Research ArticleGastroenterology Open Access | 10.1172/jci.insight.171657

Charles H. Danan,1,2,3 Kaitlyn E. Naughton,1 Katharina E. Hayer,4,5,6 Sangeevan Vellappan,7,8,9 Emily A. McMillan,1 Yusen Zhou,1,4 Rina Matsuda,3,10 Shaneice K. Nettleford,1 Kay Katada,1,3 Louis R. Parham,1,3 Xianghui Ma,1 Afrah Chowdhury,1 Benjamin J. Wilkins,6 Premal Shah,8,9 Matthew D. Weitzman,3,5,6 and Kathryn E. Hamilton1,3,11

1Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, Children’s Hospital of Philadelphia, Perelman School of Medicine;

2Medical Scientist Training Program, Perelman School of Medicine; and

3Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

4Department of Biomedical and Health Informatics, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.

5Division of Protective Immunity, Children’s Hospital of Philadelphia Research Institute, Philadelphia, Pennsylvania, USA.

6Department of Pathology and Laboratory Medicine, Perelman School of Medicine; University of Pennsylvania, Philadelphia, Pennsylvania, USA.

7Waksman Institute of Microbiology and

8Department of Genetics, Rutgers University, Piscataway, New Jersey, USA.

9Human Genetics Institute of New Jersey, Piscataway, New Jersey, USA.

10Department of Pathobiology, School of Veterinary Medicine, and

11Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Address correspondence to: Kathryn E. Hamilton, 903 Abramson Research Building, Children’s Hospital of Philadelphia, 3615 Civic Center Blvd., Pennsylvania 19104, USA. Phone: 267.426.5266; Email: hamiltonk1@chop.edu.

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1Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, Children’s Hospital of Philadelphia, Perelman School of Medicine;

2Medical Scientist Training Program, Perelman School of Medicine; and

3Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

4Department of Biomedical and Health Informatics, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.

5Division of Protective Immunity, Children’s Hospital of Philadelphia Research Institute, Philadelphia, Pennsylvania, USA.

6Department of Pathology and Laboratory Medicine, Perelman School of Medicine; University of Pennsylvania, Philadelphia, Pennsylvania, USA.

7Waksman Institute of Microbiology and

8Department of Genetics, Rutgers University, Piscataway, New Jersey, USA.

9Human Genetics Institute of New Jersey, Piscataway, New Jersey, USA.

10Department of Pathobiology, School of Veterinary Medicine, and

11Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Address correspondence to: Kathryn E. Hamilton, 903 Abramson Research Building, Children’s Hospital of Philadelphia, 3615 Civic Center Blvd., Pennsylvania 19104, USA. Phone: 267.426.5266; Email: hamiltonk1@chop.edu.

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1Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, Children’s Hospital of Philadelphia, Perelman School of Medicine;

2Medical Scientist Training Program, Perelman School of Medicine; and

3Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

4Department of Biomedical and Health Informatics, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.

5Division of Protective Immunity, Children’s Hospital of Philadelphia Research Institute, Philadelphia, Pennsylvania, USA.

6Department of Pathology and Laboratory Medicine, Perelman School of Medicine; University of Pennsylvania, Philadelphia, Pennsylvania, USA.

7Waksman Institute of Microbiology and

8Department of Genetics, Rutgers University, Piscataway, New Jersey, USA.

9Human Genetics Institute of New Jersey, Piscataway, New Jersey, USA.

10Department of Pathobiology, School of Veterinary Medicine, and

11Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Address correspondence to: Kathryn E. Hamilton, 903 Abramson Research Building, Children’s Hospital of Philadelphia, 3615 Civic Center Blvd., Pennsylvania 19104, USA. Phone: 267.426.5266; Email: hamiltonk1@chop.edu.

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1Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, Children’s Hospital of Philadelphia, Perelman School of Medicine;

2Medical Scientist Training Program, Perelman School of Medicine; and

3Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

4Department of Biomedical and Health Informatics, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.

5Division of Protective Immunity, Children’s Hospital of Philadelphia Research Institute, Philadelphia, Pennsylvania, USA.

6Department of Pathology and Laboratory Medicine, Perelman School of Medicine; University of Pennsylvania, Philadelphia, Pennsylvania, USA.

7Waksman Institute of Microbiology and

8Department of Genetics, Rutgers University, Piscataway, New Jersey, USA.

9Human Genetics Institute of New Jersey, Piscataway, New Jersey, USA.

10Department of Pathobiology, School of Veterinary Medicine, and

11Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Address correspondence to: Kathryn E. Hamilton, 903 Abramson Research Building, Children’s Hospital of Philadelphia, 3615 Civic Center Blvd., Pennsylvania 19104, USA. Phone: 267.426.5266; Email: hamiltonk1@chop.edu.

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1Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, Children’s Hospital of Philadelphia, Perelman School of Medicine;

2Medical Scientist Training Program, Perelman School of Medicine; and

3Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

4Department of Biomedical and Health Informatics, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.

5Division of Protective Immunity, Children’s Hospital of Philadelphia Research Institute, Philadelphia, Pennsylvania, USA.

6Department of Pathology and Laboratory Medicine, Perelman School of Medicine; University of Pennsylvania, Philadelphia, Pennsylvania, USA.

7Waksman Institute of Microbiology and

8Department of Genetics, Rutgers University, Piscataway, New Jersey, USA.

9Human Genetics Institute of New Jersey, Piscataway, New Jersey, USA.

10Department of Pathobiology, School of Veterinary Medicine, and

11Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Address correspondence to: Kathryn E. Hamilton, 903 Abramson Research Building, Children’s Hospital of Philadelphia, 3615 Civic Center Blvd., Pennsylvania 19104, USA. Phone: 267.426.5266; Email: hamiltonk1@chop.edu.

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1Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, Children’s Hospital of Philadelphia, Perelman School of Medicine;

2Medical Scientist Training Program, Perelman School of Medicine; and

3Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

4Department of Biomedical and Health Informatics, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.

5Division of Protective Immunity, Children’s Hospital of Philadelphia Research Institute, Philadelphia, Pennsylvania, USA.

6Department of Pathology and Laboratory Medicine, Perelman School of Medicine; University of Pennsylvania, Philadelphia, Pennsylvania, USA.

7Waksman Institute of Microbiology and

8Department of Genetics, Rutgers University, Piscataway, New Jersey, USA.

9Human Genetics Institute of New Jersey, Piscataway, New Jersey, USA.

10Department of Pathobiology, School of Veterinary Medicine, and

11Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Address correspondence to: Kathryn E. Hamilton, 903 Abramson Research Building, Children’s Hospital of Philadelphia, 3615 Civic Center Blvd., Pennsylvania 19104, USA. Phone: 267.426.5266; Email: hamiltonk1@chop.edu.

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1Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, Children’s Hospital of Philadelphia, Perelman School of Medicine;

2Medical Scientist Training Program, Perelman School of Medicine; and

3Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

4Department of Biomedical and Health Informatics, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.

5Division of Protective Immunity, Children’s Hospital of Philadelphia Research Institute, Philadelphia, Pennsylvania, USA.

6Department of Pathology and Laboratory Medicine, Perelman School of Medicine; University of Pennsylvania, Philadelphia, Pennsylvania, USA.

7Waksman Institute of Microbiology and

8Department of Genetics, Rutgers University, Piscataway, New Jersey, USA.

9Human Genetics Institute of New Jersey, Piscataway, New Jersey, USA.

10Department of Pathobiology, School of Veterinary Medicine, and

11Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Address correspondence to: Kathryn E. Hamilton, 903 Abramson Research Building, Children’s Hospital of Philadelphia, 3615 Civic Center Blvd., Pennsylvania 19104, USA. Phone: 267.426.5266; Email: hamiltonk1@chop.edu.

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1Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, Children’s Hospital of Philadelphia, Perelman School of Medicine;

2Medical Scientist Training Program, Perelman School of Medicine; and

3Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

4Department of Biomedical and Health Informatics, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.

5Division of Protective Immunity, Children’s Hospital of Philadelphia Research Institute, Philadelphia, Pennsylvania, USA.

6Department of Pathology and Laboratory Medicine, Perelman School of Medicine; University of Pennsylvania, Philadelphia, Pennsylvania, USA.

7Waksman Institute of Microbiology and

8Department of Genetics, Rutgers University, Piscataway, New Jersey, USA.

9Human Genetics Institute of New Jersey, Piscataway, New Jersey, USA.

10Department of Pathobiology, School of Veterinary Medicine, and

11Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Address correspondence to: Kathryn E. Hamilton, 903 Abramson Research Building, Children’s Hospital of Philadelphia, 3615 Civic Center Blvd., Pennsylvania 19104, USA. Phone: 267.426.5266; Email: hamiltonk1@chop.edu.

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1Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, Children’s Hospital of Philadelphia, Perelman School of Medicine;

2Medical Scientist Training Program, Perelman School of Medicine; and

3Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

4Department of Biomedical and Health Informatics, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.

5Division of Protective Immunity, Children’s Hospital of Philadelphia Research Institute, Philadelphia, Pennsylvania, USA.

6Department of Pathology and Laboratory Medicine, Perelman School of Medicine; University of Pennsylvania, Philadelphia, Pennsylvania, USA.

7Waksman Institute of Microbiology and

8Department of Genetics, Rutgers University, Piscataway, New Jersey, USA.

9Human Genetics Institute of New Jersey, Piscataway, New Jersey, USA.

10Department of Pathobiology, School of Veterinary Medicine, and

11Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Address correspondence to: Kathryn E. Hamilton, 903 Abramson Research Building, Children’s Hospital of Philadelphia, 3615 Civic Center Blvd., Pennsylvania 19104, USA. Phone: 267.426.5266; Email: hamiltonk1@chop.edu.

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1Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, Children’s Hospital of Philadelphia, Perelman School of Medicine;

2Medical Scientist Training Program, Perelman School of Medicine; and

3Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

4Department of Biomedical and Health Informatics, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.

5Division of Protective Immunity, Children’s Hospital of Philadelphia Research Institute, Philadelphia, Pennsylvania, USA.

6Department of Pathology and Laboratory Medicine, Perelman School of Medicine; University of Pennsylvania, Philadelphia, Pennsylvania, USA.

7Waksman Institute of Microbiology and

8Department of Genetics, Rutgers University, Piscataway, New Jersey, USA.

9Human Genetics Institute of New Jersey, Piscataway, New Jersey, USA.

10Department of Pathobiology, School of Veterinary Medicine, and

11Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Address correspondence to: Kathryn E. Hamilton, 903 Abramson Research Building, Children’s Hospital of Philadelphia, 3615 Civic Center Blvd., Pennsylvania 19104, USA. Phone: 267.426.5266; Email: hamiltonk1@chop.edu.

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1Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, Children’s Hospital of Philadelphia, Perelman School of Medicine;

2Medical Scientist Training Program, Perelman School of Medicine; and

3Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

4Department of Biomedical and Health Informatics, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.

5Division of Protective Immunity, Children’s Hospital of Philadelphia Research Institute, Philadelphia, Pennsylvania, USA.

6Department of Pathology and Laboratory Medicine, Perelman School of Medicine; University of Pennsylvania, Philadelphia, Pennsylvania, USA.

7Waksman Institute of Microbiology and

8Department of Genetics, Rutgers University, Piscataway, New Jersey, USA.

9Human Genetics Institute of New Jersey, Piscataway, New Jersey, USA.

10Department of Pathobiology, School of Veterinary Medicine, and

11Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Address correspondence to: Kathryn E. Hamilton, 903 Abramson Research Building, Children’s Hospital of Philadelphia, 3615 Civic Center Blvd., Pennsylvania 19104, USA. Phone: 267.426.5266; Email: hamiltonk1@chop.edu.

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1Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, Children’s Hospital of Philadelphia, Perelman School of Medicine;

2Medical Scientist Training Program, Perelman School of Medicine; and

3Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

4Department of Biomedical and Health Informatics, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.

5Division of Protective Immunity, Children’s Hospital of Philadelphia Research Institute, Philadelphia, Pennsylvania, USA.

6Department of Pathology and Laboratory Medicine, Perelman School of Medicine; University of Pennsylvania, Philadelphia, Pennsylvania, USA.

7Waksman Institute of Microbiology and

8Department of Genetics, Rutgers University, Piscataway, New Jersey, USA.

9Human Genetics Institute of New Jersey, Piscataway, New Jersey, USA.

10Department of Pathobiology, School of Veterinary Medicine, and

11Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Address correspondence to: Kathryn E. Hamilton, 903 Abramson Research Building, Children’s Hospital of Philadelphia, 3615 Civic Center Blvd., Pennsylvania 19104, USA. Phone: 267.426.5266; Email: hamiltonk1@chop.edu.

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1Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, Children’s Hospital of Philadelphia, Perelman School of Medicine;

2Medical Scientist Training Program, Perelman School of Medicine; and

3Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

4Department of Biomedical and Health Informatics, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.

5Division of Protective Immunity, Children’s Hospital of Philadelphia Research Institute, Philadelphia, Pennsylvania, USA.

6Department of Pathology and Laboratory Medicine, Perelman School of Medicine; University of Pennsylvania, Philadelphia, Pennsylvania, USA.

7Waksman Institute of Microbiology and

8Department of Genetics, Rutgers University, Piscataway, New Jersey, USA.

9Human Genetics Institute of New Jersey, Piscataway, New Jersey, USA.

10Department of Pathobiology, School of Veterinary Medicine, and

11Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Address correspondence to: Kathryn E. Hamilton, 903 Abramson Research Building, Children’s Hospital of Philadelphia, 3615 Civic Center Blvd., Pennsylvania 19104, USA. Phone: 267.426.5266; Email: hamiltonk1@chop.edu.

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1Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, Children’s Hospital of Philadelphia, Perelman School of Medicine;

2Medical Scientist Training Program, Perelman School of Medicine; and

3Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

4Department of Biomedical and Health Informatics, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.

5Division of Protective Immunity, Children’s Hospital of Philadelphia Research Institute, Philadelphia, Pennsylvania, USA.

6Department of Pathology and Laboratory Medicine, Perelman School of Medicine; University of Pennsylvania, Philadelphia, Pennsylvania, USA.

7Waksman Institute of Microbiology and

8Department of Genetics, Rutgers University, Piscataway, New Jersey, USA.

9Human Genetics Institute of New Jersey, Piscataway, New Jersey, USA.

10Department of Pathobiology, School of Veterinary Medicine, and

11Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Address correspondence to: Kathryn E. Hamilton, 903 Abramson Research Building, Children’s Hospital of Philadelphia, 3615 Civic Center Blvd., Pennsylvania 19104, USA. Phone: 267.426.5266; Email: hamiltonk1@chop.edu.

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1Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, Children’s Hospital of Philadelphia, Perelman School of Medicine;

2Medical Scientist Training Program, Perelman School of Medicine; and

3Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

4Department of Biomedical and Health Informatics, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.

5Division of Protective Immunity, Children’s Hospital of Philadelphia Research Institute, Philadelphia, Pennsylvania, USA.

6Department of Pathology and Laboratory Medicine, Perelman School of Medicine; University of Pennsylvania, Philadelphia, Pennsylvania, USA.

7Waksman Institute of Microbiology and

8Department of Genetics, Rutgers University, Piscataway, New Jersey, USA.

9Human Genetics Institute of New Jersey, Piscataway, New Jersey, USA.

10Department of Pathobiology, School of Veterinary Medicine, and

11Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Address correspondence to: Kathryn E. Hamilton, 903 Abramson Research Building, Children’s Hospital of Philadelphia, 3615 Civic Center Blvd., Pennsylvania 19104, USA. Phone: 267.426.5266; Email: hamiltonk1@chop.edu.

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1Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, Children’s Hospital of Philadelphia, Perelman School of Medicine;

2Medical Scientist Training Program, Perelman School of Medicine; and

3Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

4Department of Biomedical and Health Informatics, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.

5Division of Protective Immunity, Children’s Hospital of Philadelphia Research Institute, Philadelphia, Pennsylvania, USA.

6Department of Pathology and Laboratory Medicine, Perelman School of Medicine; University of Pennsylvania, Philadelphia, Pennsylvania, USA.

7Waksman Institute of Microbiology and

8Department of Genetics, Rutgers University, Piscataway, New Jersey, USA.

9Human Genetics Institute of New Jersey, Piscataway, New Jersey, USA.

10Department of Pathobiology, School of Veterinary Medicine, and

11Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Address correspondence to: Kathryn E. Hamilton, 903 Abramson Research Building, Children’s Hospital of Philadelphia, 3615 Civic Center Blvd., Pennsylvania 19104, USA. Phone: 267.426.5266; Email: hamiltonk1@chop.edu.

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Published October 26, 2023 - More info

Published in Volume 8, Issue 23 on December 8, 2023
JCI Insight. 2023;8(23):e171657. https://doi.org/10.1172/jci.insight.171657.
© 2023 Danan et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Published October 26, 2023 - Version history
Received: April 24, 2023; Accepted: October 25, 2023 View PDF Abstract

Intestinal epithelial transit-amplifying cells are essential stem progenitors required for intestinal homeostasis, but their rapid proliferation renders them vulnerable to DNA damage from radiation and chemotherapy. Despite these cells’ critical roles in intestinal homeostasis and disease, few studies have described genes that are essential to transit-amplifying cell function. We report that RNA methyltransferase-like 3 (METTL3) is required for survival of transit-amplifying cells in the murine small intestine. Transit-amplifying cell death after METTL3 deletion was associated with crypt and villus atrophy, loss of absorptive enterocytes, and uniform wasting and death in METTL3-depleted mice. Sequencing of polysome-bound and methylated RNAs in enteroids and in vivo demonstrated decreased translation of hundreds of methylated transcripts after METTL3 deletion, particularly transcripts involved in growth factor signal transduction such as Kras. Further investigation verified a relationship between METTL3 and Kras methylation and protein levels in vivo. Our study identifies METTL3 as an essential factor supporting the homeostasis of small intestinal tissue via direct maintenance of transit-amplifying cell survival. We highlight the crucial role of RNA modifications in regulating growth factor signaling in the intestine with important implications for both homeostatic tissue renewal and epithelial regeneration.

Graphical Abstract Introduction

The intestinal epithelium digests and absorbs nutrients, protects against pathogen invasion, and regulates interactions between mucosal immune cells and the gut lumen (1). These essential functions are made possible by the continuous renewal of differentiated intestinal epithelial cells (2). Epithelial renewal begins at the intestinal crypt base, where intestinal stem cells expressing the Wnt target gene, leucine-rich repeat-containing G protein–coupled receptor 5 (LGR5), initiate differentiation and migrate up the crypt wall. As they move up the crypt wall, LGR5+ stem cells differentiate into intestinal stem progenitors known as transit-amplifying (TA) cells (2). TA cells rapidly undergo successive cycles of proliferation to generate the bulk of intestinal epithelium. While existing research emphasizes the role of LGR5+ stem cells in tissue renewal, TA cells are the primary site of intestinal epithelial proliferation and differentiation, and they produce the majority of differentiated epithelium (3, 4). Rapid proliferation is the central defining feature of TA cells, and it renders them particularly vulnerable to DNA-damaging agents such as chemotherapeutics and radiation therapy (5–7). These routine cancer treatments cause chemotherapy-induced gastrointestinal toxicity (CIGT) and radiation-induced gastrointestinal syndrome (GIS), which together affect more than 80% of patients with cancer. CIGT and GIS are debilitating pathologies with limited treatment options (8–10). One potential therapeutic avenue would be the development of drugs that protect the TA cells preferentially damaged by these common cancer therapies. However, despite TA cells’ critical roles in intestinal homeostasis and disease, factors that maintain survival and proliferation of TA cells remain inadequately defined compared with the extensive study of LGR5+ stem cells.

Novel approaches are needed to identify factors that specifically regulate TA cell function. While numerous studies have defined transcriptional control of intestinal stem cells, posttranscriptional regulation of intestinal epithelial homeostasis is only beginning to be understood. Recent research points to important roles for the RNA modification, N6-methyladenosine (m6A), in intestinal crypts (11–15). m6A is the most common covalent modification of RNA, occurring on approximately 25% of mRNA transcripts (16, 17). It acts by recruiting RNA-binding proteins that affect mRNA fate, predominantly stability and translation. (18). Global m6A methylation patterns in the epithelium can shift with microbial and nutrient contents of the gut, and m6A-binding proteins have been implicated in intestinal regeneration and the pathogenesis of inflammatory bowel disease (11, 13, 15, 19, 20). These studies suggest critical roles for m6A in integrating environmental cues with homeostatic and regenerative processes in the intestinal epithelium.

Despite advances in the study of m6A in the gut, the effect of global depletion of m6A in the intestinal epithelium remains unclear and incomplete, particularly in the small intestine. A highly conserved m6A “writer” complex installs m6A cotranscriptionally in the nucleus of eukaryotic cells. At the core of this complex are the writer proteins methyltransferase-like 3 and 14 (METTL3 and METTL14) (18). Although METTL3 is the catalytic subunit, both METTL3 and METTL14 are thought to be essential for the methylating activity of the complex (21, 22), and both METTL proteins are deleted interchangeably to define the role of m6A in specific tissue or cell types. Recent studies reported essential functions for METTL14 in the survival of LGR5+ stem cells in the colon, with complete sparing of the small intestine (23, 24). In contrast, another very recent study found that METTL3 deletion caused defects in LGR5+ stem cells in the small intestine (25). However, in the case of METTL3 deletion, rescue of LGR5+ stem cell survival could not rescue tissue homeostasis. Therefore, the critical defect in METTL3-depleted epithelium remains unclear.

In contrast with previous reports emphasizing dysfunction of LGR5+ stem cells, we found that METTL3 deletion induced profound cell death predominantly in small intestinal TA cells. Disruption of the TA zone was associated with crypt and villus atrophy and widespread reduction in absorptive enterocytes, ultimately resulting in the death of METTL3-depleted mice. Sequencing of m6A-modified RNA in vivo and polysome-bound RNA in METTL3-depleted enteroids revealed decreased translation efficiency for methylated transcripts critical to growth factor signaling, including master growth regulator and proto-oncogene, Kras. Additional investigation verified a link between METTL3 and Kras methylation and protein expression. Our data identify METTL3 as an essential regulator of intestinal TA cell survival via direct support of growth factor signaling, including KRAS expression. By identifying epitranscriptomic regulation as an indispensable process within TA cells, we highlight the importance of an emergent gene-regulatory mechanism in this critical but poorly understood cell type.

Results

Intestinal epithelial METTL3 deletion results in complete growth failure and mortality. To determine the role of METTL3 in intestinal epithelial development and homeostasis, we paired Mettl3fl/fl mice with the pan-intestinal-epithelial Villin-Cre (Mettl3VilCreΔ/Δ) or its tamoxifen-inducible counterpart, Villin-CreERT2 (inducible Mettl3VilCreERΔ/Δ). First, we examined Mettl3VilCreΔ/Δ mice, which had constitutive Cre activation in the small intestinal and colonic epithelium beginning at embryonic day 12.5 (26). These mice were born with Mendelian distribution (χ2n = 79, P > 0.74) and appeared grossly normal at postnatal day 14, as previously described (27). However, from postnatal day 21 to 28, Mettl3VilCreΔ/Δ mice lost approximately 20% starting body weight while controls gained approximately 40% (Figure 1A). Body condition and weight loss in Mettl3VilCreΔ/Δ mice required euthanasia of 70% of mice between postnatal day 16–29 (Figure 1, B–D). To determine whether this phenotype was development specific, we next examined inducible Mettl3VilCreERΔ/Δ mice injected with tamoxifen at 8 weeks of age (Figure 2A). After their final tamoxifen injection, inducible Mettl3VilCreERΔ/Δ mice exhibited an average daily weight loss of approximately 2.5% (Figure 2B). Within 10 days, almost all mice experienced critical (>20%) weight loss requiring euthanasia (Figure 2, B and C). These data demonstrate a requirement of METTL3 for growth and survival during the postnatal period and adulthood.

Figure 1

Constitutive METTL3 deletion causes growth retardation and small intestinal epithelial distortion. (A) Growth curves from postnatal day 15 to 29. (B) Gross appearance at postnatal day 29. (C) Composite appearance and behavior score at postnatal day 29. (D) Kaplan-Meier survival curves through postnatal day 29; P value corresponds to log-rank (Mantel-Cox) test. (E) Representative small intestine and colon H&E images. Regenerative and atrophic crypts are highlighted. “L” denotes lymphocytic infiltrate. (F and G) Composite histological score for small intestine (SI) and colon. (H) Representative images of Ki67 in distal small intestine and number of hypoproliferative crypts (< 10 Ki67+ cells) per 1 mm distal half small intestine. Hypo- and hyperproliferative crypts are highlighted. (I) Representative images and quantification of TUNEL staining. (J–L) Representative images and quantification of intestinal secretory markers MUC2, LYZ, and CHGA. (M) Representative images and quantification of percentage alkaline phosphatase–positive (ALPI) villus length. Each plotted point corresponds to 1 mouse and depicts the mean of 3 representative sections imaged per mouse with bar at median value. Unless otherwise noted, P value represents unpaired parametric Student’s t test. Immunofluorescence staining and quantification performed in distal half small intestine. All scale bars 100 μm. ECAD, epithelial cadherin; MUC2, mucin 2; LYZ, lysozyme; CHGA, chromogranin A.

Figure 2

Inducible METTL3 deletion causes mortality and small intestinal epithelial disruption in adult mice. (A) Experimental schematic depicting sacrifice 9 days after final tamoxifen injection. (B) Weight curves through 9 days after tamoxifen injection, mean ± SD. (C) Kaplan-Meier survival curves through 22 days after final tamoxifen injection; P value corresponds to log-rank (Mantel-Cox) test. (D) Representative small intestine and colon H&E images. Regenerative and atrophic crypts are highlighted. “L” indicates lymphocytic infiltrate. (E and F) Composite histological score for small intestine (SI) and colon. (G) Representative images of Ki67 and quantification of hypoproliferative (< 10 Ki67+ cells) crypts per 1 mm intestine. Hypo- and hyperproliferative crypts are highlighted. (H) Representative images and quantification of TUNEL staining. (I–K) Representative images and quantification of intestinal secretory markers MUC2, LYZ, and CHGA. (L) Representative images and quantification of percentage alkaline phosphatase–positive villus length. Unless otherwise noted, each data point corresponds to 1 mouse and depicts the mean of 3 representative sections imaged per mouse with bar at median value. Unless otherwise noted, P value represents unpaired parametric Student’s t test. Immunofluorescence staining and quantification performed in distal half small intestine. All scale bars 100 μm.

METTL3 deletion induces small intestinal crypt and villus atrophy. We next examined the intestinal pathology of Mettl3VilCreΔ/Δ and inducible Mettl3VilCreERΔ/Δ mice to determine the cause of their severe growth failure and mortality. We assessed Mettl3VilCreΔ/Δ mice at postnatal day 29 and inducible Mettl3VilCreERΔ/Δ mice at 9 days after tamoxifen injection, since at this time point both cohorts exhibited daily weight loss and more than 50% of mice met the humane endpoints for euthanasia (see Methods). Western blot, in situ staining, and m6A dot blot verified depletion of METTL3 and m6A in the intestinal epithelium (Supplemental Figure 1, A–C, and Supplemental Figure 2, A and B; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.171657DS1). We also observed commensurate depletion of METTL14 in METTL3-knockout tissues, which is consistent with previous reports demonstrating stabilization of METTL14 by METTL3 (Supplemental Figure 1A and Supplemental Figure 2A) (28, 29). Using a composite histopathological score in both METTL3-knockout models, we noted histological changes throughout the small intestine and colon with only the distal colon being relatively spared (Figure 1, E–G, and Figure 2, D–F). Defects were most severe in the distal small intestine, where we observed widespread crypt atrophy alongside villus shortening. After METTL3 knockout, villi in the jejunum averaged approximately 30% of their normal length (Supplemental Figure 1D and Supplemental Figure 2C). Many distal small intestinal crypts also degenerated after METTL3 deletion, although we noted occasional hypertrophic regenerative crypts (Figure 1E and Figure 2D). Since METTL3 and METTL14 knockouts are generally considered equivalent, our findings of small intestinal destruction with distal colonic sparing were striking and unexpected given previous studies indicating METTL14 deletion spares the small intestine but induces severe distal colonic defects (23, 24). These data demonstrate that intestinal epithelial METTL3 is required for both the postnatal development and adult maintenance of full-length crypt and villus structures, particularly in the distal small intestine.

METTL3 is required for intestinal epithelial proliferation and survival. To determine the origins of crypt and villus atrophy, we next evaluated proliferation and apoptosis in Mettl3VilCreΔ/Δ and inducible Mettl3VilCreERΔ/Δ mice. Since histological changes were most severe in the distal small intestine, we focused on Ki67 and TUNEL staining in this tissue. In METTL3-knockout epithelium, atrophied crypts previously observed by H&E exhibited drastically reduced Ki67 staining. Where control crypts had an average of approximately 30 Ki67+ cells/crypt, atrophic METTL3-depleted crypts often exhibited fewer than 10 Ki67+ cells (Figure 1H and Figure 2G). We also observed some hyperproliferative crypts in both deletion models (>45 Ki67+ cells), though in inducible Mettl3VilCreERΔ/Δ mice, these were frequently METTL3+ by immunofluorescence (Supplemental Figure 2D), suggesting small areas of incomplete genetic deletion. Thus, we elected not to quantify these hyperprolifera