YTHDF 1‐mediated translation amplifies Wnt‐driven intestinal stemness
2020; Springer Nature; Volume: 21; Issue: 4 Linguagem: Inglês
10.15252/embr.201949229
ISSN1469-3178
AutoresBing Han, Sujun Yan, Saisai Wei, Jie Xiang, Kangli Liu, Zhanghui Chen, Rongpan Bai, Jinghao Sheng, Zhengping Xu, Xiangwei Gao,
Tópico(s)Cancer-related molecular mechanisms research
ResumoArticle17 February 2020free access Source DataTransparent process YTHDF1-mediated translation amplifies Wnt-driven intestinal stemness Bing Han Institute of Environmental Medicine, Sir Run-Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Sujun Yan Institute of Environmental Medicine, Sir Run-Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Saisai Wei Institute of Environmental Medicine, Sir Run-Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Jie Xiang Institute of Environmental Medicine, Sir Run-Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Kangli Liu Institute of Environmental Medicine, Sir Run-Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Zhanghui Chen Affiliated Hospital of Guangdong Medical University, Zhanjiang, China Search for more papers by this author Rongpan Bai Bioelectromagnetics Laboratory of Zhejiang Province, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Jinghao Sheng Bioelectromagnetics Laboratory of Zhejiang Province, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Zhengping Xu Bioelectromagnetics Laboratory of Zhejiang Province, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Xiangwei Gao Corresponding Author [email protected] orcid.org/0000-0002-8358-6320 Institute of Environmental Medicine, Sir Run-Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Bioelectromagnetics Laboratory of Zhejiang Province, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Bing Han Institute of Environmental Medicine, Sir Run-Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Sujun Yan Institute of Environmental Medicine, Sir Run-Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Saisai Wei Institute of Environmental Medicine, Sir Run-Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Jie Xiang Institute of Environmental Medicine, Sir Run-Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Kangli Liu Institute of Environmental Medicine, Sir Run-Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Zhanghui Chen Affiliated Hospital of Guangdong Medical University, Zhanjiang, China Search for more papers by this author Rongpan Bai Bioelectromagnetics Laboratory of Zhejiang Province, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Jinghao Sheng Bioelectromagnetics Laboratory of Zhejiang Province, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Zhengping Xu Bioelectromagnetics Laboratory of Zhejiang Province, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Xiangwei Gao Corresponding Author [email protected] orcid.org/0000-0002-8358-6320 Institute of Environmental Medicine, Sir Run-Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Bioelectromagnetics Laboratory of Zhejiang Province, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Author Information Bing Han1,‡, Sujun Yan1,‡, Saisai Wei1,‡, Jie Xiang1, Kangli Liu1, Zhanghui Chen2, Rongpan Bai3, Jinghao Sheng3, Zhengping Xu3 and Xiangwei Gao *,1,3 1Institute of Environmental Medicine, Sir Run-Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China 2Affiliated Hospital of Guangdong Medical University, Zhanjiang, China 3Bioelectromagnetics Laboratory of Zhejiang Province, Zhejiang University School of Medicine, Hangzhou, China ‡These authors contributed equally to this work *Corresponding author. Tel: +86 571 88208169; Fax: +86 571 88208169; E-mail: [email protected] EMBO Rep (2020)21:e49229https://doi.org/10.15252/embr.201949229 See also: XM Liu & SB Qian (April 2020) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract N6-methyladenosine (m6A) mRNA methylation has emerged as an important player in many biological processes by regulating gene expression. However, its roles in intestinal stem cell (ISC) homeostasis remain largely unknown. Here, we report that YTHDF1, an m6A reader, is highly expressed in ISCs and its expression is upregulated by Wnt signaling at the translational level. Whereas YTHDF1 is dispensable for normal intestinal development in mice, genetic ablation of Ythdf1 dramatically blocks Wnt-driven regeneration and tumorigenesis with reduced ISC stemness. Mechanistically, YTHDF1 facilitates the translation of Wnt signaling effectors including TCF7L2/TCF4, while this process is enhanced during Wnt activation to augment β-catenin activity. Targeting YTHDF1 in ISCs of established tumors leads to tumor shrinkage and prolonged survival. Collectively, our studies unveil YTHDF1 as an amplifier of Wnt/β-catenin signaling at the translational level, which is required for the maintenance of ISCs during regeneration and tumorigenesis. Synopsis YTHDF1, an m6A reader, is dispensable for normal intestinal development but required for Wnt-driven regeneration and tumorigenesis. YTHDF1 facilitates the translation of Wnt signaling effectors to regulate intestinal stem cell activity. YTHDF1 is highly expressed in intestinal stem cells and regulated by Wnt signaling. YTHDF1 is required for Wnt-driven regeneration and tumorigenesis. YTHDF1 promotes stemness of intestinal stem cells. YTHDF1 facilitates the translation of Wnt signaling effectors to augment β-catenin activity. Introduction The intestinal epithelium harbors remarkable self-renewal capacity driven by the intestinal stem cells (ISCs) at the intestinal crypts 1. The canonical Wnt/β-catenin (CTNNB1) signaling pathway is one of the key regulators of intestinal stemness, which is responsible for the control of intestinal homeostasis, regeneration, and tumorigenesis 2. Under physiological condition, Wnt activity is tightly regulated to form a gradient in the intestinal crypt, which is essential for the undifferentiated state of ISCs and epithelium homeostasis 2. In response to intestine damage, this signaling is activated to drive tissue regeneration 3. The aberrant activation of Wnt/β-catenin signaling, frequently observed in colorectal cancer (CRC) patient samples, leads to the development of CRC 4, 5. As the central signal transducer of Wnt pathway, β-catenin protein is tightly regulated. In the absence of a Wnt signal, the cytosolic levels of β-catenin are kept low by the destruction complex including axin, adenomatous polyposis coli (APC), and glycogen synthase kinase 3β (GSK3β). Wnt activation disrupts this complex, resulting in the translocation of β-catenin to the nucleus, where it associates with the T-cell factor/lymphoid enhancer factor (TCF/LEF) family of transcription factors to activate transcription of downstream target genes 2. Although stabilization and localization of β-catenin contribute to its activation, other layers of Wnt/β-catenin signaling regulation remain to be characterized. Recent studies point to a role of the mRNA methylation in the regulation of gene expression. In eukaryotic cells, one abundant and conserved mRNA modification is adenosine methylation at the nitrogen-6 position, N6-methyladenosine (m6A). The biological effects of m6A methylation are mediated by “writer”, “eraser”, and “reader” proteins. The RNA methyltransferase complex, mainly consisting of METTL3, METTL14, and WTAP, catalyzes the m6A methylation of mRNA 6-8. FTO and ALKBH5 have been identified to mediate the reversible removal of this methylation 9, 10. In addition, the YTH domain family proteins serve as the major m6A-binding proteins to regulate RNA metabolism, including mRNA splicing, degradation, and translation 11-14. Through regulating gene expression, m6A methylation plays important roles in a diverse array of biological processes 15. m6A mRNA methylation is emerging as a crucial modulator in regulating the pluripotency of stem cells, while the overall impact of this modification in stem cell biology is complex. For instance, knockout of Mettl3 impairs the differentiation of mouse embryonic stem cells (ESCs) as well as hematopoietic stem cells (HSCs), indicating that m6A promotes stem cell differentiation 16-18. However, m6A modification was also reported to be required for the pluripotency maintenance of human ESCs and HSCs 19, 20. These inconsistent results could be attributed to the cell-type-specific expression of the “writers”, “erasers”, and “readers”. Moreover, the effects of m6A reader proteins are pleiotropic as YTHDF1 promotes the translation whereas YTHDF2 facilitates the degradation of methylated mRNAs, making the outcome more complicated 11-14. To date, the roles of various m6A reader proteins in the maintenance of stem cell features remain largely unexplored. In this study, we systematically investigated the function of YTHDF1 in the gut. Our results indicated that YTHDF1 plays an indispensable role in the activation of β-catenin to sustain ISC characteristics during regeneration and tumorigenesis. Results Wnt signaling promotes YTHDF1 expression at the translational level m6A-dependent gene expression regulation plays critical roles in a variety of biological processes 15. To investigate the possible function of m6A modification in Wnt-regulated intestinal homeostasis, we activated Wnt signaling in mouse intestinal crypt and examined the expression of m6A machinery. Wnt3a treatment slightly increased METTL3 expression, implying the involvement of m6A in Wnt signaling. Focusing on m6A reader proteins, we found that YTHDF1 protein level was dramatically induced by Wnt3a (Fig 1A). Ythdf1 mRNA level was not affected by Wnt3a stimulation (Fig EV1A). Polysome profiling revealed that Ythdf1 mRNA distribution in polysome fractions dramatically increased after Wnt3a treatment (Fig 1B), indicating the activated translation. Inactivating mutations in APC causes constitutive activation of β-catenin. To examine the regulation of YTHDF1 expression by APC mutation, we re-introduced wild-type full-length APC using the lentivirus system in SW620 human CRC cells, which express a non-functional truncated APC protein 4. Overexpression of APC caused a significant reduction in YTHDF1 protein without affecting its mRNA level (Figs 1C and EV1B). YTHDF1 protein was relatively stable even after 12 h of translation inhibition with cycloheximide (CHX), excluding the possibility of protein degradation (Fig EV1C). Polysome profiling confirmed that APC perturbation mainly affected YTHDF1 mRNA translation (Fig 1D). The 5′ untranslated regions (5′UTRs) play essential roles in mRNA translation 21. Luciferase assay demonstrated that APC expression in SW620 cells dramatically decreased the translation mediated by the 5′UTR of YTHDF1 mRNA (Fig 1E). To test whether YTHDF1 was regulated by β-catenin, we silenced β-catenin expression in SW620 cells using siRNAs and detected no obvious change in YTHDF1 expression (Fig EV1D and E). Reciprocally, expression of a non-degradable β-catenin mutant (N90) into RKO cells, whose APC/β-catenin pathway is intact, did not affect YTHDF1 expression (Fig EV1F and G), indicating that YTHDF1 is not regulated by β-catenin. Collectively, our data demonstrated that Wnt ligand treatment or APC mutation but not β-catenin promotes YTHDF1 expression. Figure 1. Wnt signaling promotes YTHDF1 expression at the translational level Immunoblot analysis of mouse intestinal crypts treated with Wnt3a (60 ng/ml) for indicated time. The relative protein level was quantified and shown under each band. Polysome profiles of mouse intestinal crypt treated with or without Wnt3a for 30 min. The right panels show the distributions of Ythdf1 and Actb in polysome fractions. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01 (3 biological replicates, t-test). Immunoblot analysis of SW620 cells with or without APC overexpression. The relative protein level was quantified and shown under each band. Polysome profiles of SW620 cells with or without APC overexpression. The right panels show the distributions of YTHDF1 and ACTB in polysome fractions. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01 (3 biological replicates, t-test). Dual-Luciferase Assay with a construct bearing the 5′UTR of YTHDF1 in SW620 cells with or without APC overexpression. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01 (3 biological replicates, t-test). YTHDF1 protein expression in intestinal tissue from 4 pairs of Apc+/+ and Apcmin/+ mice. N, normal tissue. T, tumor tissue. A, tumor-adjacent tissue. YTHDF1 staining in matched samples of human CRC tissue and adjacent non-tumor tissue. Scale bar, 450 μm. The right panel shows the statistics of IHC scores. Chi-square test; n = 75 for each group. Source data are available online for this figure. Source Data for Figure 1 [embr201949229-sup-0008-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Wnt signaling promotes YTHDF1 expression RT–qPCR analysis of mouse intestinal crypt treated with Wnt3a (60 ng/ml) for the indicated time. Data are represented as mean ± SEM. Three biological replicates. RT–qPCR analysis of SW620 cells with or without APC overexpression. Data are represented as mean ± SEM. Three biological replicates. Immunoblot analysis of SW620 cells with or without APC overexpression treated with CHX for the indicated time. Immunoblot analysis of SW620 cells with or without β-catenin (CTNNB1) knockdown. RT–qPCR analysis of SW620 cells with or without β-catenin (CTNNB1) knockdown. Data are represented as mean ± SEM. Three biological replicates. Immunoblot analysis of RKO cells expressing a non-degradable β-catenin mutant (N90). RT–qPCR analysis of RKO cells expressing a non-degradable β-catenin mutant (N90). Data are represented as mean ± SEM. Three biological replicates. Ythdf1 mRNA expression in intestinal tissue from 4 pairs of Apc+/+ (Normal), Apcmin/+ tumor (Tumor), and Apcmin/+ tumor-adjacent tissues (Adjacent). Data are represented as mean ± SEM. Three biological replicates. YTHDF1 protein expression in intestinal tissue from 2 pairs of AOM/DSS-treated mice. T: tumor tissue; A: adjacent non-tumor tissue. YTHDF1 staining in matched samples of human CRC tissue at different stages and adjacent non-tumor tissues. Scale bar, 450 μm. IHC scoring and analysis of YTHDF1 expression in (J). Chi-square was used for analysis. Analysis of mRNA level of m6A-related genes from The Cancer Genome Atlas (TCGA) datasets. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (t-test). Source data are available online for this figure. Download figure Download PowerPoint Since mutations in APC are frequently observed in CRC 5, 22, we examined whether YTHDF1 expression was altered in mouse and human intestinal tumors. We found that YTHDF1 protein was weakly expressed in the mouse intestinal tissue while sharply upregulated following Apc mutation (Apcmin/+). High YTHDF1 expression was maintained in intestinal adenomas from the Apcmin/+ mice but not in adjacent non-tumor tissue (Figs 1F and EV1H). Immunohistochemical staining revealed that YTHDF1 protein level is ubiquitously higher in CRC tissues than that in adjacent non-tumor tissues (Figs 1G and EV1J and K). TCGA datasets revealed upregulation of YTHDF1 expression in CRC (Fig EV1L). These results suggested that an increase in YTHDF1 might play an oncogenic role in intestinal tumorigenesis. YTHDF1 is required for intestinal epithelial regeneration In order to assess the function of intestinal epithelial YTHDF1, we generated a Ythdf1 conditional knockout mouse strain under the villin promoter (VillinCre/+:Ythdf1fl/fl, termed as Ythdf1cKO) by targeting exon 4 (Fig EV2A–C). To determine the in vivo implications of YTHDF1 in normal intestinal homeostasis, intestinal architecture and body weight were evaluated in wild-type (Ythdf1CTL) and Ythdf1cKO mice at 8 weeks of age. The ablation of Ythdf1 in intestinal epithelium did not affect body mass (Fig EV2D), crypt–villus architecture, and epithelial cell proliferation (Figs 2A and EV2E–H). Alkaline phosphatase staining (enterocytes) and Alcian blue staining (goblet cells) did not show significant difference between Ythdf1CTL and Ythdf1cKO intestine (Fig EV2I). Thus, YTHDF1 is largely dispensable for intestinal homeostasis under physiological condition. Click here to expand this figure. Figure EV2. YTHDF1 is dispensable for intestinal development Generation of Ythdf1 conditional knockout (Ythdf1cKO) mouse. Genotyping of wild-type (Ythdf1CTL), heterogeneous (Ythdf1Heter), and Ythdf1cKO mice. Immunoblot analysis of intestinal epithelia from Ythdf1CTL and Ythdf1cKO mice. Bodyweight of Ythdf1CTL and Ythdf1cKO mice at the age of 2 months. Data are represented as mean ± SEM. (15 mice for each group, t-test) H&E staining of duodenum, ileum, and colon from wild-type (Ythdf1CTL) and Ythdf1cKO mice. The quantification of crypt height in (E). Scale bar, 50 μm. Data are represented as mean ± SEM. (9 mice for each group, t-test) BrdU staining of duodenum, ileum, and colon from wild-type (Ythdf1CTL) and Ythdf1cKO mice. The quantification of BrdU+ cells per crypt in (G). Scale bar, 50 μm. Data are represented as mean ± SEM. (9 mice for each group, t-test) Alkaline phosphatase staining (ALPI) and Alcian blue staining (Alcian blue) of small intestine from wild-type (Ythdf1CTL) and Ythdf1cKO mice. Scale bar, 50 μm. Source data are available online for this figure. Download figure Download PowerPoint Figure 2. YTHDF1 is required for efficient intestinal regeneration The representative jejunum from wild-type (Ythdf1CTL) and Ythdf1cKO mice. Left: hematoxylin and eosin staining (H&E); middle: BrdU staining; right: quantification of crypt height and BrdU+ cells. Scale bar, 50 μm. Data are represented as mean ± SEM. (9 biological replicates, t-test). Immunoblot analysis of small intestines from Ythdf1CTL and Ythdf1cKO mice after 12 Gy IR. YTHDF1 (left) and Ki67 (right) staining of small intestine from wild-type mice 72 h after 12 Gy IR. Scale bar, 50 μm. Red arrows indicate the proliferating cells. Small intestines from Ythdf1CTL and Ythdf1cKO mice 72 h after 12 Gy IR. Left: H&E staining; middle: Ki67 staining; right: quantification of crypt height and Ki67+ cells. Scale bar, 50 μm. Vertical bars indicate the length of the crypts. Data are represented as mean ± SEM. ****P < 0.0001 (9 biological replicates, t-test). RT–qPCR analysis of small intestines from Ythdf1CTL and Ythdf1cKO mice 72 h after 12 Gy IR. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01 (5 biological replicates, t-test). Source data are available online for this figure. Source Data for Figure 2 [embr201949229-sup-0009-SDataFig2.pdf] Download figure Download PowerPoint The ability of the intestinal epithelium to regenerate after challenge is a Wnt-driven process that mimics the proliferation observed after Apc deletion 22, 23. We, therefore, examined YTHDF1 expression in response to intestinal injury. Mice were exposed to 12 Gy whole-body γ-irradiation (IR), and then, YTHDF1 expression was examined at various time points during the recovery phase. YTHDF1 protein level was markedly upregulated 3 days post-IR during which regenerative proliferation from the radio-resistant ISCs initiated, and then returned to baseline levels within 1 week (Fig 2B). The expression pattern of YTHDF1 was much similar to c-Myc, a proliferation marker (Fig 2B). Immunohistochemistry revealed that YTHDF1-expressing cells were located in the regenerative foci known to exhibit high Ki67 expression (Fig 2C). Together, these data suggested the induction of YTHDF1 during regeneration. Given the dynamic changes of YTHDF1 expression in response to irradiation, we considered that YTHDF1 may play a role during intestinal regeneration. In Ythdf1CTL mice, small intestine regeneration was characterized by a rapid renewal of intestinal crypts, which could be seen 72 h following 12 Gy whole-body IR (Fig 2D). By comparison, Ythdf1cKO mice showed a clear defect in recovery capability which was highlighted by a significant reduction in both the amount and size of regenerating crypts (Fig 2D). Moreover, a remarkably reduced amount of Ki67-positive cells was manifested within the Ythdf1cKO intestinal crypts (Fig 2D). Knockout of Ythdf1 significantly reduced the expression of Wnt target genes including Lgr5, Fzd7, and Myc (Fig 2E) 24, 25. Taken together, these data suggested that YTHDF1 within the intestinal epithelium is critical for Wnt-driven intestinal regeneration. Ythdf1 deletion reduces Wnt-driven tumorigenesis in vivo Given that YTHDF1 was upregulated in intestinal tumors, we examined the function of YTHDF1 in Wnt-driven tumorigenesis using the Apcmin/+ mouse model. Deficiency of YTHDF1 dramatically reduced intestinal tumor formation (Fig 3A). Both tumor number and tumor load were significantly diminished in Ythdf1-deleted mice (Fig 3B and C). Furthermore, analysis of tumor-size distribution showed that most of the tumors in Ythdf1-deleted mice were smaller in size (Fig 3D). Figure 3. Ythdf1 deletion reduces Wnt-driven tumorigenesis in vivo A. Small intestines from 24-week-old Apcmin/+; Ythdf1CTL and Apcmin/+; Ythdf1cKO mice. B. H&E staining of small intestines from 24-week-old Apcmin/+; Ythdf1CTL and Apcmin/+; Ythdf1cKO mice. Scale bar, 1 mm. The black arrows indicate the tumors. C, D. Tumor number (C) and size distribution (D) from the small intestines of 24-week-old Apcmin/+; Ythdf1CTL and Apcmin/+; Ythdf1cKO mice. Data are shown as mean ± SEM (6 mice for each group). **P < 0.01, ***P < 0.001 (t-test). E. Workflow of the AOM/DSS-induced colitis-associated cancer model. F. Colons from Ythdf1CTL and Ythdf1cKO mice on day 84 of AOM/DSS induction. G. H&E staining of colons from Apcmin/+; Ythdf1CTL and Apcmin/+; Ythdf1cKO mice after AOM/DSS induction. Scale bar, 200 μm. H, I. Colon tumor number (H) and size distribution (I) from Ythdf1CTL and Ythdf1cKO mice on day 84 of induction. Data are shown as mean ± SEM (6 mice for each group). **P < 0.01 (t-test). Download figure Download PowerPoint To further confirm the results in Apcmin/+ mouse model, we employed another CRC model induced by azoxymethane (AOM)/dextran sulfate sodium (DSS) (Fig 3E). In this model, mice develop CRC as the result of mutations in several genes including β-catenin pathways 26. In AOM/DSS-induced tumor, YTHDF1 expression is upregulated (Fig EV1I). Similar to the Apcmin/+ model, decreased tumor number, tumor load, and size were observed in Ythdf1cKO mice compared to the wild-type group (Fig 3F–I). These data highlighted the necessity of YTHDF1 in Wnt-driven intestinal tumor development. YTHDF1 is required for maintenance of mouse intestinal stem cells To start exploring the mechanism of YTHDF1-promoted intestinal tumorigenesis, we examined the YTHDF1 expression pattern in the intestine. In situ hybridization revealed that Ythdf1 mRNA was predominantly localized in crypts, where the ISCs reside (Fig 4A). Consistently, fractionating primary intestinal tissue into villus-enriched and crypt-enriched populations also revealed gradient expression of YTHDF1 from intestinal stem cell localized crypts to the differentiated villi (Fig 4B). Lineage tracing has identified the Wnt target gene leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) as an ISC marker 27. Taken advantage of the Lgr5-EGFP-IRES-creERT2 mouse, we examined the expression of YTHDF1 in epithelial cells with various degrees of stemness. The YTHDF1 expression level was high in the Lgr5-GFPhigh stem cells, intermediate in the Lgr5-GFPlow transit-amplifying cell population, and low in the Lgr5-GFPneg population (Fig 4C). Figure 4. YTHDF1 is required for maintenance of intestinal stem cells Fluorescent in situ hybridization for Ythdf1 mRNA in Ythdf1CTL and Ythdf1cKO small intestines. Scale bar, 50 μm. Immunoblot analysis of fractionated murine intestine enriched for villus cells and crypt cells. Flow cytometry analysis of YTHDF1 protein expression in GFPhigh/low/neg populations from Lgr5-GFP-IRES-creERT2 mice. Morphology of organoids from Lgr5-creERT2:Ythdf1fl/fl mice in Wnt3a-conditioned medium without or with 4-OHT induction and infected with lentivirus expressing YTHDF1 or YTHDF1 mutant. Scale bar, 250 μm. Quantification of differentiated versus undifferentiated organoids from (D). Data are represented as mean ± SEM. **P < 0.01 (4 biological replicates, t-test). Relative mRNA levels of intestinal stem cell and differentiation marker genes in organoids described in (D). Data are represented as mean ± SEM. 4 biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001 (t-test). Source data are available online for this figure. Source Data for Figure 4 [embr201949229-sup-0010-SDataFig4.pdf] Download figure Download PowerPoint Given the high expression of YTHDF1 in ISCs, we asked whether Ythdf1 deletion-induced intestinal defects are related to the dysfunction of ISCs. To this end, we deleted Ythdf1 in Lgr5+ ISCs using tamoxifen-inducible Lgr5-creERT (Lgr5-creERT2:Ythdf1fl/fl) and performed organoid culture. Ythdf1-deficient organoids grew comparably to the control organoids but only with less budding (Fig EV3A and B). Next, we cultured organoids with Wnt3a-conditioned medium. Wnt-treated organoids formed fast-growing “spheroids” displaying a cyst-like morphology lacking the budding crypts (Fig 4D and E). Surprisingly, Ythdf1 deletion induced extensive budding by day 5, implying the loss of stemness (Fig 4D and E). Consistently, Ythdf1 deletion in ISCs dramatically reduced the expression of stem cell markers Lgr5, Ascl2, and Olfm4, while increased the expression of differentiation markers ChgA, Anpep, and Fabp2 (Fig 4F). Click here to expand this figure. Figure EV3. The role of m6A in the maintenance of intestinal stemness Morphology of organoids from wild-type (Ythdf1CTL) and Ythdf1cKO mice at day 6. Scale bar, 250 μm. Quantification of percentage of organoid budding in (A) (3 independent replicates). Immunoblot analysis of organoids infected with lentivirus expression Flag-tagged YTHDF1 (Flag-Y1) and YTH domain-deleted mutant (Flag-Y1-mut). Immunoblot analysis of organoids with or without METTL3 knockdown. Morphology of organoids in Wnt3a-conditioned medium with or without METTL3 knockdown. Scale bar, 200 μm. Quantification of differentiated versus undifferentiated organoids from (E). Data are represented as mean ± SEM. Three biological replicates. Spheroid diameter analysis from (E). Data are represented as mean ± SEM. ***P < 0.001 (3 biological replicates, t-test). Source data are available online for this figure. Download figure Download PowerPoint To confirm that the observed defects resulted from loss of YTHDF1, we performed rescue experiments. We delivered lentivirus expressing either YTHDF1, YTH domain-deleted mutant, or a control vector (Fig EV3C). Re-expression of YTHDF1 in Ythdf1-deleted organoids substantially enhanced the spheroid features and restored the expressions of stem cell markers. However, the YTH domain-deleted YTHDF1 mutant could not rescue the phenotypes by Ythdf1 deletion (Fig 4D–F). To substantiate the role of m6A in the maintenance of stemness, METTL3 was silenced. Knockdown of METTL3 dramatically inhibited the growth of organoids and induced differentiation of organoids under Wnt3a treatment (Fig EV3D–G). Transcriptome-wide identification of YTHDF1-regulated mRNAs To identify potential targets regulated by YTHDF1, we performed antibody-based m6A profiling and RNA sequencing (m6A-seq) as well as RNA immunoprecipitation and sequencing (RIP-seq) in colorectal cancer cell line HCT116, harboring a constitutively activating mutation of β-catenin. m6A-seq revealed the expected distribution of m6A within the transcriptome and enrichment around the stop codon within transcripts (Fig EV4A). The analysis yielded 17,707 m6A peaks within 8,076 transcripts (Fig EV4B and C, Dataset EV1). By combining with the RIP-seq data, we identified 3,388 m6A-containing transcripts that are bound by YTHDF1 (referred to YTHDF1 targets, Fig EV4C and Dataset EV2). Given that YTHDF1 is known to affect mRNA translation 12, we assessed the changes in translational efficiency by ribosome profiling (Ribo-seq) after YTHDF1 knockdown using two shRNAs 12, 28 (Fig EV4D and E, Dataset EV3). Data revealed 1015 decreased mRNAs and 387 increased mRNAs in translational efficiency after YTHDF1 knockdown, with slight changes in transcription (Fig 5A). As expected, we observed an elevated m6A methylation level in the translational downregulated genes after YTHDF1 knockdown (Fig 5B). Reciprocally, we analyzed the translational changes based on m6A modification. Data revealed a notable
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