Transcriptional and epigenomic profiling identifies YAP signaling as a key regulator of intestinal epithelium maturation

Abstract

During intestinal organogenesis, equipotent epithelial progenitors mature into phenotypically distinct stem cells that are responsible for lifelong maintenance of the tissue. While the morphological changes associated with the transition are well characterized, the molecular mechanisms underpinning the maturation process are not fully understood. Here, we leverage intestinal organoid cultures to profile transcriptional, chromatin accessibility, DNA methylation, and three-dimensional (3D) chromatin conformation landscapes in fetal and adult epithelial cells. We observed prominent differences in gene expression and enhancer activity, which are accompanied by local changes in 3D organization, DNA accessibility, and methylation between the two cellular states. Using integrative analyses, we identified sustained Yes-Associated Protein (YAP) transcriptional activity as a major gatekeeper of the immature fetal state. We found the YAP-associated transcriptional network to be regulated at various levels of chromatin organization and likely to be coordinated by changes in extracellular matrix composition. Together, our work highlights the value of unbiased profiling of regulatory landscapes for the identification of key mechanisms underlying tissue maturation.

Fig. 1.. Fetal and adult intestinal organoid cultures are transcriptionally distinct and recapitulate their in vivo counterparts.

(A) Schematic representation of the fetal (E16.5) and adult small intestinal epithelium (top) and their respective in vitro derivatives (FEnS and aOrg) maintained in ENR and ENR + ChNic (bottom). Scale bars, 100 μm. (B) Volcano plot of CAGE differential expression analysis aggregated to the gene level of cultures maintained in ENR + ChNic. The x axis shows aOrg versus FEnS log₂ fold changes (FC), and the y axis shows log₁₀-transformed FDR values. Colors: red, enriched in FEnS (FDR < 0.05 and log₂FC < −1); blue, enriched in aOrg (FDR < 0.05 and log₂FC > 1); and gray, not differentially expressed. Previously described fetal- and adult-specific genes are labeled in the plot. (C) GO term enrichment analysis among genes FEnS- and aOrg-specific genes compared to all expressed genes. Color indicates the significant level of enrichment (−log₁₀ FDR). (D) Density plots of gene expression of in vivo E16.5 epithelium versus E16.5-derived FEnS (left) and in vivo adult crypt versus in vitro aOrg (right). Spearman correlation coefficient is indicated with ρ. (E) Gene set enrichment analysis (GSEA) of the in vivo E16.5 genes in CAGE data sorted by aOrg versus FEnS fold change. (F) GSEA of the in vivo adult crypt genes in CAGE data sorted by aOrg versus FEnS fold change. (G) Heatmap of gene expression (top 50 most variable genes) with hierarchical clustering of in vivo E16.5 and adult epithelium and in vitro FEnS and aOrg.

Fig. 2.. Transcriptional changes reflect differences at various levels of chromatin regulation.

(A) Schematic view of typical enhancers and promoters defined as ATAC-seq peaks with CAGE signal. (B) CAGE differential expression at enhancers (left) and promoters (right) with aOrg versus FEnS log₂ fold changes (logFC) on the x axis, and log₁₀-transformed FDR values on the y axis colored by differential expression (FEnS, red; aOrg, blue; static, gray). (C) Distribution of number of CpGs within 1-kb windows from enhancer center (yellow) or promoter summit (black). (D) Number of differentially expressed CpG-sparse (<30 CpGs per 1 kb; left) and CpG-dense promoters (right). (E) Correlation between ATAC logFC and CAGE logFC at enhancers and CpG-sparse and -dense promoters colored by differential expression. ρ = Spearman’s correlation coefficient. (F) Correlation between CpG methylation logFC and CAGE logFC as in (E). (G) Top, representative Hi-C interaction heatmap with chr17 coordinates as axis. Bottom, eigenvalues of the first principal component (PC1) of 500-kb bins along chromosome 17 for each state colored by state (FEnS, red and aOrg, blue) and compartment (A, dark and B, light). Both plots represent averages of two biological replicates. (H) CAGE signal from + and − strand, ATAC, CpG methylation, eigenvalues of Hi-C PC1, and RefSeq annotation along chr3. Zoom-in contains B_FEnS ≥ A_aOrg transitioning region indicated by a yellow arrow. (I) Faction of differentially expressed genes and enhancers within bins constitutively in A or B compartment, or A_FEnS ≥ B_aOrg or B_FEnS ≥ A_aOrg compartment. (J) Right, differential interactions between 100-kb bins with mean interaction counts per million (CPMs) on the x axis and interaction logFC on the y axis, colored by differential interactions (aOrg, blue; FEnS, red; static, gray). Left, fraction of differentially expressed enhancers and promoters overlapping the differentially interacting bins on the x axis and differential expression of the enhancers and promoters on the y axis, split by direction of differential interactions.

Fig. 3.. Distinct TF networks drive fetal and adult state–specific promoter and enhancer activity.

(A) MA plot of TF gene level aggregated CAGE expression log₂ CPMs over log₂ fold changes (FC). The TFs with motifs enrichment [as in (B)] in FEnS- or aOrg-specific promoters and enhancers [as in (B)] are labeled in red and blue, respectively. (B) Transcription factor binding site (TFBS) enrichment at enhancers and promoters. Each row represents one TF or a group of TFs sharing a binding motif. Color represents enrichment odds ratios (ORs) over all expressed enhancers and promoters, respectively. Enrichment with FDR value <0.05 are marked by asterisks (*). (C) TFBS enrichment at differentially methylated, accessible, interacting, or compartment switching enhancers compared to all state-specific enhancers. Color represents enrichment odds ratios over all expressed enhancers and promoters, respectively. Enrichment with FDR value <0.05 are marked by asterisks (*). (D and E) Venn diagrams depicting overlap of differential methylation (WGBS), accessibility (ATAC), interactions (Hi-C), and compartment status (Hi-C) among FEnS (D) and aOrg (E) specific enhancers.

Fig. 4.. Fetal cells are maintained by sustainably high levels of YAP activity.

(A) GSEA showing enrichment of active YAP-associated gene signatures and (B) bar plots depicting mRNA expression levels of direct YAP target genes in FEnS and aOrg cultures (ENR medium). P values calculated by unpaired t test are shown. (C) Immunofluorescent analysis showing YAP subcellular localization in FEnS and aOrg grown in ENR medium. Nuclear signal is observed in most fetal cells, while adult cells show mostly cytoplasmic localization. Scale bar, 100 μm. (c) Higher magnification of regions of interest (ROI) highlighted in (C). Scale bar, 25 μm. (D) Bar plotting showing percentage of cells with high YAP nuclear signal. Each dot represents an individual FEnS or aOrg structure. P value defined by unpaired t test is shown. (E) Representative phase contrast images of FEnS and aOrg cultures treated with YAP signaling inhibitors. Images from MGH-CP1 treatments are shown. Scale bar, 200 μm. (F) Bar plots depicting replating efficiency following treatment with the indicated inhibitors. Each dot represents a technical replica of at least three independent experiments. n.s., not significant.

Fig. 5.. Activation of YAP induces fetal-like features in aOrg cultures.

(A) Left, representative images of TetON-YAP aOrg cultures in ENR medium with the absence or presence of DOX (±DOX). Right, bar plot depicting quantification of organoid circularity. Scale bar, 200 μm. (B) Analysis of SCA1 protein expression by flow cytometry in tetON-YAP aOrg cultures. (C) Volcano plot showing differentially expressed genes upon DOX treatment. Representative adult- and fetal-specific genes are highlighted in blue and red, respectively. Representative known YAP target genes are highlighted in yellow. (D) Principal components analysis (PCA) from RNA-seq profiling data from FEnS and TetON-YAP aOrg (±DOX) cultures in ENR medium. PCA was carried out on the basis of the top 1% most variable genes (i.e., 137) across all samples. (E) Venn plot depicting fraction of shared up-regulated genes in FEnS (versus aOrg −DOX) and aOrg +DOX (versus aOrg −DOX) cultures. (F) GSEA showing enrichment of FEnS-associated gene signature upon YAP induction (+DOX). (G) Uniform Manifold Approximation and Projection (UMAP) visualization of single cells from FEnS and aOrg cultures colored by sample (left) or cell type (right). Cell-type clusters were annotated on the basis of expression of known marker genes. (H) Average expression of YAP-associated gene signature overlaid on the UMAP cell-type plot. (h) Violin plot depicting enrichment of the YAP signature in each cell type cluster. See Materials and Methods and associated manuscript for further details on the single-cell RNA analyses in (G) and (H).

Fig. 6.. FEnS and aOrg express different ECM genes.

(A) Heatmap depiction of mRNA expression levels of ECM genes (GO: 0031012 - ECM) that are differentially expressed consistently between FEnS and aOrg and freshly isolated fetal and adult epithelial cells. (B) Western blot analysis of ECM proteins fibronectin and collagen IV in FEnS and aOrg (n = 3 independent passages each). Blot against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control. (C and D) Replating assay of FEnS and aOrg cultures treated with the pharmacological inhibitors of FAK (C) and SRC (D) kinases. Each dot represents a technical replica of at least three independent experiments. ****P > 0.0001 by two-way ANOVA following multicomparison (each dose versus 0 μM for FEnS or aOrg). Scale bar, 200 μm.