1.
Comparison of expression patterns of TCF4 splice variants in mouse tissues and ES cells. (A) Simplified structural schemes for TCF4 proteins (top) and the mouse Tcf7l2 gene (bottom). The Tcf7l2 gene consists of 17 exons, some of which are subject to alternative splicing (exons 4, 13–16; labelled in red). Constitutive exons are labelled in green. Additional variation of Tcf7l2 mRNA sequences is generated due to the use of alternative splice acceptor and donor sites, respectively, in exons 7, 8 and 9. Affected regions are also indicated by red boxes. The position of the start codon in exon 1 is marked. Which of the potential stop codons following exon 8 or within exons 15, 16 and 17 is used, depends on the particular exon combination present in the final mRNA. Retention of sequences following exon 8 (labelled N) in the final mRNA generates the TCF4N variant. Exon lengths are drawn approximately to scale. Horizontal arrowheads indicate the positions and the orientation of primers used in RT-PCR reactions. Some of the structural features of TCF4 proteins are indicated and linked with connectors to the exons by which they are encoded. Dashed lines skirt the highly variable C-terminal parts of TCF4 derived from exons 13–17. β-cat: β-catenin binding domain; Grg: binding motif for groucho-related gene (Grg) products (73); HMG: HMG box; NLS: nuclear localization signal. (B) Expression analyses of TCF4 splice variants in different mouse tissues and ES cells. Upon isolation of RNA from mouse ES cells and tissue samples from 3-days-old mice, cDNA was synthesized, and TCF4 splice variants were amplified by PCR using exon-specific primer combinations as indicated. Amplification of GAPDH sequences served to control RNA and cDNA integrity. PCR products were separated by gel electrophoresis, and visualized by ethidium bromide staining. To determine the exon combination represented by the various DNA fragments, PCR products were subcloned and sequenced. The deduced size of the PCR products and their assignment to TCF4 E, S and M groups of splice variants is indicated on the right side of the panels. PCR products from brain tissue labelled by asterisks represent isoforms S3 and S6. (C) Summary of the C-terminal TCF4 splice variants identified. Red exons are alternatively spliced whereas green exons are constitutively used. Blue areas indicate untranslated regions. Positions of stop codons used are shown. Splice variants were grouped depending upon whether the resulting translation products contain no C-clamp or complete and incomplete versions thereof, respectively (Figure 2). Accordingly, the isoform formerly denoted S5 (26) was reclassified as M3.
Figure 2.
Figure 2.
Amino acid sequence diversity at the C-terminus of TCF4 generated by alternative splicing. A schematic comparison of the C-terminal amino acid sequences derived from TCF4 splice variants identified in Figure 1 is shown. TCF4 splice variants are denoted, and their particular combination of exons is indicated by the numbers in brackets. Amino acid sequences encoded by alternative and constitutive exons are shaded green and red as before, and exon numbers are shown above the amino acid sequences. Exon 15 is differentially translated when it is preceded by exons 13 or 14, respectively. To facilitate the sequence comparison, amino acid sequences derived from exons 14 and 15, respectively, are either shown as pile up in the same column (variants E1–E4 and S3, 4, 6) or consecutively (variants S1, 2, 8). The four conserved cysteines of the C-clamp are highlighted in dark orange. Variable CRALF and CRARF motives are shaded yellow. White shading indicates amino acid sequences diverging among the splice variants.
Figure 3.
Figure 3.
Structure and expression of TCF4 protein isoforms chosen for functional analyses. (A) Schematic representation of TCF4 protein isoforms E2, E2ex4, E4, S2, and M1. The location of binding domains for β-catenin (β-cat), Grg and CtBP corepressors, the HMG box, and the nuclear localization signal (NLS) are indicated. The presence of a complete or incomplete (ΔC) C-clamp is denoted. The TCF4E2, TCF4E2ex4 and TCF4S2 variants contain a CRARF motif as shown whereas in TCF4E4 a CRALF motif is present. The variant TCF4E2ex4 incorporates additional amino acid sequences derived from exon 4 (orange box). TCF4M1 does not share amino acid sequences with TCF4E and TCF4S variants C-terminally to the NLS as shown by the grey box. (B) Expression analyses of TCF4 isoforms. HEK293 cells were transfected with expression vectors for TCF4E2, TCF4E2ex4, TCF4E4, TCF4M1 and TCF4S2. Control samples received the empty expression vector (lane 1). TCF4 proteins were detected in total cell lysates 40 h after transfection by western blotting. The same blot was subsequently probed with antibodies against α-Tubulin (α-Tub) as loading control. Asterisks label residual TCF4S2 and TCF4M1 signals from the previous detection round. (C) U2-OS cells were transfected with expression vectors for TCF4E2, TCF4E2ex4, TCF4E4, TCF4S2 and TCF4M1, respectively. Control samples received the empty expression vector. TCF4 isoforms were detected by indirect immunofluorescence using a monoclonal antibody to TCF4 and an Alexa-555-labelled secondary antibody. Nuclei were counterstained with DAPI. Phase contrast images (left) and an overlay (right) of Alexa-555 (αTCF4) and DAPI stainings are shown. Bars: 20 µm.
Figure 4.
Figure 4.
TCF4 splice variants differentially activate Wnt/β-catenin target gene promoters. HEK293 cells were cotransfected with combinations of firefly and Renilla luciferase reporter genes, control vector, expression vector for a constitutively active form of β-catenin, and increasing amounts of expression vectors for TCF4 splice variants as indicated. Firefly luciferase expression was driven by promoters from the Wnt/β-catenin target genes Axin2 (A), Cyclin D1 (B), Siamois (C) and Cdx1 (D). Reporter gene activities were determined 40 h post transfection. Bars represent relative luciferase activities (RLA) compared to controls transfected with luciferase reporter plasmids and empty expression vectors only. Average values and standard deviations from at least three independent experiments are given.
Figure 5.
Figure 5.
Differential activation of the Cdx1 promoter in HEK293 cells (A) and mouse embryonic stem (ES) cells (B) by recombinant Wnt3a. Cells were cotransfected with combinations of firefly and Renilla luciferase reporter genes, control vector, expression vector for a constitutively active form of β-catenin, and constructs coding for TCF4E2, TCF1B, TCF1E, LEF1 and TCF3 as indicated. A cDNA construct for the human TCF4E2 variant was used because it is present in the same vector backbone as the other TCF family members. Firefly luciferase expression was driven by the Cdx1 promoter. To induce reporter gene expression by endogenous pathway components transfected cells received 200 ng/ml recombinant Wnt3a (rWnt3a) 16 h prior to harvest. Control cells remained untreated. Reporter gene activities were determined 40 h post transfection. Bars represent relative luciferase activities (RLA) compared to controls transfected with luciferase reporter plasmids and empty expression vectors only. Average values and standard deviations from at least three independent experiments are given. hu: human; m: mouse. (C) Expression of endogenous LEF/TCF proteins in different cell lines. Nuclear extracts from HEK293 cells, ES cells, C17.2 neural progenitors (NPC) and C2C12 myoblasts were used for western blotting and immunodetection of TCF4, TCF1, LEF1 and TCF3. Antibodies against GSK3β were used to monitor equal loading. As a control for antibody specificity, epitope-tagged TCF1E, LEF1, TCF3 and TCF4E were used (controls). For this, HEK293 cells were transfected with expression vectors for HA-tagged LEF/TCF proteins, whole cell lysates were prepared 40 h after transfection, and aliquots containing roughly equal amounts of the reference proteins were analysed in parallel with the nuclear extracts. The presence of the HA-tag accounts for size differences between controls and cellular TCFs. Mw: molecular-weight standard; n.a.: not analysed.
Figure 6.
Figure 6.
TCF4 isoforms differentially bind to Wnt/β-catenin target gene promoters. (A) DNA probes used for EMSAs. Top: The extended consensus TBE sequence derived from CASTing experiments and the 5′-RCCG-3′ motif recognized by the TCF1 C-clamp (24). Nucleotide sequences representing the heptameric TBE core (74,75) and the 5′-RCCG-3′ element are underlined. Promoter fragments from the Cdx1 and Siamois genes are shown schematically. TBEs and their relative positions are indicated. Coordinates of the DNA fragments are given relative to the transcription start site (+1; marked by an arrow at the Siamois promoter). Grey boxes indicate 5′-RCCG-3′ elements in the vicinity of the Cdx1 TBEs. Capital letters: matches between core consensus and the actual TBEs, lower case: mismatches. Asterisks indicate the positions which were mutated in core TBE or 5′-RCCG-3′ motifs. (B–E) In vitro transcribed and translated TCF4 isoforms E2, M1 and S2, or a mock programmed wheat germ extract (C) were used in EMSAs as indicated. A PCR fragment with Siamois promoter sequences (SiaP), or synthetic oligonucleotides harbouring Cdx1 promoter sequences with single, double or triple combinations of TBEs 3′, 3 and 4, were used as probes as schematically indicated on top of each panel. The Cdx1 probes contained all wild-type sequences (WT) or base pair exchanges in every TBE (mTBE) or 5′-RCCG-3′ motif (mCCG) present as indicated. At the Siamois promoter, two different protein amounts were used (black boxes). For supershifts in panel E, a goat polyclonal TCF4 antibody (N-20) was added to the binding reactions (αTCF4). Specific protein::DNA complexes and positions of the unbound DNA probes are marked. Asterisks indicate non-specific bands.
Figure 7.
Figure 7.
Promoter-binding capacity of TCF4 isoforms in transfected cells. (A) Schemes of the Cdx1 and Siamois (Sia) promoter regions present in the pGL3 basic luciferase vector. Vertical bars mark the location of TBEs. Positions of primers used for quantitative PCR are shown (demi-arrows). Coordinates of primers and DNA fragment end points are given relative to the transcription start sites (+1; arrows) of the Cdx1 and Siamois promoters. DNA sequences used as EMSA probes are marked by gray boxes. At the pGL3 basic backbone positions of primers refer to the parental vector. (B) HEK293 cells were cotransfected with combinations of expression vectors for TCF4 isoforms and reporter gene constructs harbouring Siamois or Cdx1 promoter sequences as indicated. Control samples received the empty expression vector and a promoter-less construct, respectively. To determine promoter occupancy, cells were crosslinked 40 h post transfection and solubilized fragmented chromatin was prepared. ChIP was done with a goat polyclonal antibody against TCF4 (N-20) or non-specific goat control IgGs. Promoter DNA recovered by ChIP was quantified by real-time PCR. Relative enrichments represent the ratio of DNA amounts obtained by specific versus non-specific precipitations. Results from at least three independent experiments and the corresponding standard deviations are shown. (C) Abundance of TCF4 splice variants in solubilized fragmented chromatin from transfected HEK293 cells and their recovery by ChIP. Levels of TCF4 splice variants were monitored by western blotting using fractions of the input chromatin and of the material immunoprecipitated by either goat control IgGs or the goat polyclonal anti-TCF4 antibody N-20. Detection was performed with the mouse monoclonal anti-TCF4 antibody 6H5-3 and appropriate peroxidase-conjugated secondary antibodies. Results from one representative experiment are shown.

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