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The transcription factor GATA6 enables self-renewal of colon adenoma stem cells by repressing BMP gene expression

Abstract

Aberrant activation of WNT signalling and loss of BMP signals represent the two main alterations leading to the initiation of colorectal cancer (CRC). Here we screen for genes required for maintaining the tumour stem cell phenotype and identify the zinc-finger transcription factor GATA6 as a key regulator of the WNT and BMP pathways in CRC. GATA6 directly drives the expression of LGR5 in adenoma stem cells whereas it restricts BMP signalling to differentiated tumour cells. Genetic deletion of Gata6 from mouse colon adenomas increases the levels of BMP factors, which signal to block self-renewal of tumour stem cells. In human tumours, GATA6 competes with β-catenin/TCF4 for binding to a distal regulatory region of the BMP4 locus that has been linked to increased susceptibility to development of CRC. Hence, GATA6 creates an environment permissive for CRC initiation by lowering the threshold of BMP signalling required for tumour stem cell expansion.

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Figure 1: The transcription factor GATA6 directly regulates LGR5 expression.
Figure 2: GATA6 is the only GATA factor expressed in colon.
Figure 3: Gata6 deletion in an Apc-null background led to prolonged survival, lower tumour burden and a decrease in LGR5-positive cells in colon adenomas.
Figure 4: Gata6 deletion in colon adenoma cells resulted in increased BMP signalling.
Figure 5: BMP inhibitors rescued the morphological, proliferative and clonogenic potential of Gata6-null tumour organoids.
Figure 6: In vivo treatment of mice with BMP inhibitor LDN-193189 increased tumour burden and rescued Gata6-null phenotype.
Figure 7: Regulation of BMP4 gene expression by GATA6 through distal enhancer elements.
Figure 8: GATA6 inhibited β-catenin/TCF4 binding to the BMP4 R.31 enhancer.

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Acknowledgements

We thank H. Clevers and J. Schuijers for help interpreting the β-catenin/TCF4 ChIP-seq data, M. Beato for support, G. Stassi for tumour organoid material, the IRB Barcelona Biostatistics Unit for analysis of the genomics and transcriptomics data, E. Carrillo (CNIO) for bioinformatic support, the IRB Barcelona Functional Genomics Core Facility for technical assistance in microarray hybridization experiments, B. Dominguez for assistance with histology and the Batlle laboratory for discussions and support. This work has been supported by grants to E.B. from the European Research Council (FP7-EU), Ministerio de Economía y Competitividad (OncoBIO Consolider, Plan Nacional and RTICC) and The Josef Steiner Foundation, and by grants to F.X.R. (SAF2011-29530, OncoBIO Consolider from Ministerio de Ciencia e Innovación, and Red Temática de Investigación Cooperativa en Cáncer—RTICC).

Author information

Authors and Affiliations

Authors

Contributions

E.B. and G.W. conceived the project and G.W. generated most key data and prepared figures. E.M. carried out BMP and WNT analysis on adenoma organoids and mice. P.M. and F.X.R. carried out ChIP and ChIP-seq experiments. M.S. and X.H-M. gave technical support in histological and mouse work respectively. A.S.N. and G.P.V. carried out 3C experiments and analysis of chromatin marks. A.A., S.C-B. and A.C. provided patient material and carried out linkage disequilibrium analysis. C.S-O.A. did bioinformatic and statistical analysis. A.C., C.C. and P.J. gave support in multiple experiments including molecular biology and experiments with organoids. E.S. and E.B. wrote the manuscript.

Corresponding author

Correspondence to Eduard Batlle.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Gata6 deficiency in colonic adenomas decreased levels of the ISC marker genes EPHB3 and LRIG1.

Representative serial immunohistochemical staining of LGR5 (GFP), EPHB3 and LRIG1. Red dashed lines delineate normal (N) and adenoma (Ad) tissue. Cells expressing high levels of these three marker genes were positioned at the base of tumour glands (black arrowheads) in Gata6 wt (left) adenomas. Notably, the number of cells expressing these markers was markedly reduced in Apc/Gata6 compound mutants (right). Images are representative of n > 50 murine colon adenomas observed in 8 animals/genotype. Scale bars in panels are 25 μm.

Supplementary Figure 2 Conditional deletion of Gata6 using the Villin CreERT2 driver resulted in proliferative defects and loss of AdSC markers LGR5 and CLDN2.

(a) Quantification of proliferating cells in the colon of VillinCreERT2Gata6+/+Apcfl/fl (purple bars) and VillinCreERT2Gata6fl/flApcfl/fl (red bars) using phospho-histone-H3 (pHH3). Data are mean ± s.e.m. of positive cells per crypt in n = 150 crypts counted in 6 mice/genotype. *P < 0.05 by Student’s t-test. (b) Representative staining of LGR5 (GFP) from VillinCreERT2Lgr5eGFPCreERT2Gata6+/+Apcfl/fl (left) and VillinCreERT2Lgr5eGFPCreERT2Gata6fl/flApcfl/fl (right) mice. Black dashed lines delineate AdSC zone. The size of the AdSC zone is reduced in compound mutants (dashed lines). Note the mosaic expression of the Lgr5–GFP transgene in the colon. (c) Representative staining of β-catenin and CLDN2 in colonic crypts from VillinCreERT2Gata6+/+Apc+/+ (top), VillinCreERT2Gata6+/+Apcfl/fl (middle) and VillinCreERT2Gata6fl/flApcfl/fl (bottom) mice 4 days after tamoxifen treatment (Methods). Note the sharp decrease in CLDN2 staining in compound mutants. Images are representative of n > 50 murine colon adenomas observed in 3 mice/genotype. Scale bars in panels are 25 μm.

Supplementary Figure 3 Effects of Gata6 deletion in the small intestine, compared with the colon.

(a,b) Quantification of proliferating cells, using phospho-histone-H3 labeling, in the small intestine (a) and colon (b) of VillinCreERT2Gata6+/+Apcfl/fl (purple bars) and VillinCreERT2Gata6fl/flApcfl/fl (red bars) 4 days after Tamoxifen treatment (Methods). Note that only the colon displayed a significant decrease in proliferating cells on Gata6 deletion. Data are mean ± s.e.m. of positive cells in n = 150 small intestine or colon crypts counted in 6 mice/genotype. *P < 0.05 by Student’s t-test. (c) Epithelial cells from the small intestine of Lgr5eGFPCreERT2Gata6+/+Apcfl/fl and Lgr5eGFPCreERT2Gata6fl/flApcfl/fl mice were disaggregated 4 days post tamoxifen administration and analysed by flow cytometry. Single GFP high (blue), low (red) and negative (green) cells were gated using log GFP fluorescence intensity. (d) Quantification of %GFP high cells from the small intestine and colon of Lgr5eGFPCreERT2Gata6+/+Apcfl/fl (purple bars) and Lgr5eGFPCreERT2Gata6fl/flApcfl/fl (red bars) cells. Data are shown as mean ± s.e.m. of n = 6 mice/genotype. **P < 0.01 by Student’s t-test. (e) Representative staining of LGR5 (GFP), β-catenin and CLDN2 in adenomas from the small intestine of Apc null (top) and compound Apc/Gata6 mutant (bottom) mice. Note that staining for all markers was comparable in the two genotypes. Images are representative of n > 50 murine colon adenomas observed in 3 mice/genotype. Scale bars in panels are 25 μm.

Supplementary Figure 4 Growth deficiency induced by loss of Gata6 is not rescued by overexpression of LGR5 or ectopic activation of the WNT pathway.

(a) Colon tumour organoids from VillinCreERT2Gata6+/+Apcfl/fl and VillinCreERT2Gata6fl/flApcfl/fl mice were either engineered to overexpress LGR5 (right) or were cultured in control media (left) or media supplemented with RSPO1 (1 μg ml−1) plus recombinant WNT3a (100 ng ml−1) (middle panels) every day for 7 days. Note that neither supplementation with RSPO1 + WNT3a nor overexpression of LGR5 restored spheroid shape and size in compound mutant organoids. Pictures are representative images of adenoma organoids observed in 5 independent cultures from each genotype and condition. Scale bars in panels are 100 μm. (b) Organoid size (diameter) was assessed from tumour organoids cultured in the above mentioned conditions. Gata6 wt (purple) and compound mutant (red) tumour organoids did not grow better in the presence of RSPO1/WNT3a (dark orange and light orange) or when LGR5 was overexpressed (dark grey and light grey). Data are mean ± s.e.m. of measurements in n organoids growing in 5 independent wells per condition (n = 38 Gata6+/+; Apcfl/fl, 23 Gata6+/+; Apcfl/fl+ RSPO1/WNT3a, 18 Gata6+/+; Apcfl/fl− LGR5, 31 Gata6fl/fl; Apcfl/fl, 29 Gata6fl/fl; Apcfl/fl+ RSPO1/WNT3a, 28 Gata6fl/fl; Apcfl/fl− LGR5); *** indicates P < 0.001 by Willconox test with exact p-value. n.s is not significant. (c) Overexpression of exogenous human LGR5 in the organoids of a,b determined by qRT-PCR. (d) Endogenous Lgr5 levels were not affected by overexpression of exogenous LGR5 in Gata6 wt (dark grey) nor compound mutants (light grey) compared to control vector infected cells, (purple and red respectively). Endogenous Lgr5 levels were assessed after overexpression of human LGR5 by qRT-PCR using mouse specific Taqman probe. Data in panel c and d are mean ± s.e.m. of n = 4 independent cell dishes. n.s. is not significant by Student’s t-test. Original data for c and d are provided in the statistical source data (Supplementary Table 4).

Supplementary Figure 5 Conditional deletion of Gata6 using the Villin CreERT2 driver resulted in BMP pathway marker expansion in Adenoma crypts.

Representative staining of β-catenin (top), P-SMAD1/5/8 (middle) and ID1 (bottom) in colonic crypts from VillinCreERT2Gata6+/+Apcfl/fl (left) and VillinCreERT2Gata6fl/flApcfl/fl (right) mice. P-SMAD1/5/8 and ID1 were intensely expressed in adenoma cells from the upper half of crypts in Apc null (left panels). Note that compound mutants (right panels) exhibited expanded P-SMAD1/5/8 and ID1 domains that extended into the crypt base (arrowheads). Dashed lines identify the upper third of the crypt and arrowheads (black) point to P-SMAD1/5/8 and ID1 positive cells. Images in all panels are representative of the intestine of n = 5 mice/genotype. Scale bars in panels are 25 μm.

Supplementary Figure 6 Treatment of mice with LDN-193189 blocked BMP signalling in adenomas and reduced AdSC numbers.

Representative staining of P-SMAD1/5/8 (left), LGR5–GFP (middle) and EPHB3 (right) in serial sections of Lgr5eGFPCreERT2Gata6+/+Apcfl/fl (top rows) and Lgr5eGFPCreERT2Gata6fl/flApcfl/fl (bottom rows) colonic tissues. LDN-193189 treatment reduced P-SMAD1/5/8 (left) levels in both Gata6 wild-type and compound mutant tumours. Numbers of LGR5–GFP+ and EPHB3+ tumour cells increased in adenomas of Gata6 wild-type mice on LDN-193189 treatment. In Gata6 mutant adenomas, the frequency of EPHB3+ cells also increased on LDN-193189 treatment yet only few LGR5–GFP+ cells were observed restricted to base of adenoma glands. Green dashed lines delineate the AdSC compartment at the base of adenoma glands. Green arrowheads depict cells expressing AdSCs marker genes. Red dashed lines delineate normal (N) from adenoma tissue. Images are representative of n > 50 adenomas observed in 3 mice per condition and genotype. Scale bars in panels are 25 μm.

Supplementary Figure 7 GATA6 competed with β-catenin/TCF4 for binding at BMP7 enhancers.

(a) Relative gene expression of BMP7 determined by qRT-PCR in SW403 cells expressing control shRNA or shRNA targeting GATA6. (b) BMP7 levels determined by qRT-PCR in SW403 cells stably transduced with a 4-hydroxytamoxifen (4OHT)-inducible N-terminus domain of TCF4 (NTCF) fused to the modified ligand-binding domain of the estrogen receptor (ERT2) compared to cells transduced with ERT2 alone, both in the presence of 4OHT. Data in panel a and b are mean ± s.e.m. of n = 4 independent cell dishes. *P < 0.05 and **P < 0.01 by Student’s t-test in both cases. Original data for c and d are provided in the statistical source data (Supplementary Table 4). Data is representative of two independent experiments. (c) GATA6, β-catenin and TCF4 occupancy of the BMP7 locus by ChIP-seq are overlaid. The single GATA6 peak located at 5.1 Kb from the BMP7 TSS (R1) overlapped a β-catenin and TCF4-binding peak (in red). R1 displayed optimal GATA6 and TCF4 consensus separated by 23 nucleotides. (d) ChIP of LS174T cells followed by PCR to amplify R1 was performed. Cells stably transduced with a doxycycline-inducible shGATA6 were treated (shG6) or not (CON) with doxycycline for 4 days. Chromatin was immunoprecipitated using anti-GATA6, TCF4 and β-catenin or non-specific IgGs. Data are represented as relative enrichment compared to an unrelated genomic control region (CON). Note that GATA6 knockdown led to increased-binding of β-catenin and TCF4 to the BMP7 R1 enhancer. Data in the three panels are mean ± s.e.m. of 3 independent experiments. *P < 0.05 and **P < 0.01 by Student’s t-test in all cases. Original data for a, b and d are provided in the statistical source data (Supplementary Table 4).

Supplementary Figure 8 Overlapping of TCF4 and GATA6 binding and the regulation of expression of β-catenin/TCF4 targets by GATA6.

(a) Enriched motives in GATA6 bound regions defined by ChIPSeq. The Meme-ChIP algorithm from the Meme-suit returned the GATA1/GATA6 motif as the most enriched transcription factor (TF) among a large TF database (Jaspar Vertebrates and UNIProbe Mouse). The optimal TCF7L2 (aka TCF4)-binding sequence was also very significantly enriched in these regions. (b) Genome-wide overlap between peaks from TCF4 and GATA6 ChIPSeq experiments. (c) Analysis of relative expression (Z-scores) of β-catenin/TCF4 target genes on GATA6 knockdown in LS174T cells. Boxes contain data within the first and third quartiles with the horizontal line at the median. Notches correspond to 95% confidence interval for the difference of the medians. Both upregulated genes (Fold change >1.5, p-value <0.05; left panel) and downregulated genes (Fold change < −1.5, p-value <0.05; right panel) by GATA6 shRNA showed largest fold changes when contained in overlapping TCF4 and GATA6 peaks in their regulatory regions. * is P < 0.01, ** is P < 0.001, *** is P < 0.0001 and n.s. is not significant by Wilcoxon test. Blox plots show. (d) We identified β-catenin/TCF4 target genes by global transcriptomic analysis of LS174T cells before and after 36 h of induction of a dominant negative TCF4 (NTCF-ERT2) (Fold change < −1.5, p-value <0.05,n = 1,146 genes). We classified the β-catenin/TCF4 geneset in 5 categories according to absence or presence of single or overlapping TCF4 and GATA6 ChIP-seq peaks in their regulatory regions (promoters, intergenic or distal enhancers) and studied their behavior on GATA6 knockdown. Figure shows MA-plots of expression of β-catenin/TCF4 regulated genes on GATA6 shRNA-mediated silencing. Indicated p-values refer to the comparison with genes containing no peaks (Wilcoxon test). Note that the subset of genes with GATA6 peaks in their regulatory regions does not change significantly on GATA6 knockdown compared to those without peaks. In contrast, genes with GATA6 peaks overlapping TCF4 peaks in regulatory regions showed significant differences on GATA6 downregulation. Genes with non-overlapping peaks GATA6 and TCF4 peaks in regulatory regions showed moderate but still significant changes.

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Whissell, G., Montagni, E., Martinelli, P. et al. The transcription factor GATA6 enables self-renewal of colon adenoma stem cells by repressing BMP gene expression. Nat Cell Biol 16, 695–707 (2014). https://doi.org/10.1038/ncb2992

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