Journal: bioRxiv

Article Title: ONECUT2 restricts Microfold cell numbers in the small intestine; a multi-omics study

doi: 10.1101/2022.09.01.506202

Figure Lengend Snippet: ONECUT2 signalling in M cell precursor cluster. a On the left, UMAP embedding overlay showing three partitions, group of interest 1 (GOI1), 2 (GOI2) and 3 (GOI3). On the right, top four transcription factors expressed within GOI1, with the highest positive correlation between transcript factor expression and motif activity. The logos for the motif of HNF4G (GM.5.0.Nuclear_receptor.0068), KLF5 (GM.5.0.C2H2_ZF.0006), ONECUT2 (GM.5.0.CUT_Homeodomain.0005) and TCF7L2 (GM.5.0.Mixed.0092) are displayed. b Gene-set enrichment analysis showing ONECUT1-, ONECUT2- and ONECUT3-specific gene signatures from Data ref: enriched in RANKL-treated organoids compared to control organoids (dark grey). Normalized enrichment score (NES) and padj values are described. Signatures enriched in control compared to RANKL condition (light grey) have a negative NES. c Gene-regulatory network of Onecut2 drivers predicted by ANANSE. Interaction scores (arbitrary cut-off ≥ 0.83) are depicted. d Trajectory plot overlaid with cluster identity of individual cells from RANKL-treated clusters in GOI1. Three major branches are shown along the plot. On the right, same trajectory plot with cells ordered along a pseudotime. Below, log 10 expression values of Onecut2 and top genes from each branching point are shown. e Co-regulated modules of differentially expressed genes by cells from RANKL-treated clusters in GOI1. The transcription factors predicted by ANANSE as Onecut2 drivers and ONECUT2 targets, that are differentially expressed (FDR < 0.05) by cells from RANKL-treated clusters within GOI1 and defined as pseudotime-dependent genes are highlighted.

Article Snippet: Then, chromatin of each replicate was incubated with 1 μg of antibody against H3K27ac (Diagenode, C15410196, RRID:AB_2637079) or 1 μg of antibody against ONECUT2 (Proteintech, 21916-1-AP, AB_2848180) in dilution buffer (1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris pH 8, 167 mM NaCl, 1x Protease Inhibitor Cocktail (Roche)), rotating overnight at 4°C.

Techniques: Expressing, Activity Assay, Control

Journal: bioRxiv

Article Title: ONECUT2 restricts Microfold cell numbers in the small intestine; a multi-omics study

doi: 10.1101/2022.09.01.506202

Figure Lengend Snippet: ONECUT2 restricts M cell lineage specification in vitro . a Bright-field images of organoids treated with RANKL, ONECUT2 inhibitor CSRM617 or a combination of both, and control organoids. Microscope magnification is depicted. Below, graph with organoid circularity quantification (AU, arbitrary units) and mean ± SEM. Control n=40, CSRM617 n=35, RANKL n=33 and RANKL + CSRM617 = 70. b Heatmap showing the relative change in the mRNA expression of transcripts upon co-treatment with RANKL and CSRM617, compared to RANKL-alone. Rows show Z scores of normalized, log 2 -transformed values from significantly changing genes (padj < 0.05). c Bar graph representing the mean ± SEM of at least four differentiation experiments. Below, density plots depicting fluorescent intensities in FITC (LGR5) and PE (GP2) channels, determined by flow cytometry. Percentage of gated populations is described. d Geneset enrichment analysis (GSEA) showing RANKL-treated cluster 12-specific gene signature, enriched in organoids treated as in a, compared to control. Dotplot shows the normalized enrichment score (NES, colour) and FDR (size). e Label transferring from RANKL-treated cell clusters within GOI1 to cells cotreated with RANKL and CSRM617. Bar graph on the right shows the fold change (FC) number of cells with RANKL clusters’ identity (RANKL + CSRM617 over RANKL). f GSEA showing the HNF4G- and ONECUT-ChIP signatures, enriched in organoids growing in EN medium, compared to control medium. NES and padj values are described. e Relative mRNA expression of Onecut2, Hnf4g, Klf5 and Klf6 detected in organoids growing on EN medium alone or with RANKL. f Ratio of LGR5 + or GP2 + cell numbers in organoids growing on EN or control medium with RANKL or EN medium alone, over control conditions, determined by flow cytometry. Bar graph representing the mean ± SEM of three differentiation experiments. For b, c, e and f, unpaired t test p < 0.05 is represented by (*), p < 0.01 by (**) and p < 0.001 by (***).

Article Snippet: Then, chromatin of each replicate was incubated with 1 μg of antibody against H3K27ac (Diagenode, C15410196, RRID:AB_2637079) or 1 μg of antibody against ONECUT2 (Proteintech, 21916-1-AP, AB_2848180) in dilution buffer (1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris pH 8, 167 mM NaCl, 1x Protease Inhibitor Cocktail (Roche)), rotating overnight at 4°C.

Techniques: In Vitro, Control, Microscopy, Expressing, Transformation Assay, Flow Cytometry, Transferring

Journal: bioRxiv

Article Title: ONECUT2 restricts Microfold cell numbers in the small intestine; a multi-omics study

doi: 10.1101/2022.09.01.506202

Figure Lengend Snippet: Transcriptional responses to co-treatment with RANKL and CSRM617 in vivo . a Heatmap showing the relative change in the mRNA expression of transcripts upon co-treatment with RANKL and CSRM617, compared to RANKL-alone, CSRM617 alone and control conditions. Rows show Z scores of normalized, log 2 -transformed values from significantly changing genes (padj < 0.05). b UMAP embedding of single cell transcriptome from RANKL and CSRM617 co-treatment in organoids. Each dot represents a single cell. Cell colours represent cluster identity. c Venn diagram with regulons in the single-cell RNA-sequencing dataset of RANKL-treated organoids and that of organoids co-treated with RANKL and CSRM617. Treatment-specific regulons are listed and ONECUT target genes are highlighted on red. d Work plan for in vivo experiments with ONECUT2 inhibitor CSRM617. Four treatment groups were designed: One group of mice treated with 50⍰mg⍰kg-1 CSRM617 for 7 days (n=6) and another group for 14 days (n=6), a recovery group consisted of mice treated with 50⍰mg⍰kg-1 CSRM617 for 7 days followed by 7 days treatment with phosphate buffered saline (PBS) and 0.2% dimethyl sulfoxide (n=6), and a control group treated with PBS and 0.2% DMSO for 7 days (n=6). e RT-qPCR analysis showing relative mRNA expression levels of Dclk1, Defa6 and Reg4 in small intestine sections from mice treated as described in d. f Body weight of mice treated as described in d.

Article Snippet: Then, chromatin of each replicate was incubated with 1 μg of antibody against H3K27ac (Diagenode, C15410196, RRID:AB_2637079) or 1 μg of antibody against ONECUT2 (Proteintech, 21916-1-AP, AB_2848180) in dilution buffer (1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris pH 8, 167 mM NaCl, 1x Protease Inhibitor Cocktail (Roche)), rotating overnight at 4°C.

Techniques: In Vivo, Expressing, Control, Transformation Assay, RNA Sequencing, Saline, Quantitative RT-PCR

Journal: bioRxiv

Article Title: ONECUT2 restricts Microfold cell numbers in the small intestine; a multi-omics study

doi: 10.1101/2022.09.01.506202

Figure Lengend Snippet: ONECUT2 regulates M cell number and mucosal immunity in vivo . a Immunohistochemistry analysis for GP2 and SPIB protein expression in Peyer’s patches of small intestine sections from mice treated with PBS control or CSRM617 for 14 days. Nuclear counterstaining with hematoxylin (bright field) or DAPI (fluorescence) is shown. Scale bars are annotated. Dotted rectangles show amplified areas and yellow arrow heads show GP2 + cells, b RT-qPCR analysis showing relative mRNA expression of M-cell markers Spib, Gp2 and Tnfaip2 in small intestine sections from mice treated as described in Additional file 1: . Unpaired t test p < 0.05 is represented by (*) and p < 0.01 by (**). c Relative mRNA expression of ONECUT2-targeted genes Klf5, Klf6, Hoxb8, Foxn3, Tfcp2l1 and Hnf4g detected in tissue from b. Unpaired t test p < 0.05 is represented by (*), p < 0.01 by (**) and p < 0.001 by (***). d Secretory IgA (SIgA) levels in faecal samples from mice treated as described in Additional file 1: . Unpaired t test p < 0.05 is represented by (*). e Relative mRNA expression of markers from IgA + B cells Ccr9 and Itgb7 detected in tissue from b.

Article Snippet: Then, chromatin of each replicate was incubated with 1 μg of antibody against H3K27ac (Diagenode, C15410196, RRID:AB_2637079) or 1 μg of antibody against ONECUT2 (Proteintech, 21916-1-AP, AB_2848180) in dilution buffer (1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris pH 8, 167 mM NaCl, 1x Protease Inhibitor Cocktail (Roche)), rotating overnight at 4°C.

Techniques: In Vivo, Immunohistochemistry, Expressing, Control, Fluorescence, Amplification, Quantitative RT-PCR