Colonic stem cell from severe ulcerative colitis maintains environment-independent immune activation by altering chromatin accessibility and global m6A loss

Abstract

Ulcerative colitis (UC) is a chronic inflammatory disease of colon, which is characterized by cryptarchitectural distortion. Alternation of colonic stem cell (CoSC) contributed to the occurrence of UC, yet the regulatory mechanisms remain unclear. To investigate the dysregulation of transcriptional and post-transcriptional regulation, we performed RNA-seq, ATAC-seq, and m⁶A meRIP-seq analysis of the cultured CoSCs that were isolated from UC patients. The transcriptome analysis revealed distinct expression signatures of UC patients in mild and severe stages. We observed abnormal activation of immune and extracellular matrix-related genes in patients affected by severe UC. The chromatin accessibility at the promoter regions of these genes was also specifically increased in the severe stage. In addition, we identified that a global loss of RNA m⁶A modification in the severe stage was accompanied by higher expression of the m⁶A demethylase FTO. The aberrant activation of a large number of immune and extracellular matrix-related genes, including IL4R, HLA-DPA1, and COL6A1, was related to both the gain of chromatin accessibility and the loss of m⁶A in severe UC patients. Our finding revealed an environment-independent immune activation of CoSCs in UC and provided FTO as a potential therapeutic target.

Keywords: chromatin accessibility; colonic stem cell; m6A; FTO; ulcerative colitis.

Figure 1.

Bulk RNA-seq revealed abnormal activation of immune and extracellular matrix-related genes in patients affected by UC disease, particularly evident in severe cases. (A) Diagram showing the process used to acquire CoSCs and perform multi-omics sequencing. (B) Representative staining of CoSCs colonies stained with indicated antibodies for proliferative marker Ki-67 or colon epithelial stem cells marker SOX9 (red) merged with DAPI (blue). Scale bar, 50 μm. (C) PCA of top 500 variant genes in CoSCs from healthy individuals and UC patients. The upper panel shows groups determined by PCA and the corresponding Mayo scores. (D) Hierarchical clustering of dynamic genes distinguishing CoSCs from healthy individuals and UC patients. Heatmap colors represent z-score normalized transcripts per million (TPM). The bars represent group definition or transcriptional changes. (E) Left, volcano plot showing genes significantly altered in severe UC CoSCs compared to healthy individuals, with upregulated genes in red and downregulated genes in blue. Right, volcano plot of genes in mild UC CoSCs. Genes associated with the highlighted pathway are listed. (F) GSEA analysis showing differences in selected enriched pathways between mild and severe UC. Colors indicate the NES (Normalized Enrichment Score). Black squares around the values indicate P-value < 0.05.

Figure 2.

Chromatin accessibility of immunity and ECM-related genes at promoter regions. (A) Scatter plots comparing ATAC-seq signal at promoter regions in severe (left panel) or mild (right panel) UC CoSCs to that in healthy CoSCs. Pie charts show the proportion of genes with up- or down-regulated ATAC-seq signaling. Up-regulated genes are marked in red and down-regulated ones in blue. (B) Heatmap showing ATAC peaks specific to mild and severe UC patients identified by hierarchical clustering. Four ATAC-peaks groups are identified. (C) Enriched KEGG pathways of genes regulated by ATAC peaks from each cluster values are negative log₁₀ transformed P-values. (D) Left, SeqLogo plot showing the top 10 enriched motifs found in ATAC-gain or ATAC-loss peaks in severe and mild CoSCs. Right, dot plot showing the statistical significance of all the top enriched motifs at ATAC-gain or ATAC-loss peaks in severe and mild CoSCs. The size of the dots refers to the negative log-transformed P-value. (E) Scatter plots showing the relationship between fold changes of ATAC signal at promoter regions and fold change of expression level in severe (left panel) or mild (right panel) UC CoSCs compared to healthy individuals.

Figure 3.

RNA m⁶A levels showing a global down-regulation in severe UC CoSCs accompanied by higher expression of FTO. (A) Scatter plots comparing m⁶A in severe (left panel) or mild (right panel) UC CoSCs to that in healthy CoSCs. Pie charts show the proportion of the genes with up- or down-regulated m⁶A signaling. Up-regulated genes are marked in red and down-regulated ones in blue. (B) Left, immunoblotting of METTL3, METTL14, FTO, and YTHDC1 in healthy individuals, mild and severe UC patients. Right, bar plot representing the differential expression levels of indicated protein calculated from the grayscale values of the protein bands shown in the left panel. Error bars indicate the standard deviation. *P-value < 0.05; **P-value < 0.01. (C) Scatter plots showing the relationship between m⁶A signal difference at gene body regions and fold change of expression level in severe (left panel) or mild (right panel) UC CoSCs compared to healthy individuals.

Figure 4.

RNA m⁶A and chromatin accessibility determine the abnormality of the downstream transcriptome in UC patients. (A) Pie chart showing the ratio of the four types of genes. Promoter open +: promoters with ATAC signal; m⁶A marked +: genes with m⁶A signal at gene body region. (B) Bar plots showing the pathways enriched in severe and mild CoSCs compared to the healthy ones. Left panel refers to the ATAC-gain genes in [+−] cases. Right panel refers to the m⁶A-loss genes in [−+] cases. Values are negative log₁₀ transformed P-values. (C) Genes co-regulated by m⁶A and chromatin accessibility at promoter regions classified into seven groups by k-means clustering shown in the upper heatmap. The middle bar graph shows the percentage of up- or down-regulation of m⁶A or ATAC signals on genes in each clustering group. The boxplot below presents the difference values of m⁶A signal and fold changes of ATAC signal and RNA expression in each group of genes in severe UC patients compared to the healthy ones. (D) Bar plot showing the pathways enriched by the genes of the selected groups in severe and mild UC patients compared to healthy individuals on genes with open promoters and m⁶A signal. Values are negative log₁₀ transformed P-values. (E) IGV tracks displaying RNA and m⁶A abundances of IL4R transcripts in CoSCs.

Figure 5.

Graphical summary. The synergistic effect of chromatin accessibility and RNA m⁶A modification leads to the aberrant activation of immune and ECM-related genes in serve CoSCs.