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. 2020 Oct;159(4):1328-1341.e3.
doi: 10.1053/j.gastro.2020.06.012. Epub 2020 Jun 15.

The Transcription Co-Repressors MTG8 and MTG16 Regulate Exit of Intestinal Stem Cells From Their Niche and Differentiation Into Enterocyte vs Secretory Lineages

Anna Baulies  1 , Nikolaos Angelis  1 , Valentina Foglizzo  1 , E Thomas Danielsen  1 , Harshil Patel  1 , Laura Novellasdemunt  1 , Anna Kucharska  1 , Joana Carvalho  1 , Emma Nye  1 , Paolo De Coppi  2 , Vivian S W Li  3
Affiliations

The Transcription Co-Repressors MTG8 and MTG16 Regulate Exit of Intestinal Stem Cells From Their Niche and Differentiation Into Enterocyte vs Secretory Lineages

Anna Baulies et al. Gastroenterology. 2020 Oct.

Abstract

Background & aims: Notch signaling maintains intestinal stem cells (ISCs). When ISCs exit the niche, Notch signaling among early progenitor cells at position +4/5 regulates their specification toward secretory vs enterocyte lineages (binary fate). The transcription factor ATOH1 is repressed by Notch in ISCs; its de-repression, when Notch is inactivated, drives progenitor cells to differentiate along the secretory lineage. However, it is not clear what promotes transition of ISCs to progenitors and how this fate decision is established.

Methods: We sorted cells from Lgr5-GFP knockin intestines from mice and characterized gene expression patterns. We analyzed Notch regulation by examining expression profiles (by quantitative reverse transcription polymerase chain reaction and RNAscope) of small intestinal organoids incubated with the Notch inhibitor DAPT, intestine tissues from mice given injections of the γ-secretase inhibitor dibenzazepine, and mice with intestine-specific disruption of Rbpj. We analyzed intestine tissues from mice with disruption of the RUNX1 translocation partner 1 gene (Runx1t1, also called Mtg8) or CBFA2/RUNX1 partner transcriptional co-repressor 3 (Cbfa2t3, also called Mtg16), and derived their organoids, by histology, immunohistochemistry, and RNA sequencing (RNA-seq). We performed chromatin immunoprecipitation and sequencing analyses of intestinal crypts to identify genes regulated by MTG16.

Results: The transcription co-repressors MTG8 and MTG16 were highly expressed by +4/5 early progenitors, compared with other cells along crypt-villus axis. Expression of MTG8 and MTG16 were repressed by Notch signaling via ATOH1 in organoids and intestine tissues from mice. MTG8- and MTG16-knockout intestines had increased crypt hyperproliferation and expansion of ISCs, but enterocyte differentiation was impaired, based on loss of enterocyte markers and functions. Chromatin immunoprecipitation and sequencing analyses showed that MTG16 bound to promoters of genes that are specifically expressed by stem cells (such as Lgr5 and Ascl2) and repressed their transcription. MTG16 also bound to previously reported enhancer regions of genes regulated by ATOH1, including genes that encode Delta-like canonical Notch ligand and other secretory-specific transcription factors.

Conclusions: In intestine tissues of mice and human intestinal organoids, MTG8 and MTG16 repress transcription in the earliest progenitor cells to promote exit of ISCs from their niche (niche exit) and control the binary fate decision (secretory vs enterocyte lineage) by repressing genes regulated by ATOH1.

Keywords: Chromatin Remodeling; Lateral Inhibition; Lineage Specification; Niche Exit.

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Figures

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Graphical abstract
Figure 1
Figure 1
Expression of Mtg8 and Mtg16 in the +4/5 early progenitor cells. (A) FACS isolation of GFP-high and GFP-low cells from the Lgr5-EGFP-IRES-CreERT2 intestinal crypts. qRT-PCR analysis of the indicated genes in the 2 populations. Data represent mean ± SEM from biologically independent animals (n = 3). ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, 2-sided t-test. (B) RNAscope brown staining of Mtg8, Mtg16 and Mtgr1 in intestinal crypts from WT mice. (C) Quantification of Mtg8 and Mtg16 RNAscope staining in (B) along the crypt. Data represent mean ± SEM from biologically independent animals (n = 3). (D, E) RNAscope duplex staining of Mtg16 (D) or Mtg8 (E) (blue) with Atoh1 or Lgr5 (red) in WT intestinal crypts. Empty arrows indicate exclusive staining, black arrows indicate colocalized staining. (F) Quantification of Mtg16 RNAscope signal (area of dots) in Paneth cells and progenitor cells. Data represent mean ± SEM from biologically independent animals (n = 3). ∗∗P < .01, 2-sided t-test. (G) Quantification of Mtg16+Atoh1+ and Mtg16+Atoh1− cell populations in early progenitors (+3–5 positions) from the RNAscope staining in (D). Scale bars, 20 μm.
Figure 2
Figure 2
Mtg8 and Mtg16 are regulated by Notch signaling. (A, B) qRT-PCR analysis of WT mouse intestinal organoids treated with Notch inhibitor DAPT for 2 and 3 days. Data represent mean ± SEM from biologically independent small intestinal organoid isolations (n = 3). The experiment was performed twice. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, 2-sided t-test. (C) qRT-PCR analysis of intestinal epithelium from Villin CreER and Villin CreER;Rbpjfl/fl mice collected at indicated days after tamoxifen induction. Data represent mean ± SEM from biologically independent animals (n = 4 per group). Three independent experiments were performed. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, 2-way analysis of variance (ANOVA). (D) RNAscope brown staining of Mtg8, Mtg16, and Mtgr1 in intestinal tissue obtained from Villin CreER and Villin CreER;Rbpjfl/fl mice collected at day 4 post-tamoxifen induction. Arrows indicate Mtg8+ cells. Scale bars, 50 μm. (E) RNAscope duplex staining of Mtg16 (blue) and Lgr5 (red) in intestinal tissues of the indicated genotypes at day 6 post-tamoxifen induction. (F) qRT-PCR analysis of intestinal epithelium from WT mice collected at the indicated days after DBZ or vehicle treatment. Data represent mean ± SEM from biologically independent animals (n = 3 per group). ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, 2-way ANOVA. (G) qRT-PCR analysis of DAPT-treated Villin CreER and Villin CreER;Atoh1fl/fl organoids induced with 4-OHT. Data represent mean ± SEM. The experiment was performed 4 times. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, compared with untreated control group; #P < .05, ##P < .01, compared with DAPT-treated control group, 2-sided t-test.
Figure 3
Figure 3
Loss of Mtg8 and Mtg16 increases ISC numbers and proliferation. Intestinal tissues were collected from newborn (P0) (n = 4–5 for each genotype) (A, B) or adult mice (n = 3–6 mice per group) (C–E) for analysis. (A) EdU staining showing increased proliferation in Mtg16−/− and Mtg8−/− Mtg16−/− animals compared with WT. Graphs showing EdU+ cells distribution along the crypt and quantitation of EdU+ cells per inter-villus region in WT, Mtg16−/− and Mtg8−/−Mtg16−/− intestine. Data represent mean ± SEM of 3 independent experiments. At least 10 representative crypts per animal have been analyzed. (B) Lgr5 RNAscope staining and qRT-PCR showing increased ISC gene expression in newborn Mtg16−/− and Mtg8−/−Mtg16−/− tissues. Data represent mean ± SEM of 3 independent experiments (n = 4 per group). ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, 1-way analysis of variance (ANOVA). (C) EdU staining in WT and Mtg16−/− adult intestine. (D) Lgr5 and Olfm4 RNAscope brown staining in small intestinal tissue from WT and Mtg16−/− adult mice. (E) Colony formation assay of small intestine organoids isolated from WT and Mtg16−/− adult mice. Data represent mean ± SEM of 2 independent experiments (n = 6 mice per group). ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, 2-sided t-test. Scale bars, 100 μm (B–D), 1000 μm (E). (F) Colony formation assay of small intestine organoids derived from WT, Mtg16−/− and Mtg16−/−Mtg8−/− newborn pups. Data represent mean ± SEM of 3 independent experiments (n = 2–4 mice per group). ∗∗∗P < .001, 1-way ANOVA. (G) Representative images showing newborn organoids of the indicated genotypes cultured in 5% or 2% RSPO conditions for 3 to 4 days. Scale bar, 1000 μm. Right, quantification of the organoid health status maintained in 2% of RSPO condition. Data represent mean ± SEM of 2 independent experiments (n = 2–4 mice per group). ∗∗P < .01, ∗∗∗P < .001 compared with WT, 1-way ANOVA. #P < .05, ##P < .01 compared to Mtg16−/−, 1-way ANOVA.
Figure 4
Figure 4
Mtg8 and Mtg16 deletion impairs intestinal lineage specification. Intestinal tissues were collected from newborn (P0) (n = 4–5 for each genotype) (A) Heatmap showing genes differentially expressed in WT and Mtg16−/− intestine. (B, D, E) Gene Set Enrichment Analysis (GSEA) probing (B) Wnt/Stem cell signature genes, (D) intestinal absorption and digestion, and (E) secretory signature genes. (C) FAPB1 and APOA4 immunostaining in adult WT and Mtg16−/− intestinal tissue. (F) Scheme showing the disaccharidase assay performed in organoids. (G) qRT-PCR of mature enterocyte markers in adult WT and Mtg16−/− organoids. (H) Glucose levels detected in the supernatant of intestinal organoids of the indicated genotypes after 1-hour sucrose incubation. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, 2-sided t-test.
Figure 5
Figure 5
MTG16 binds to ISC- and secretory lineage-signature gene loci. (A) Genome-wide distribution of 7843 MTG16-binding sites. (B) Gene ontology analysis identified ontology terms associated significantly with MTG16 targetome, including Wnt, Notch, and Ephrin pathways, as well as in histone-modifying genes. (C) ChIP-seq data showing MTG16 binding signal (per million reads) to ISC gene loci (Lgr5 and Ascl2). (D, E) Composite profile (D) and heatmap (E) showing striking overlap between ATOH1 and MTG16 binding sites (7843 sites). (F) ChIP-seq data showing MTG16 binding signal (per million reads) to previously reported ATOH1-enhancer regions (asterisk) of the indicated gene. Reduced levels of DHS in enterocyte progenitors (EP) versus secretory progenitors (SP) are indicated by dotted box. (G) MTG16 de novo motif matches with previously reported ATOH1 and ASCL1/2 motif.
Figure 6
Figure 6
Proposed model for intestinal stem cell hierarchy. (A) Updated ISC fate model. See text for details. (B) qRT-PCR analysis of the indicated genes after 1, 2, or 3 days of DAPT treatment. On the right, illustration of expression kinetics of the stem cell, secretory and enterocyte markers upon time-course Notch inhibition. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, 2-sided t-test.
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