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First published online 26 May 2004
doi: 10.1242/dev.01176

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Sox17 and ß-catenin cooperate to regulate the transcription of endodermal genes

Débora Sinner^*, Scott Rankin^*, Monica Lee and Aaron M. Zorn

Cincinnati Children’s Hospital Medical Center, Division of Developmental Biology and The Department of Pediatrics, University of Cincinnati College of Medicine, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA

View larger version (41K): [in a new window]   Fig. 3. Structure-function mapping of the transactivation domain of Sox17ß. (A) The Gal4 DNA-binding domain (Gal4DBD) was fused to various parts of the Sox17ß open reading frame. COS-1 cells were co-transfected with the indicated Gal4-fusion constructs (300 ng), UAS:luciferase reporter (100 ng) containing five Gal4 binding sites and a pTK-Renilla Luciferase plasmid (50 ng). The average relative luciferase activity normalized to renilla activity, from a triplicate experiment is shown. The Gal4 DNA-binding domain alone (Gal4DBD) is a negative control and Gal4:VP16 is a positive control containing the viral VP16 transactivation domain. (B) 200 pg of RNA encoding the indicated HA-tagged Sox17ß deletion constructs was injected into two-cell stage embryos, animal cap tissue was isolated at blastula stage, cultured until gastrula stage and assayed by real-time RT-PCR for the expression of Sox17 target genes. The histogram shows the relative gene expression normalized to the loading control ODC. (C) The schematic shows the transactivation domain of Sox17ß contains a sequence motif conserved in all members of the SoxF subfamily of Sox proteins. A sequence alignment of this conserved motif from representative proteins is shown. Identical amino acids are white on black, conserved resides are in bold. Below the schematic the ‘3G’ and ‘TA’ mutations generated in both Sox17 and Sox17ß are shown. (D) 200 pg of RNA encoding either wild-type or mutant versions of Sox17ß (top three constructs) or Sox17 (bottom three constructs) were injected into two-cell stage embryos, animal cap tissue was isolated at blastula stage, cultured until gastrula stage and assayed by real-time RT-PCR for the expression of Sox17 target genes. The histogram shows the relative gene expression normalized to the loading control ODC.

View larger version (38K): [in a new window]   Fig. 1. Transcriptional targets of Sox17ß. Embryos were injected at the two-cell stage with increasing doses mRNA of encoding Sox17ß (62 pg, 125 pg, 250 pg, 500 pg), animal cap explants were isolated at blastula (stage 9), cultured until gastrula (stage 11) and assayed for endodermal gene expression by RT-PCR. None of the endodermal genes was expressed in uninjected animal cap tissue (–), but all were expressed in gastrula whole embryos. Ef1, is a loading control; –RT, without reverse transcriptase; +RT, with reverse transcriptase.

View larger version (92K): [in a new window]   Fig. 2. Direct targets of Sox17ß. (A) A schematic of the GR:Sox17ß fusion protein consisting of the hormone-binding domain of the human glucocorticoid receptor fused to Sox17ß. (B) Isolated blastula animal cap explants either injected with mRNA encoding GR:Sox17ß (150 pg) or uninjected were each cultured in three conditions: 1xMBS, 1xMBS + 10–6 dexamethasone (DEX) or in 1xMBS with 10–6 dexamethasone + 10 µg/ml cycloheximide (CHX). Dexamethasone activates the GR:Sox17ß fusion protein and cycloheximide blocks translation. Control animal caps were treated with 5 ng/ml human activin A either with or without 10 µg/ml cycloheximide. At gastrula (stage 11) the explants were assayed by RT-PCR. GR:Sox17ß induced Hnf1ß, Foxa1, Foxa2 and Sox17 transcription when translation was blocked and are therefore direct Sox17 targets. A repeat of this experiment assayed by real-time RT-PCR is presented in Figs S1-S3 at http://dev.biologists.org/supplemental. (C) Whole-mount in situ hybridization to bisected gastrula (stage 11) embryos with the indicated probes confirms that Sox17 target genes are expressed in the endoderm. Dorsal/anterior towards the left.

View larger version (41K): [in a new window]   Fig. 4. Interaction of endogenous ß-catenin and Sox17. (A) A 160 µM mid-sagittal optical z-series of anti-ß-catenin immunofluorescence in a Xenopus midgastrula shows both membrane bound and nuclear ß-catenin in the deep endodermal cells. The intense staining throughout the animal cap and mesoderm is the result of the cells in this region of the embryo being much smaller than in the endoderm (therefore the optical stack is several cells thick in these regions, resulting in an almost uniform staining). The right panel shows a higher magnification of the endoderm (white box) with the stained nuclei clearly visible. Dorsal/anterior towards the left. (B) Western blotting of whole cell, cytosolic and nuclear extracts (10 µg of protein each) from human SW480 colorectal cancer cells with anti-tubulin (cytosol antigen), anti-histone H1 (nuclear antigen), anti-Sox17 and anti-ß-catenin antibodies. SW480 cells express both endogenous Sox17 and ß-catenin in the nuclear fraction. (C) After immunoprecipitation of the nuclear extract with either anti-Sox17 or anti-HA (as a negative control) antibodies, associated ß-catenin protein was detected by western blotting. Endogenous ß-catenin co-immunoprecipitated with nuclear Sox17. The precipitation of Sox17/ß-catenin complexes can be competed by the addition of Sox17 peptide recognized by the anti-Sox17 antibody but not by peptides to other regions of Sox17.

View larger version (36K): [in a new window]   Fig. 5. The transactivation domain of Sox17ß is required for ß-catenin binding. COS-1 cells were transfected with (A) 3 µg of DNA encoding the indicated V5-epitope tagged Sox17ß constructs or (B) 3 µg of DNA encoding the indicated V5-epitope tagged Sox17 constructs. The resulting cell extracts were either incubated with GST-agarose or GST-ß-catenin-agarose. The input and bound V5-tagged proteins were visualized by anti-V5 immunoblotting. Mutations or deletion of the conserved transactivation motif in either Sox17ß or Sox17 impairs or abolishes ß-catenin binding.

View larger version (16K): [in a new window]   Fig. 6. Sox17 and ß-catenin co-operate to transcribe endodermal genes. Embryos were injected at the 2-cell stage with Sox17ß mRNA (20 pg, 60 pg, 180 pg) either with or without co-injection of RNA encoding a stabilized ß-catenin (pt-ß-catenin, 100 pg) (Yost et al., 1996). At blastula stage, animal cap tissue was explanted and cultured for 3-4 hours until gastrula stage when it was assayed by real-time RT-PCR for the expression of Sox17 target genes. The histograms show the relative expression levels normalized to the loading control ODC. Plakoglobin (Plako) is control gene that is neither a target of Sox17 nor ß-catenin.

View larger version (26K): [in a new window]   Fig. 7. Sox17ß requires ß-catenin to robustly activate target gene transcription in animal caps. (A) Embryos were injected at the two-cell stage with the indicated combinations of: Sox17ß mRNA (200 pg), RNA encoding a N-terminal deleted form of stabilized ß-catenin (N-ß-catenin, 100 pg) (Yost et al., 1996), or an antisense ß-catenin morpholino oligos (ßcat-MO; 20 ng). At blastula stage, animal cap tissue was explanted and cultured for 3-4 hours until gastrula stage, when it was assayed by real-time RT-PCR for the expression of Sox17 target genes. The histograms show the relative expression levels normalized to the loading control, ODC. Plakoglobin (Plako) is control gene that is neither a Sox17 nor ß-catenin target. (B) A proportion of each sample from the same experiment was assayed by immunoblotting with anti-ß-catenin, anti-Sox17ß or anti-tubulin antibodies. Injected N-ß-catenin protein has a higher molecular weight than endogenous ß-catenin because of the presence of an epitope tag. Tubulin is a loading control.

View larger version (43K): [in a new window]   Fig. 8. ß-catenin is required for the expression of Sox17 targets genes at gastrula stage. (A) Two-cell embryos were vegetally injected with either 20 ng of antisense ß-catenin morpholino oligos (MO) and/or 100 pg of RNA encoding a N-terminal deleted form of stabilized ß-catenin (N-ß-cat) (Yost et al., 1996), which does not contain the sequence targeted by the antisense oligo. At a series of stages throughout gastrulation (stages 10, 11 and 12) whole embryos were harvested and assayed by real-time RT-PCR for the expression of Sox17 target genes as well as several other control genes. The histograms show the relative expression levels normalized to the loading control, ODC. For simplicity only the stage 11 data (stage 10 for Xnr1, Xnr2, Xnr4 and Derriere) is shown. Edd, Hnf1ß, Foxa1 and Foxa2 are direct Sox17 target genes. Siamois, Hex and Cerberus are known ß-catenin target genes. Xnr1, Xnr2, Xnr4, Derreire and Mixer are endodermal genes that are not Sox17 targets. Plakoglobin (Plako) and Ef1 are control genes that are neither Sox17 nor ß-catenin targets. (B) A proportion of each sample from the same experiment was assayed by immunoblotting with either anti-ß-catenin or anti-tubulin antibodies. Injected N-ß-catenin protein has a higher molecular weight than endogenous ß-catenin because of the presence of an epitope tag.

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