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A reinforcing HNF4–SMAD4 feed-forward module stabilizes enterocyte identity

Abstract

BMP/SMAD signaling is a crucial regulator of intestinal differentiation1,2,3,4. However, the molecular underpinnings of the BMP pathway in this context are unknown. Here, we characterize the mechanism by which BMP/SMAD signaling drives enterocyte differentiation. We establish that the transcription factor HNF4A acts redundantly with an intestine-restricted HNF4 paralog, HNF4G, to activate enhancer chromatin and upregulate the majority of transcripts enriched in the differentiated epithelium; cells fail to differentiate on double knockout of both HNF4 paralogs. Furthermore, we show that SMAD4 and HNF4 function via a reinforcing feed-forward loop, activating each other’s expression and co-binding to regulatory elements of differentiation genes. This feed-forward regulatory module promotes and stabilizes enterocyte cell identity; disruption of the HNF4–SMAD4 module results in loss of enterocyte fate in favor of progenitor and secretory cell lineages. This intersection of signaling and transcriptional control provides a framework to understand regenerative tissue homeostasis, particularly in tissues with inherent cellular plasticity5.

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Fig. 1: HNF4A and HNF4G are redundantly required to drive intestinal differentiation.
Fig. 2: HNF4 binding is required to activate enhancer chromatin and stimulate genes required for intestinal differentiation.
Fig. 3: HNF4 and BMP/SMAD reinforce each other’s expression.
Fig. 4: HNF4 and SMAD4 cobind genomic enhancers, activating enterocyte identity genes.
Fig. 5: Disruption of an HNF4–SMAD4 regulatory loop compromises enterocyte identity.

Data availability

All RNA-seq, ChIP-seq and ATAC-seq data of this study have been deposited in GEO (GSE112946). The following datasets from GEO were reanalyzed with our sequencing data: the accession numbers for the transcriptome of Smad4KO and HNF4A ChIP in CaCo-2 cells from our previous studies are GSE102171(ref. 52) and GSE23436 (ref. 23), respectively. GSE51336 (ref. 7) and GSE36025 (ref. 14) were used to analyze chromatin accessibility and RNA-seq data across different tissues. GSE57919 (ref. 53) and GSE98724 (ref. 54) were used to mark active chromatin. GSE53545, GSE70766 (ref. 55) and GSE102171 (ref. 52) were used to perform RNA-seq analysis of villus-enriched genes and crypt-enriched genes.

References

  1. 1.

    Davis, H. et al. Aberrant epithelial GREM1 expression initiates colonic tumorigenesis from cells outside the stem cell niche. Nat. Med. 21, 62–70 (2015).

    CAS  Article  Google Scholar 

  2. 2.

    Haramis, A. P. et al. De novo crypt formation and juvenile polyposis on BMP inhibition in mouse intestine. Science 303, 1684–1686 (2004).

    CAS  Article  Google Scholar 

  3. 3.

    He, X. C. et al. BMP signaling inhibits intestinal stem cell self-renewal through suppression of Wnt-beta-catenin signaling. Nat. Genet. 36, 1117–1121 (2004).

    CAS  Article  Google Scholar 

  4. 4.

    Auclair, B. A., Benoit, Y. D., Rivard, N., Mishina, Y. & Perreault, N. Bone morphogenetic protein signaling is essential for terminal differentiation of the intestinal secretory cell lineage. Gastroenterology 133, 887–896 (2007).

    CAS  Article  Google Scholar 

  5. 5.

    Kim, T. H. et al. Broadly permissive intestinal chromatin underlies lateral inhibition and cell plasticity. Nature 506, 511–515 (2014).

    CAS  Article  Google Scholar 

  6. 6.

    Iwafuchi-Doi, M. & Zaret, K. S. Cell fate control by pioneer transcription factors. Development 143, 1833–1837 (2016).

    CAS  Article  Google Scholar 

  7. 7.

    Vierstra, J. et al. Mouse regulatory DNA landscapes reveal global principles of cis-regulatory evolution. Science 346, 1007–1012 (2014).

    CAS  Article  Google Scholar 

  8. 8.

    UK IBD Genetics Consortium, et al. Genome-wide association study of ulcerative colitis identifies three new susceptibility loci, including the HNF4A region. Nat. Genet. 41, 1330–1334 (2009).

    Article  Google Scholar 

  9. 9.

    Cancer Genome Atlas Network. Comprehensive molecular characterization of human colon and rectal cancer. Nature 487, 330–337 (2012).

    Article  Google Scholar 

  10. 10.

    Zhang, B. et al. Proteogenomic characterization of human colon and rectal cancer. Nature 513, 382–387 (2014).

    CAS  Article  Google Scholar 

  11. 11.

    Babeu, J. P., Darsigny, M., Lussier, C. R. & Boudreau, F. Hepatocyte nuclear factor 4alpha contributes to an intestinal epithelial phenotype in vitro and plays a partial role in mouse intestinal epithelium differentiation. Am. J. Physiol. Gastrointest. Liver Physiol. 297, G124–G134 (2009).

    CAS  Article  Google Scholar 

  12. 12.

    Cattin, A. L. et al. Hepatocyte nuclear factor 4alpha, a key factor for homeostasis, cell architecture, and barrier function of the adult intestinal epithelium. Mol. Cell Biol. 29, 6294–6308 (2009).

    CAS  Article  Google Scholar 

  13. 13.

    San Roman, A. K., Aronson, B. E., Krasinski, S. D., Shivdasani, R. A. & Verzi, M. P. Transcription factors GATA4 and HNF4A control distinct aspects of intestinal homeostasis in conjunction with transcription factor CDX2. J. Biol. Chem. 290, 1850–1860 (2015).

    Article  Google Scholar 

  14. 14.

    Lin, S. et al. Comparison of the transcriptional landscapes between human and mouse tissues. Proc. Natl Acad. Sci. USA 111, 17224–17229 (2014).

    CAS  Article  Google Scholar 

  15. 15.

    Stark, R. & Brown, G. D. DiffBind: differential binding analysis of ChIP-seq peak data. Bioconductor http://bioconductor.org/packages/release/bioc/html/DiffBind.html (2011).

  16. 16.

    Fang, B., Mane-Padros, D., Bolotin, E., Jiang, T. & Sladek, F. M. Identification of a binding motif specific to HNF4 by comparative analysis of multiple nuclear receptors. Nucl. Acids Res. 40, 5343–5356 (2012).

    CAS  Article  Google Scholar 

  17. 17.

    Gerdin, A. K. et al. Phenotypic screening of hepatocyte nuclear factor (HNF) 4-gamma receptor knockout mice. Biochem. Bioph. Res. Co. 349, 825–832 (2006).

    CAS  Article  Google Scholar 

  18. 18.

    Baraille, F. et al. Glucose tolerance is improved in mice invalidated for the nuclear receptor HNF-4gamma: a critical role for enteroendocrine cell lineage. Diabetes 64, 2744–2756 (2015).

    CAS  Article  Google Scholar 

  19. 19.

    Verzi, M. P., Shin, H., San Roman, A. K., Liu, X. S. & Shivdasani, R. A. Intestinal master transcription factor CDX2 controls chromatin access for partner transcription factor binding. Mol. Cell. Biol. 33, 281–292 (2013).

    CAS  Article  Google Scholar 

  20. 20.

    Merlos-Suarez, A. et al. The intestinal stem cell signature identifies colorectal cancer stem cells and predicts disease relapse. Cell Stem Cell 8, 511–524 (2011).

    CAS  Article  Google Scholar 

  21. 21.

    Boyer, L. A. et al. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122, 947–956 (2005).

    CAS  Article  Google Scholar 

  22. 22.

    Qi, Z. et al. BMP restricts stemness of intestinal Lgr5(+) stem cells by directly suppressing their signature genes. Nat. Commun. 8, 13824 (2017).

    CAS  Article  Google Scholar 

  23. 23.

    Verzi, M. P. et al. Differentiation-specific histone modifications reveal dynamic chromatin interactions and partners for the intestinal transcription factor CDX2. Dev. Cell 19, 713–726 (2010).

    CAS  Article  Google Scholar 

  24. 24.

    Haber, A. L. et al. A single-cell survey of the small intestinal epithelium. Nature 551, 333–339 (2017).

    CAS  Article  Google Scholar 

  25. 25.

    Moor, A. E. et al. Spatial reconstruction of single enterocytes uncovers broad zonation along the intestinal villus axis. Cell 175, 1156–1167.e15 (2018).

    CAS  Article  Google Scholar 

  26. 26.

    Yousefi, M., Li, L. & Lengner, C. J. Hierarchy and plasticity in the intestinal stem cell compartment. Trends Cell Biol. 27, 753–764 (2017).

    CAS  Article  Google Scholar 

  27. 27.

    Tetteh, P. W. et al. Replacement of Lost Lgr5-positive stem cells through plasticity of their enterocyte-lineage daughters. Cell Stem Cell 18, 203–213 (2016).

    CAS  Article  Google Scholar 

  28. 28.

    el Marjou, F. et al. Tissue-specific and inducible Cre-mediated recombination in the gut epithelium. Genesis 39, 186–193 (2004).

    CAS  Article  Google Scholar 

  29. 29.

    Hayhurst, G. P., Lee, Y. H., Lambert, G., Ward, J. M. & Gonzalez, F. J. Hepatocyte nuclear factor 4alpha (nuclear receptor 2A1) is essential for maintenance of hepatic gene expression and lipid homeostasis. Mol. Cell Biol. 21, 1393–1403 (2001).

    CAS  Article  Google Scholar 

  30. 30.

    Yang, X., Li, C., Herrera, P. L. & Deng, C. X. Generation of Smad4/Dpc4 conditional knockout mice. Genesis 32, 80–81 (2002).

    CAS  Article  Google Scholar 

  31. 31.

    Wong, V. W. et al. Lrig1 controls intestinal stem-cell homeostasis by negative regulation of ErbB signalling. Nat. Cell Biol. 14, 401–408 (2012).

    CAS  Article  Google Scholar 

  32. 32.

    Jadhav, U. et al. Dynamic reorganization of chromatin accessibility signatures during dedifferentiation of secretory precursors into Lgr5+ intestinal stem cells. Cell Stem Cell 21, 65–77.e5 (2017).

    CAS  Article  Google Scholar 

  33. 33.

    Trapnell, C. et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. Protoc. 7, 562–578 (2012).

    CAS  Article  Google Scholar 

  34. 34.

    Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl Acad. Sci. USA 102, 15545–15550 (2005).

    CAS  Article  Google Scholar 

  35. 35.

    Babicki, S. et al. Heatmapper: web-enabled heat mapping for all. Nucl. Acids Res. 44, W147–W153 (2016).

    CAS  Article  Google Scholar 

  36. 36.

    Perekatt, A. O. et al. YY1 is indispensable for Lgr5+ intestinal stem cell renewal. Proc. Natl Acad. Sci. USA 111, 7695–7700 (2014).

    CAS  Article  Google Scholar 

  37. 37.

    Buenrostro, J. D., Wu, B., Chang, H. Y. & Greenleaf, W. J. ATAC-seq: a method for assaying chromatin accessibility genome-wide. Curr. Protoc. Mol. Biol. 109, 21.29.1–21.29.9 (2015).

    Article  Google Scholar 

  38. 38.

    Buenrostro, J. D., Giresi, P. G., Zaba, L. C., Chang, H. Y. & Greenleaf, W. J. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat. Methods 10, 1213–1218 (2013).

    CAS  Article  Google Scholar 

  39. 39.

    Ramirez, F. et al. deepTools2: a next generation web server for deep-sequencing data analysis. Nucl. Acids Res. 44, W160–W165 (2016).

    CAS  Article  Google Scholar 

  40. 40.

    Robinson, J. T. et al. Integrative genomics viewer. Nat. Biotechnol. 29, 24–26 (2011).

    CAS  Article  Google Scholar 

  41. 41.

    Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).

    Article  Google Scholar 

  42. 42.

    Quinlan, A. R. BEDTools: the Swiss-Army tool for genome feature analysis. Curr. Protoc. Bioinformatics 47, 11.12.1–11.12.34 (2014).

    Article  Google Scholar 

  43. 43.

    Pinello, L., Farouni, R. & Yuan, G. C. Haystack: systematic analysis of the variation of epigenetic states and cell-type specific regulatory elements. Bioinformatics 34, 1930–1933 (2018).

    CAS  Article  Google Scholar 

  44. 44.

    Ross-Innes, C. S. et al. Differential oestrogen receptor binding is associated with clinical outcome in breast cancer. Nature 481, 389–393 (2012).

    CAS  Article  Google Scholar 

  45. 45.

    Shin, H., Liu, T., Manrai, A. K. & Liu, X. S. CEAS: cis-regulatory element annotation system. Bioinformatics 25, 2605–2606 (2009).

    CAS  Article  Google Scholar 

  46. 46.

    Liu, T. et al. Cistrome: an integrative platform for transcriptional regulation studies. Genome Biol. 12, R83 (2011).

    CAS  Article  Google Scholar 

  47. 47.

    Huang da, W., Sherman, B. T. & Lempicki, R. A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57 (2009).

    Article  Google Scholar 

  48. 48.

    Heinz, S. et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell 38, 576–589 (2010).

    CAS  Article  Google Scholar 

  49. 49.

    Shaked, H., Guma, M. & Karin, M. Analysis of NF-kappaB activation in mouse intestinal epithelial cells. Method. Mol. Biol. 1280, 593–606 (2015).

    CAS  Article  Google Scholar 

  50. 50.

    Sato, T. et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009).

    CAS  Article  Google Scholar 

  51. 51.

    Tamayo, P., Steinhardt, G., Liberzon, A. & Mesirov, J. P. The limitations of simple gene set enrichment analysis assuming gene independence. Stat. Methods Med. Res. 25, 472–487 (2016).

    Article  Google Scholar 

  52. 52.

    Perekatt, A. O. et al. SMAD4 suppresses WNT-driven dedifferentiation and oncogenesis in the differentiated gut epithelium. Cancer Res. 78, 4878–4890 (2018).

    CAS  Article  Google Scholar 

  53. 53.

    Camp, J. G. et al. Microbiota modulate transcription in the intestinal epithelium without remodeling the accessible chromatin landscape. Genome Res. 24, 1504–1516 (2014).

    CAS  Article  Google Scholar 

  54. 54.

    Saxena, M. et al. Transcription factor-dependent ‘anti-repressive’ mammalian enhancers exclude H3K27me3 from extended genomic domains. Gene. Dev. 31, 2391–2404 (2017).

    CAS  Article  Google Scholar 

  55. 55.

    San Roman, A. K., Tovaglieri, A., Breault, D. T. & Shivdasani, R. A. Distinct processes and transcriptional targets underlie CDX2 requirements in intestinal stem cells and differentiated villus cells. Stem Cell Rep. 5, 673–681 (2015).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This research was funded by a grant from the National Institutes of Health (NIH; R01CA190558) to M.P.V. The Verzi laboratory is also supported by the Intestinal Stem Cell Consortium funded by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) and the National Institute of Allergy and Infectious Diseases (NIAID) of the NIH under grant number U01 DK103141. L.C. was supported by New Jersey Commission on Cancer Research grant DFHS18PPC051. N.H.T., S.L., R.P.V. and A.P. were supported by MacMillan Summer Undergraduate Research Fellowships. R. Shivdasani, R. Hart, T. Nakamura and B. Nickels provided helpful discussions. The research was supported by the Genome Editing shared resource of Rutgers Cancer Institute of New Jersey (P30CA072720), next-generation sequencing services of RUCDR, flow cytometry/cell sorting core facility at Environmental and Occupational Health Sciences Institute (EOHSI) and imaging core facility of Human Genetics Institute of New Jersey.

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Authors

Contributions

L.C. conceived, designed and performed the animal, cellular, molecular, biochemical and bioinformatic experiments; collected and analyzed the data; and wrote the manuscript. N.H.T. contributed to the staining and organoid experiments. R.P.V. contributed to the staining experiments. S.L. and A.P. contributed to the mouse experiments. R.L.F. performed the Diffbind analysis. A.O.P. performed the human SMAD4 ChIP. M.P.V. conceived, designed and supervised the study and wrote the manuscript.

Corresponding author

Correspondence to Michael P. Verzi.

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The authors declare no competing interests.

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Integrated supplementary information

Supplementary Figure 1 HNF4A/G motif is the most enriched in intestine-specific enhancer chromatin.

a, Heatmap of DNase I hypersensitive sites (GSE51336) across different tissues was plotted at regions of accessible chromatin of intestinal villi defined by ATAC-seq (MACS P value ≤ 10-5). We identified 2 clusters of putative enhancers: 1) those with broadly accessible chromatin across all tissues (4,620 sites); and 2) intestine-specific accessible chromatin (4,575 sites). HNF4A and HNF4G DNA-binding motifs (HOMER de novo) are the most enriched motifs at intestine-specific open chromatin regions. b, DNase-seq enrichment at gene loci of Hnf4α, Hnf4γ and Actb (reference gene) among different tissues (GSE51336) shows that HNF4G harbors robust chromatin accessibility only in the intestine. c, RNA-seq (GSE36025) tracks of Hnf4α, Hnf4γ and Actb (reference gene) in different tissues show intestine-particular expression of Hnf4γ. d, We verified these findings via qRT-PCR on mouse tissues. The data are presented as mean ± s.e.m. (n=3 biologically independent mice).

Supplementary Figure 2 HNF4A and HNF4G bind epithelial chromatin in a strikingly similar pattern.

a, Western blot shows the specificity of Hnf4α and Hnf4γ antibodies using single HNF4 knockout mice to monitor the possibility of cross-reactivity (n=3 independent experiments). Uncropped western blots are shown in Supplementary Fig. 11. b, Heatmap shows a near-identical pattern of Hnf4α and Hnf4γ binding in WT or single mutant epithelium, which is consistent with the redundancy of these factors. c, Examples of the few differentially bound sites (DiffBind analysis) between Hnf4α and Hnf4γ ChIP-seq experiments (n=2 biologically independent mice each), revealing that many of the uniquely-called peaks actually harbor some overlapping binding activity between the two paralogs. d, HOMER motif enrichment analysis at HNF4 binding regions (MACS P value ≤ 10-3) reveals that the top two motifs present are remarkably similar to previously defined HNF4A and HNF4G binding sequence preferences. These data highlight the remarkable similarity of the cistromes of Hnf4α and Hnf4γ. Subsequent analysis of HNF4 binding sites consists of all peaks at MACS P value ≤ 10-3 in each of the 4 wild-type replicates merged into a set of 7,287 binding regions.

Supplementary Figure 3 Loss of HNF4G has a modest effect on intestinal morphology or gene expression.

a, Strategy for CRISPR/Cas9 targeting to generate Hnf4γKO. b, 4 bp (TACC) deletion confirmed by Sanger sequencing. c, Genotyping of WT and Hnf4γKO with allele specific primers. d, Immunostaining of HNF4γ in WT and Hnf4γKO (representative of 3 biologically independent mice). e-f, Transcriptional profiling of the duodenal epithelium of Hnf4γ mutants showed modest changes compared to the littermate controls (n=3 biologically independent mice), with 560 genes significantly changed (FDR < 0.05, log2(fold change) > 1 or < -1). e, Volcano plot of gene expression changes in Hnf4γKO. Green color: genes with log2(fold change) < -1 or > 1, FDR < 0.05. Statistical tests were embedded in Cuffdiff. f, Functional annotation (DAVID) of downregulated genes in Hnf4γKO (log2(fold change) < -1, FDR < 0.05). Benjamini P values were calculated using DAVID.

Supplementary Figure 4 HNF4A and HNF4G are redundantly required for intestinal homeostasis and regulate thousands of transcripts in the intestinal epithelium.

a, Hnf4αγDKO shows rapid weight loss upon tamoxifen treatment, unlike single mutants or controls. Data are presented as mean ± s.e.m. (WT and single mutants: n=4 biologically independent mice; double mutants: n=6 biologically independent mice, one-way ANOVA followed by Dunnett’s post test at P < 0.001***). b, Immunostaining of KRT20 (differentiation marker, brown color; representative of 4 biologically independent mice). c, Lumenal view of the intact epithelium and d, isolated crypt epithelium, via whole mount (representative of 4 biologically independent mice). e, Immunostaining of Ki67 (proliferation marker, brown nuclei, representative of 4 biologically independent mice). The Hnf4αγDKO mice show a significant expansion of the crypt at the expense of the villus. f, Heatmap shows all significantly regulated genes detected in RNA-seq on epithelial cells of duodenum collected from mice 2 or 3 days after injection of tamoxifen or vehicle (FDR < 0.05, n=3 biologically independent mice). Only the Hnf4αγDKO mice show a striking alteration of gene expression. Statistical tests were embedded in Cuffdiff. g, Functional annotation (DAVID) of downregulated genes in Hnf4αγDKO (log2(fold change) < -1, FDR < 0.05, n = 3 biologically independent mice) indicates that genes associated with differentiated intestinal cell functions are dependent upon HNF4 for expression. Benjamini P values were calculated using DAVID. h, GSEA (Kolmogorov-Smirnov test, one-sided for positive and negative enrichment scores, p<0.001, n=3 biologically independent mice) confirms that proliferation genes are elevated while differentiation genes are reduced upon loss of HNF4A and HNF4G. i, RNA-seq (n=3 biologically independent mice) shows that a differentiation gene, Slc6a19, is specifically lost in the double mutants, whereas a proliferation gene Ccnd1 is elevated. HNF4 ChIP-seq (n=2 biologically independent mice) shows that both HNF4α and HNF4γ bind to the differentiation gene locus (arrows), suggesting direct regulation. Conversely, the proliferation gene is unbound, suggesting indirect regulation.

Supplementary Figure 5 Individual HNF4 single mutant phenotypes are similar to each other and subtle in comparison to the doube mutant phenotype, yet some differences are observed between HNF4A and HNF4G single mutants.

Heatmap of (a) differentiation and (b) proliferation genes detected by RNA-seq on epithelial cells of duodenum collected from mice 2 or 3 days after injection of tamoxifen or vehicle (n=3 biologically independent mice). c, Immunostaining of Ki67 and BrdU (proliferation marker, brown nuclei; representative of 3 biologically independent mice). For BrdU immunohistochemistry, mice were injected with 1 mg of BrdU 1 h before euthanasia. 50 crypts per mouse (n=3 biologically independent mice) were counted for quantification of (d) Ki67 positive cells and (e) BrdU positive cells. f, Proliferation zone of different genotypes (n=3 biologically independent mice) was measured with Image J. All the data are presented as mean ± s.e.m., and statistical comparisons were performed using one-way ANOVA followed by Dunnett’s post test at P < 0.001*** and P < 0.01**. g, Primary organoid culture assay of the indicated genotypes (representative of 3 independent experiments). Compared to WT, Hnf4αγDKO shows a spherical morphology, whereas Hnf4αKO shows longer crypts and Hnf4γKO shows retarded growth. The onset of HNF4 paralog inactivation could contribute to the observed differences, as the HNF4G knockout is germline, while the HNF4A is conditional, induced in the mature animal.

Supplementary Figure 6 HNF4 binding is required for active enhancer chromatin, and chromatin structure at active promoters is less affected in Hnf4αγDKO.

a, In the duodenal epithelium of Hnf4αγDKO, H3K27ac signal is dramatically decreased at enhancer regions of HNF4 binding sites. The signals of open chromatin (ATAC-seq in this study; DNase-seq, GSE57919) and active enhancer histone markers (H3K4me1 and H3K4me2, GSE98724) are stronger at the sites with strong HNF4 binding, and unsurprisingly, repressive chromatin markers (H3K9me3 and H3K27me3, GSE98724) are weaker at those sites. The factors involved in the activation of gene transcription are also active at the sites with strong HNF4 binding, including RNA Polymerase II (RNAPII, GSE98724) and its co-activator (Mediator 1, GSE98724), components of the SWI/SNF chromatin-remodeling complex (PBRM1 and SMARCC1, GSE98724), and histone demethylase (KDM6A, GSE98724). b, HNF4-dependent active-enhancer chromatin is correlated with HNF4-dependent gene expression. GSEA shows genes nearby regions losing H3K27ac signal (clusters 1 and 2) are downregulated in Hnf4αγDKO, but regions with minimal change in H3K27ac (cluster 3) do not show significant changes in gene expression (within 10 kb of the H3K27ac-marked regions). Kolmogorov-Smirnov test, one-sided for positive and negative enrichment scores, n=3 biologically independent mice. c, H3K27ac signal does not change at promoter regions in Hnf4αγDKO vs WT mice intestinal epithelium, including HNF4 direct-bound promoters, promoters unbound by HNF4 but with nearby HNF4 sites, and HNF4-independent promoters (a distance of 20 kb is used to define whether a promoter has nearby HNF4 binding), indicating that HNF4 functions in maintaining active enhancer chromatin rather than at promoter chromatin.

Supplementary Figure 7 HNF4 activates villus-enriched genes.

a, RNA-seq analysis identifies villus-enriched and crypt-enriched genes (n = 5 crypts, 3 villi, GSE53545, GSE70766 and GSE102171). b, HNF4 factors are more likely to bind and activate villus-enriched genes than crypt-enriched genes (HNF4 bound genes defined as genes with a TSS within 30kb of a binding site; significantly regulated genes consider those with FDR < 0.05, n=3 biologically independent mice). Significant genes were called via Cuffdiff.

Supplementary Figure 8 HNF4 and BMP/SMAD reinforce each other’s expression and activate a common transcriptional program.

a, ChIP-seq tracks (n=2 biologically independent mice) show that HNF4 factors bind to BMP/SMAD genes and loss of HNF4 results in reduced H3K27ac signal at these sites (see dashed rectangles). BMP1 is reported to activate BMP2 and BMP4 by cleaving Chordin, an extracellular antagonist of BMP signaling. Active chromatin structure is also lost upon Hnf4αγDKO at HNF4-bound regions at the Bmp1 locus. b, qRT-PCR shows Smad4 depletion (4 days after tamoxifen injection) can cause significant downregulation of Hnf4α and Hnf4γ transcripts. The data are presented as mean ± s.e.m. (n=4 biologically independent mice, Student’s t-test, two-sided at P < 0.01** and P < 0.05*). c, Elevated HNF4G protein levels were observed in WT organoids after 24h of BMP2 treatment (n=3 independent experiments). d, To knockout of Hnf4α, organoids were treated with tamoxifen (1 µM) on Day 2 after seeding. Elevated HNF4G protein levels were observed in Hnf4αKO organoids after 6h of BMP2 treatment (n=2 independent experiments). Uncropped western blots are shown in Supplementary Fig. 11. e, qPCR shows BMP2 suppresses stem cell genes (n=3 independent organoid cultures). f, mRNA expression levels of Hnf4α isoforms in presence of BMP2 (100 ng/ml) treatment (n=3 independent organoid cultures). All the qPCR data are presented as mean ± s.e.m. Transcript levels are relative to BMP 0h, and statistical comparisons were performed using one-way ANOVA followed by Dunnett’s post test at P < 0.001***, P < 0.01** and P < 0.05*. All the primary organoids were harvested at Day 6 after seeding. g, Co-staining of HNF4A and SMAD4 in intestinal epithelial cells by immunofluorescence confocal microscopy (n=3 biologically independent mice). h, The common downregulated genes (black dashed line in Fig. 4i) between Hnf4αγDKO and Smad4KO show more striking changes in Hnf4αγDKO than in Smad4KO mouse epithelium. The line in the middle of the box is plotted at the median, and the whiskers are drawn down to the 10th percentile and up to the 90th percentile (Mann-Whitney test, two-sided at P < 0.001***; n=3 biologically independent mice).

Supplementary Figure 9 Increased numbers of goblet cells are observed upon loss of HNF4 paralogs.

a, Flow cytometry shows an increase in the percentage of goblet cells (UEA-1+CD24-) among dissociated villus epithelial cells derived from Hnf4αγDKO mice than in WT littermates. The data are presented as mean ± s.e.m. (n=4 biologically independent mice, Student’s t-test, two-sided at P < 0.05*). The number of cells expressing goblet cell markers are also elevated in the villus of Hnf4αγDKO, as evidenced by immunohistochemistry staining (n=3 biologically independent mice) of (b) MUC2 and (c) TFF3. d, No differences in goblet cell (PAS positive) numbers were observed in crypts in the experiments in which BrdU (48h pulse-chase) and PAS co-staining were performed (See Fig. 5d-f), suggesting that the increased numbers of goblet cells is initiated on the villus. The data are presented as mean ± s.e.m. (n=3 biologically independent mice, Student’s t-test, two-sided). e, Immunohistochemistry staining of TFF3 shows increased numbers of goblet cells in Hnf4αKOSmad4KO (n=3 biologically independent mice, expanded panel from Fig. 5h). f-h, Single cell RNA-seq on dissociated villus epithelium of WT and mutants shows a gain in the expression levels of Goblet cell markers in mutant enterocytes, along with a reduction in the expression levels of enterocyte markers (expanded panel from Fig. 5i-k; GC: Goblet Cells). Estimated number of cells in each experiment was WT, n=4,100; Hnf4αγDKO, n=4,200; Hnf4αKOSmad4KO, n=4,900.

Supplementary Figure 10 HNF4 and SMAD4 function via a reinforcing regulatory module to promote and maintain enterocyte identity.

Disruption of the HNF4-SMAD4 circuit results in loss of enterocyte fate in favor of progenitor and goblet cell identities. Upon loss of the HNF4-SMAD4 regulatory module, progenitor cells fail to transition onto the differentiated villus compartment, resulting in elongated crypts. The remaining villus-resident cells in the mutant exhibit goblet cell features at the expense of enterocyte gene expression.

Supplementary Figure 11

Uncropped western blots with size-marker indications.

Supplementary information

Supplementary Information

Supplementary Figures 1–11 and Supplementary Notes 1–4

Reporting Summary

Supplementary Table 1

De novo motifs of common vs. intestine-enriched chromatin-accessible regions as defined by HOMER

Supplementary Table 2

Gene signature lists used in this study

Supplementary Table 3

Gene ontology of downregulated genes

Supplementary Table 4

Exact P values calculated in this study

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Chen, L., Toke, N.H., Luo, S. et al. A reinforcing HNF4–SMAD4 feed-forward module stabilizes enterocyte identity. Nat Genet 51, 777–785 (2019). https://doi.org/10.1038/s41588-019-0384-0

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