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Supplementary Materials for
The E3 ligase RNF43 inhibits Wnt signaling downstream of mutated catenin by sequestering TCF4 to the nuclear membrane
Anke Loregger, Martina Grandl, Raquel Mejías-Luque, Michael Allgäuer,
Kathrin Degenhart, Verena Haselmann, Christina Oikonomou, Pantelis Hatzis,
Klaus-Peter Janssen, Ulrich Nitsche, Dietmar Gradl, Olaf van den Broek, Olivier Destree,
Kurt Ulm, Michael Neumaier, Behnam Kalali, Andreas Jung, Ignacio Varela,
Roland M. Schmid, Roland Rad, Dirk H. Busch, Markus Gerhard*
*Corresponding author. E-mail: [email protected]
Published 8 September 2015, Sci. Signal. 8, ra90 (2015)
DOI: 10.1126/scisignal.aac6757
The PDF file includes:
Fig. S1. RNF43 expression.
Fig. S2. RNF43 subcellular localization.
Fig. S3. RNF43 is heterogeneously expressed in stage II colon tumors.
Fig. S4. RNF43 is a Wnt target gene.
Fig. S5. RNF43 inhibits Wnt signaling in vivo.
Fig. S6. Mode of action of RNF43-mediated Wnt inhibition.
Fig. S7. Model of RNF43 mode of action and RNF43 mutations.
Fig. S8. RNF43 overexpression does not affect cell viability.
Fig. S1. RNF43 expression. (A) Rnf43 mRNA expression in murine small intestine as well as
in adenomas and tumors of APCmin mice detected by in situ hybridization. Rnf43 expression
in murine small intestine is restricted to a limited number of cells, being stronger at the socalled “+4 position”, where a more quiescent stem cell type population is located, as well as
in crypt base columnar cells (left). High expression of Rnf43 was detected in tumors of
APCmin mice (right). Scale bars, 100 µm. (B) In situ hybridization showing RNF43 mRNA
expression in aberrant crypt foci and small intestine of adult APCmin mice. Scale bars, 100
µm. (C) RNF43 mRNA expression in organs of adult mice. RT-PCR was performed two
times in duplicates. Data shown are means+SD of duplicates of one representative RT-PCR.
(D) Expression of RNF43 at different embryonic stages. Mouse embryos at 8.5 dpc showed
Rnf43 in a posterior expression pattern, which is typical for Wnt target genes. At 9.5 dpc,
Rnf43 was expressed in the developing stomach and intestine. Furthermore, a strong staining
in the trunk region was observed. (E) mRNA expression in embryonic murine tissue from
14.5 dpc and 18.5 dpc. RT-PCR was performed two times in duplicates. Data shown are
means+SD of duplicates of one representative RT-PCR. (F) RNF43 expression detected by
immunocytochemistry in HT-29 shcontrol or shRNF43 cells. Scale bar 10µm.
Fig. S2. RNF43 subcellular localization. (A) RNF43-HA localizes at the nuclear envelope.
HCT116 cells were transiently transfected with wild-type RNF43-HA and stained for
confocal immunofluorescence imaging. Scale bar, 10 µm. (B) RNF43 co-localizes with
Lamin B receptor and Calnexin at the nuclear membrane as detected by confocal
immunofluorescence. Scale bars, 10 µm. (C) RNF43 co-localizes with PSF at the nuclear
envelope. SW480 cells were transiently transfected with wild-type RNF43 or mutant RNF43H292R and PSF constructs and stained for confocal immunofluorescence imaging. Scale bar,
10 µm.
Fig. S3. RNF43 is heterogeneously expressed in stage II colon tumors. RNF43 mRNA
expression in stage II colon tumor samples analyzed by quantitative PCR. mRNA levels were
normalized to that of GADPH.
Fig. S4. RNF43 is a Wnt target gene. (A) RNF43 gene displaying putative TCF4 binding
sites. (B) ChIP assays showing binding of TCF4 and β-catenin to the enhancing region
(Intenh) of RNF43 located at chr17:53,823,655-53,824,478 (hg18 assembly on UCSC
genome browser) in LS174T cells. (C) Luciferase assay from TCF4 and RNF43 promoter
/enhancer regions containing putative TCF4 binding sites (n=3). (D) Relative RNF43 mRNA
levels after shRNA knockdown in HT-29 colon cancer cells (n=3). (E) Relative RNF43
mRNA levels after siRNA knockdown in HT-29 colon cancer cells (n=2). (F) TCF
transcriptional activity after knocking down wild type RNF43 in HT-29 colon cancer cells
(n=2). **p ≤ 0.01. Student’s t-test or ANOVA for multiple comparison test were performed.
Fig. S5. RNF43 inhibits Wnt signaling in vivo. (A) WNT1 mRNA was injected into ventral
blastomeres of a 4-cell X. laevis embryo alone (n=20) or in combination with wild-type
RNF43 (n=14) or RNF43-H292R (n=20) mutant. The inhibitory activity of RNF43 was
already apparent at an early developmental stage just after gastrulation. (B) Phenotypical
changes detected in X. laevis embryos injected with mRNA encoding wild-type or mutant
RNF43. Embryos injected with RNF43 mRNA alone exhibited a loss of posterior structures,
while embryos injected with the mutant RNF43-H292R mRNA were phenotypically normal at
later stages of development. Representative images are shown. (C) Expression of the
molecular markers chordin, which is a dorsalizing factor, and MyoD, a Wnt target gene
expressed in cells derived from the ventral-lateral portion, after injection of X. laevis embryos
with wild-type or mutant RNF43. A lower amount of MyoD mRNA was detected upon
RNF43 injection. Chordin staining at the dorsal marginal zone was reduced in RNF43 mRNAinjected embryos as compared to the non-injected control embryos. In embryos injected with
RNF43-H292R mRNA, the signal of chordin staining was extended laterally along the
marginal zone, indicating increased or accelerated dorsalizing activity. (figure S5 continued
next page)
Fig. S5, continued: (D) Amino acid sequence alignment of the RNF43 protein sequence of
different species: Homo sapiens, Mus musculus, Rattus norvegicus, Bos Taurus, Equus
caballus, Pan troglodytes, Loxodonta Africana and Xenopus tropicalis. IBUVU PRALINE
sequence alignment program (http://www.ibi.vu.nl/programs/pralinewww/) was used.
Residues highlighted in red represent identical residues, similar residues in orange and green
and non-conserved residues appear in blue.
A very high conservation (over 80%) is
noticeable over the entire sequence of the protein, while 41.17% identity was detected
between human and Xenopus tropicalis RNF43.
Fig. S6. Mode of action of RNF43-mediated Wnt inhibition. (A) TCF luciferase reporter
assay after expression of RNF43 wild type or mutant RNF43H292R in the presence of the
porcupine inhibitor LGK974. Results normalized to renilla values from two independent
experiments performed in duplicates are shown as mean ± S.D. (B) NF-kB reporter assay
performed in HCT116 cells after transfection of RNF43 wild type or the ring domain mutant
H292R. Cells were stimulated with 20ng/ml of TNFα. Results from three independent
experiments expressed as mean ± S.D. are shown. (C) HCT116 cells were transiently
transfected with a plasmid expressing RNF43-HA or RNF43H292R-HA. Whole cell lysates
were subjected to immunoprecipitation with anti-HA antibody or mouse IgG as a control. The
immunoprecipitates were analyzed by Western blot using the indicated antibodies. (D)
RNF43 interacts with PSF. HCT116 cells were transfected with RNF43-Flag and PSF-HA
and immunoprecipitation experiments were performed using the indicated antibodies. (E)
Truncated N-terminal RNF43-245-783 and TCF4-HA as well and TCF4-HA expression in
HCT116 cells. Scale bar, 10 µm. (F) TOP/FOP luciferase reporter assay in DLD1 cells
transfected with wild type RNF43 and RNF43-245-783. Results were normalized to renilla
values. Data are shown as mean± S.D. of five independent experiments. (G) TCF luciferase
reporter assay after expression of RNF43-R437A-K655A lacking the NLS. Results
normalized to renilla values from three independent experiments are shown as mean ± S.D.
(H) TCF4 subcellular localization detected by immunofluorescence in HT-29 shcontrol and
HT-29 shRNF43 cells. Scale bar, 10 µm. (I) HCT116 cells were transfected with RNF43 and
the expression of LGR5 and MMP7 was assessed by qPCR. Data from three replicates are
shown. Student’s t-test or ANOVA for multiple comparisons were performed. *p≤ 0.05; **p
≤ 0.01 ***; p < 0.005.
Fig. S7. Model of RNF43 mode of action and RNF43 mutations. (A) RNF43 mutations
described in the literature. The x-axis shows RNF43 amino acid sequence, while the number
of samples showing a specific mutation is shown on the y-axis (mutation hits). (B) Proposed
model of Wnt signaling regulation by RNF43. Wild type RNF43 tethers the TCF4
transcription complex to the nuclear membrane, preventing its transcriptional activity.
Mutations in RNF43 result in an impaired binding to TCF4, which remains in the
nucleoplasm to activate Wnt signaling.
Fig. S8. RNF43 overexpression does not affect cell viability. (A) Representative pictures of
HCT116 cells transfected for 24 hours (upper panel) or 48 hours (lower panel) with the
indicated amounts of wild type and mutant RNF43-H292R. Images were obtained using
Nikon DS-L3 camera control unit at 10x magnification. (B) HCT116 cells were transfected
with the indicated plasmids for 48 hours and cell viability was measured by MTT cell
viability assay.