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- W3136409253 abstract "Article17 March 2021free access Source DataTransparent process N6-methyladenosine modification of lncRNA Pvt1 governs epidermal stemness Jimmy Lee Jimmy Lee orcid.org/0000-0002-5547-4858 Ben May Department for Cancer Research, The University of Chicago, Chicago, IL, USA Search for more papers by this author Yuchen Wu Yuchen Wu Ben May Department for Cancer Research, The University of Chicago, Chicago, IL, USA Department of Colorectal Surgery, Fudan University Shanghai Cancer Center, Shanghai, China Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China Search for more papers by this author Bryan T Harada Bryan T Harada orcid.org/0000-0002-9683-782X Department of Chemistry, Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, USA Department of Biochemistry and Molecular Biology, Howard Hughes Medical Institute, The University of Chicago, Chicago, IL, USA Search for more papers by this author Yuanyuan Li Yuanyuan Li Ben May Department for Cancer Research, The University of Chicago, Chicago, IL, USA Search for more papers by this author Jing Zhao Jing Zhao Ben May Department for Cancer Research, The University of Chicago, Chicago, IL, USA Search for more papers by this author Chuan He Corresponding Author Chuan He [email protected] orcid.org/0000-0003-4319-7424 Department of Chemistry, Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, USA Department of Biochemistry and Molecular Biology, Howard Hughes Medical Institute, The University of Chicago, Chicago, IL, USA Search for more papers by this author Yanlei Ma Corresponding Author Yanlei Ma [email protected] orcid.org/0000-0002-0632-5258 Department of Colorectal Surgery, Fudan University Shanghai Cancer Center, Shanghai, China Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China Search for more papers by this author Xiaoyang Wu Corresponding Author Xiaoyang Wu [email protected] orcid.org/0000-0001-6378-3207 Ben May Department for Cancer Research, The University of Chicago, Chicago, IL, USA Search for more papers by this author Jimmy Lee Jimmy Lee orcid.org/0000-0002-5547-4858 Ben May Department for Cancer Research, The University of Chicago, Chicago, IL, USA Search for more papers by this author Yuchen Wu Yuchen Wu Ben May Department for Cancer Research, The University of Chicago, Chicago, IL, USA Department of Colorectal Surgery, Fudan University Shanghai Cancer Center, Shanghai, China Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China Search for more papers by this author Bryan T Harada Bryan T Harada orcid.org/0000-0002-9683-782X Department of Chemistry, Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, USA Department of Biochemistry and Molecular Biology, Howard Hughes Medical Institute, The University of Chicago, Chicago, IL, USA Search for more papers by this author Yuanyuan Li Yuanyuan Li Ben May Department for Cancer Research, The University of Chicago, Chicago, IL, USA Search for more papers by this author Jing Zhao Jing Zhao Ben May Department for Cancer Research, The University of Chicago, Chicago, IL, USA Search for more papers by this author Chuan He Corresponding Author Chuan He [email protected] orcid.org/0000-0003-4319-7424 Department of Chemistry, Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, USA Department of Biochemistry and Molecular Biology, Howard Hughes Medical Institute, The University of Chicago, Chicago, IL, USA Search for more papers by this author Yanlei Ma Corresponding Author Yanlei Ma [email protected] orcid.org/0000-0002-0632-5258 Department of Colorectal Surgery, Fudan University Shanghai Cancer Center, Shanghai, China Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China Search for more papers by this author Xiaoyang Wu Corresponding Author Xiaoyang Wu [email protected] orcid.org/0000-0001-6378-3207 Ben May Department for Cancer Research, The University of Chicago, Chicago, IL, USA Search for more papers by this author Author Information Jimmy Lee1, Yuchen Wu1,2,3, Bryan T Harada4,5, Yuanyuan Li1, Jing Zhao1, Chuan He *,4,5, Yanlei Ma *,2,3 and Xiaoyang Wu *,1 1Ben May Department for Cancer Research, The University of Chicago, Chicago, IL, USA 2Department of Colorectal Surgery, Fudan University Shanghai Cancer Center, Shanghai, China 3Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China 4Department of Chemistry, Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, USA 5Department of Biochemistry and Molecular Biology, Howard Hughes Medical Institute, The University of Chicago, Chicago, IL, USA *Corresponding author. Tel: +1 773 702 5061; E-mail: [email protected] *Corresponding author. Tel: +86 13122680635; E-mail: [email protected] *Corresponding author. Tel: +1 773 702 1110; Fax: +1 773 702 4476; E-mail: [email protected] The EMBO Journal (2021)40:e106276https://doi.org/10.15252/embj.2020106276 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 Figures & Info Abstract Dynamic chemical modifications of RNA represent novel and fundamental mechanisms that regulate stemness and tissue homeostasis. Rejuvenation and wound repair of mammalian skin are sustained by epidermal progenitor cells, which are localized within the basal layer of the skin epidermis. N6-methyladenosine (m6A) is one of the most abundant modifications found in eukaryotic mRNA and lncRNA (long noncoding RNA). In this report, we survey changes of m6A RNA methylomes upon epidermal differentiation and identify Pvt1, a lncRNA whose m6A modification is critically involved in sustaining stemness of epidermal progenitor cells. With genome-editing and a mouse genetics approach, we show that ablation of m6A methyltransferase or Pvt1 impairs the self-renewal and wound healing capability of skin. Mechanistically, methylation of Pvt1 transcripts enhances its interaction with MYC and stabilizes the MYC protein in epidermal progenitor cells. Our study presents a global view of epitranscriptomic dynamics that occur during epidermal differentiation and identifies the m6A modification of Pvt1 as a key signaling event involved in skin tissue homeostasis and wound repair. SYNOPSIS The impact of RNA modification pathways on skin biology has remained poorly explored. Here, combined mouse genetics and multi-layered profiling identifies methyltransferase Mettl14-dependent N6-methyladenosine (m6A) modification as a critical regulator of epidermal stem cell differentiation and homeostasis. Tissue-specific depletion of Mettl14 reduces global m6A levels and perturbed skin development. Depletion of Mettl14 decreases epidermal stem cell function. Epidermal differentiation is associated with significant methylome changes in mRNAs and long non-coding RNAs (lncRNAs). m6A modification on lncRNA Pvt1 enhances its association with MYC and supports epidermal stemness in vivo. Introduction Chemical modifications of RNA are critically involved in many biological processes. To date, over 150 different RNA modifications have been identified with a wide variety of chemical diversities (http://rna-mdb.cas.albany.edu/RNAmods/) (Cantara et al, 2011). Besides abundant and diverse modifications on tRNA and rRNA, some of these modifications also occur on mammalian mRNA and lncRNA (long noncoding RNA). These post-transcriptional changes are crucial to many fundamental processes including cell differentiation, tissue development, metabolism, neural functions, immune response, and viral infection. N6-methyladenosine (m6A) is one of the most abundant modifications found in eukaryotic mRNA and lncRNA. The m6A modification is ubiquitous among all higher eukaryotes and among viruses that replicate inside host nuclei. Each mammalian mRNA contains ~3 m6A modifications within a consensus sequence of Pu[G>A]m6AC[A/C/U] (Harper et al, 1990). Antibody-based m6A-profiling has revealed human and mouse m6A RNA methylomes (Dominissini et al, 2012; Meyer et al, 2012), indicating that these modifications are preserved across different tissues and are subject to dynamic modulation, further suggesting the regulatory roles of m6A. The recent extensive functional characterizations of m6A in eukaryotes uncovered it as a basic mechanism to control processing, transport, translation, and stability of mRNAs and lncRNAs (Yue et al, 2015; Zhao et al, 2017; Roundtree et al, 2017a; Zhao et al, 2018). This modification is installed on mRNA by a dedicated methyltransferase complex, with Mettl3 (methyltransferase like 3) and Mettl14 (Methyltransferase like 14) forming the core catalytic complex, but also with a number of other protein cofactors (Yue et al, 2015; Wen et al, 2018; Yue et al, 2018). It can be removed by two erasers, FTO (fat mass and obesity-associated protein/α-ketoglutarate-dependent dioxygenase) and ALKBH5 (AlkB homolog 5). There are a number of proteins that have been identified as reader proteins that could recognize m6A-methylated RNA (Wang et al, 2014; Wang et al, 2015; Liu et al, 2015b; Hsu et al, 2017; Shi et al, 2017; Roundtree et al, 2017b; Huang et al, 2018), including the YTH (YT521-B homology) domain family of proteins (Theler et al, 2014; Xu et al, 2014). Studies from the past few years suggest that m6A as an mRNA chemical mark allows cells to group hundreds to thousands of transcripts for coordinated translation and transcriptome turnover during stem cell differentiation or in response to cellular and environmental signals (Zhao et al, 2017; Roundtree et al, 2017a). Mammalian skin protects us from many different environmental damages (Fuchs, 2008). The epidermal progenitor/stem cells that localize at the basal layer of the epidermis play a critical role in tissue homeostasis of skin. During epidermal stratification, basal progenitor cells can migrate upward from the basement membrane and differentiate to replenish the tissue with new differentiated epidermal keratinocytes (Fuchs, 2008; Lopez-Pajares et al, 2013; Perdigoto et al, 2014). Differentiation of basal progenitor cells involves the synthesis and modification of different lipid and protein molecules of differentiated corneocytes, the controlled disruption, and degradation of subcellular organelles and the nucleus, as well as the permanent withdrawal from the cell cycle. Deregulation of skin differentiation can lead to many different skin diseases, such as inflammatory skin diseases and skin cancers. Despite our understanding of the transcriptional changes and signal transductions involved in skin differentiation (Fuchs, 2008; Lopez-Pajares et al, 2013; Perdigoto et al, 2014), the potential role of m6A modification in skin development and adult skin tissue homeostasis remains largely unclear. Using a mouse genetics approach, we found that conditional ablation of Mettl14 (Yue et al, 2015), the core subunit of the RNA methyltransferase in skin epidermis, can lead to diminished stemness of basal progenitor cells and aberrant epidermal differentiation in vivo. With transcriptome-wide profiling, we further demonstrated profoundly altered RNA m6A methylomes upon epidermal differentiation. Gene Ontology (GO) term and enrichment analysis unraveled various enriched RNA clusters after skin differentiation, including both mRNAs and lncRNAs. LncRNAs represent an emerging class of noncoding RNAs that integrate a myriad of external signaling to regulate gene expression and epigenetic reprogramming of cells (Flynn & Chang, 2014). Increasing evidence have implicated lncRNAs in epidermal development and differentiation (Hombach & Kretz, 2013; Wan & Wang, 2014); however, the complexity of the signaling networks involved in these processes in skin remains poorly defined. Our data revealed that the lncRNA Pvt1 (plasmacytoma variant translocation gene 1) displayed significantly reduced m6A modification upon epidermal differentiation. In the human genome, Pvt1 is located in the well-established cancer risk region 8q24 on chromosome 8 and is immediately adjacent to the oncogene MYC (myelocytomatosis oncogene) (Tseng et al, 2014; Colombo et al, 2015). 8q24 has frequent genetic aberrations, including translocation, amplification, and viral integration in different types of malignancies in humans. The copy number of Pvt1 was co-amplified in more than 98% of MYC-copy-increase cancers, and a gain of Pvt1 expression is required for a high MYC oncogene level in 8q24-amplifed cancer cells. It has been shown that Pvt1 can physically interact with MYC and protect it from protein degradation (Tseng et al, 2014). Adding to this, the MYC-Pvt1 genes are syntenic not only in the human genome, but also in other mammalian species, including the mouse (chromosome 15) and rat (chromosome 7). In the skin epidermis, mounting evidence has demonstrated a central role of MYC in controlling tissue homeostasis, stemness of basal progenitor cells, and skin carcinogenesis (Arnold & Watt, 2001; Waikel et al, 2001; Frye et al, 2003; Zanet et al, 2005; Oskarsson et al, 2006; Watt et al, 2008). Interestingly, in addition to regulation of translation or RNA stability, the m6A modification can also lead to profound changes in the mRNA or lncRNA secondary structure, and thus alter their interaction with proteins, a process known as an “m6A switch” (Liu et al, 2015b; Zhou et al, 2016). For instance, the lncRNA MALAT1 (metastasis associated lung adenocarcinoma transcript 1) can undergo a conformational change to expose a U5-tract for recognition and binding by HNRNPC (heterogeneous nuclear ribonucleoprotein C) upon m6A modification (Liu et al, 2015b; Zhou et al, 2016). Our data showed that the m6A modification on five key residues of Pvt1 is essential for its interaction with MYC and protects MYC protein stability. Overall, our results unraveled RNA methylome dynamics upon epidermal differentiation and identified an important molecular pathway whereby m6A modification of Pvt1 regulates differentiation of somatic stem cells in skin. Results Conditional ablation of Mettl14 leads to aberrant skin development and inhibits skin wound healing RNA modifications can coordinate complex translation and transcriptome turnover during stem cell differentiation (Zhao et al, 2017; Roundtree et al, 2017a). Western blot analysis indicates a specific decrease in Mettl14 protein upon calcium-induced epidermal differentiation in vitro (Appendix Fig S1A), suggesting a potentially significant role of RNA m6A modification in this process. To further investigate RNA m6A modification in skin development and tissue homeostasis, we developed a skin cKO (conditional knockout) mouse model of Mettl14. A targeting cassette with two loxP sites was engineered into the Mettl14 locus in mouse chromosome 3 by homologous recombination (Yoon et al, 2017). To conditionally delete Mettl14 in the skin epithelium, we bred Mettl14fl/fl mice with transgenic mice carrying K14-Cre recombinase, which can efficiently excise floxed genomic DNA by embryonic day E15.5 (Vasioukhin et al, 1999; Wu et al, 2008) (Fig 1A and Appendix Fig S1B). cKO mice are born in the expected Mendelian ratio but exhibit significant perinatal lethality. The cKO animals are smaller and often have tighter, smoother, and shiny skin compared with their littermates (Fig 1A). Immunohistochemistry confirms loss of Mettl14 in skin epithelial cells in the cKO animals (Fig 1B). Mass spectrometry analysis shows a dramatic decrease of global mRNA m6A modification in cKO skin epidermis (Appendix Fig S1C). Figure 1. Mettl14 regulates skin tissue homeostasis and wound healing Mettl14 cKO leads to perinatal lethality. Newborn cKO pups are smaller with tight and shiny skin. Immunohistochemistry confirms loss of Mettl14 expression in skin in cKO animals. H/E staining of newborn skin sections from WT and Mettl14 cKO mice. Expression of p63 in skin epidermis was examined by immunofluorescence staining in WT and cKO mice. Number of p63-positive cells in WT and cKO skin was quantified and shown as box and whisker plots. The plot indicates the mean (solid diamond within the box), 25th percentile (bottom line of the box), median (middle line of the box), 75th percentile (top line of the box), 5th and 95th percentile (whiskers), 1st and 99th percentile (solid triangles), and minimum and maximum measurements (solid squares). n = 6 (biological repeats), P < 0.05 (Student’s t-test). Skin stratification in WT and cKO skin was determined by immunofluorescence staining with different antibodies as indicated. Krt14: Keratin 14; Krt10: Keratin 10; β4: β4-integrin; DAPI for nucleus staining. The dashed line denotes the basement membrane that separates dermis and epidermis (Epi). Wound healing as monitored by wound size 8 days post-injury. n = 3; P < 0.01 (Student’s t-test). Error bar represents s.d. (standard deviation). Histological staining of skin sections at the wound edges. Halves of wound sections are shown. Note significant reduction of HPE (hyperproliferative epidermis) in cKO skin. Es: eschar. Dotted lines denote epidermal boundaries. Quantification of Ki67-positive cells present in wound HPE. The plot indicates the mean (open circles within the box), 25th percentile (bottom line of the box), median (middle line of the box), 75th percentile (top line of the box), 5th and 95th percentile (whiskers), 1st and 99th percentile (solid triangles), and minimum and maximum measurements (solid squares). n = 19, P < 0.01 (Student’s t-test). Download figure Download PowerPoint To first investigate the role of Mettl14 in skin development, we analyzed WT and cKO skin from newborn pups. Skin histology shows that the epidermis is notably thicker upon Mettl14 deletion (Fig 1C, and quantification in Appendix Fig S1D). Transcription factor p63 is a master regulator controlling epidermal morphogenesis and stemness (Botchkarev & Flores, 2014; Melino et al, 2015). Immunofluorescence staining demonstrates a significantly reduced number of p63-positive basal cells in cKO skin (Fig 1D and quantification in Fig 1E). Along with decreased and disorientated basal progenitor cells, Mettl14 cKO skin epidermis has an expanded spinous layer as determined by staining with the early differentiation marker, Keratin 10 (Fig 1F, quantification in Appendix Fig S1E). Wounding in skin can mobilize quiescent epidermal progenitor cells for proliferation and migration. To circumvent the issue of perinatal lethality in Mettl14 cKO animals, we bred the Metttl14l/fl mice with transgenic mice carrying the K14-Cre-ERT2 allele (Vasioukhin et al, 1999; Wu et al, 2008). The resultant inducible cKO animals can grow to adulthood with no apparent changes in skin epidermis or hair coat. However, when challenged to skin injury after administration of tamoxifen, Mettl14 cKO skin exhibited a significant delay in repairing full-thickness wounds as compared to WT skin (Fig 1G, Appendix Fig S1F). Histological analysis revealed that the area of hyperproliferative epithelium that typically proliferates and migrates into the wound site was significantly diminished following injury (Fig 1H). Wound-induced hyperproliferation of epidermal cells was also inhibited in cKO skin (Fig 1I). Together, our results strongly suggest that m6A modification of RNA mediated by Mettl14 plays a critical role in skin tissue homeostasis and wound repair in vivo. Loss of Mettl14 impairs epidermal stemness Although wound-induced hyperproliferation in adult epidermis is inhibited upon ablation of Mettl14, epidermal proliferation is not significantly affected in newborn skin of the cKO mice (Appendix Fig S2A). Potential markers for long-term epidermal progenitor cells are not clearly defined in vivo, and different models have been proposed for epidermal tissue maintenance (Lavker & Sun, 1982; Potten et al, 1982; Morris et al, 1985; Loeffler et al, 1987; Potten & Loeffler, 1987; Jones et al, 1995; Clayton et al, 2007; Mascre et al, 2012; Rompolas et al, 2016; Sada et al, 2016; Mesa et al, 2018; Dekoninck et al, 2020). However, accumulating evidence reveals the existence of distinct basal cell populations with hierarchical organization and proliferation dynamics in skin epidermis, including slow-cycling progenitor cells and committed progenitor cells with limited proliferative potential (Lavker & Sun, 1982; Potten et al, 1982; Morris et al, 1985; Loeffler et al, 1987; Potten & Loeffler, 1987; Jones et al, 1995; Mascre et al, 2012). With classical label-retaining analysis (Appendix Fig S2B), we found that loss of Mettl14 in skin leads to dramatic decrease in the slow cycling, label-retaining cells in the basal layer (Fig 2A and quantification in 2B). Figure 2. Loss of Mettl14 impairs epidermal stemness A. EdU staining of WT or Mettl14 cKO skin after pulse-chase labeling. Skin samples were counterstained with antibody against β4-integrin. Note reduced EdU label-retaining cells in cKO skin epidermis. Arrows indicate Edu-positive cells. B. Label-retaining cells in WT or Mettl14 cKO skin were quantified and shown as box plots. The plot indicates the mean (open circle within the box), 25th percentile (bottom line of the box), median (middle line of the box), 75th percentile (top line of the box), 5th and 95th percentile (whiskers), 1st and 99th percentile (solid triangles), and minimum and maximum measurements (solid squares). n = 6 (biological repeats), P < 0.05 (Student’s t-test). n = 19, P < 0.01 (Student’s t-test). C. Number of holoclones derived from WT and Mettl14 cKO skin was quantified and shown as box and whisker plots. The plot indicates the mean (solid diamond within the box), 25th percentile (bottom line of the box), median (middle line of the box), 75th percentile (top line of the box), 5th and 95th percentile (whiskers), 1st and 99th percentile (solid triangles), and minimum and maximum measurements (solid squares). n = 6 (biological repeats), P < 0.05 (Student’s t-test). n = 24, P < 0.01 (Student’s t-test). D. Morphology of primary keratinocytes isolated from WT or Mettl14 cKO skin. E. Expression of Mettl14 and p63 in WT and KO cells was determined by immunoblots with respective antibodies. Immunoblot for GAPDH was used as loading control. F. Proliferation of WT and Mettl14 null cells in vitro was quantified and shown as dot plots. n = 3, P < 0.01 (Student’s t-test) for Days 6, 9, and 12, and P < 0.05 (Student’s t-test) for Day 3. Error bar represents s.d. G, H. CFE (colony formation efficiency) of WT and Mettl14 null cells was determined in vitro. Results were quantified and shown as bar graph (H). n = 19, P < 0.01 (Student’s t-test). Error bar represents s.d. I. Fluorescence microscopy demonstrates different survival capability of WT and Mettl14 inducible KO cells with or without Tamoxifen (TAM) treatment. J. Ratio of WT and Mettl14-inducible KO cells in the co-culture model was quantified and shown as dot plots. n = 8, P < 0.01 (Student’s t-test) for KO cells with TAM treatment compared with WT cells or KO cells without TAM stimulation at both Days 7 and 14. Error bar represents s.d. Download figure Download PowerPoint Upon culture in vitro, primary epidermal keratinocytes will generate three different types of colonies, named holoclones, meroclones, and paraclones, with only holoclones containing undifferentiated epidermal progenitor cells (Rheinwald & Green, 1975; Green et al, 1977; Blanpain & Fuchs, 2006). Deletion of Mettl14 led to a dramatic reduction in holoclones when primary cells were cultured on 3T3 feeder layers (Fig 2C). Whereas WT holoclone cells are typically small and tightly packed with undifferentiated morphology, the KO colonies usually have cells that are large and multinucleated with senescent or differentiated morphology (Fig 2D). Immunoblots confirmed loss of Mettl14 expression in KO cells. Expression of p63 was also significantly reduced in KO cells (Fig 2E). When passaged in vitro, the KO cells proliferate slower with significantly longer doubling time (Fig 2F) and reduced EdU (5-ethynyl-2’-deoxyuridine) incorporation (Appendix Fig S2C) compared with WT controls. The colony formation efficiency (CFE) was markedly decreased upon loss of Mettl14 (Fig 2G and quantification in 2H), and re-expression of WT Mettl14 but not Mettl14 R298P mutant that is deficient in methyltransferase activity (Liu et al, 2018) can restore the CFE in vitro (Appendix Fig S2D). To further investigate the change of epidermal stemness, we carried out a clonal competition and lineage trace analysis (Siegle et al, 2014) using our skin organoid culture and transplantation model (Yue et al, 2017; Li et al, 2019). As Mettl14-deficient cells failed to sustain long-term culture in vitro, we isolated primary epidermal keratinocytes from Mettl14-inducible KO strain. Before treatment with tamoxifen, the inducible KO cells grow at a comparable rate with their WT counterparts. To monitor their epidermal regeneration capacity and trace their fate upon engraftment, we transduced the WT and inducible KO cells with lentivirus encoding either H2B-RFP or H2B-GFP. Fluorescently labeled cells were mixed at a 1:1 ratio to generate skin organoids. Interestingly, whereas clone size measurement with fluorescent microscopy revealed no significant changes between WT and inducible KO cells before tamoxifen treatment, induction of Cre recombination with tamoxifen led to continual loss of Mettl14 KO cells in the skin, which became barely detectible 14 days post-tamoxifen treatment (Fig 2I and quantification in 2J). N6-methyladenosine modification of Pvt1 regulates stemness of epidermal progenitor cells In order to uncover how changes in the RNA methylomes regulate self-renewal and differentiation of epidermal progenitor cells, we performed m6A sequencing and RNA sequencing of polyadenylated RNAs isolated from undifferentiated and differentiated cells (Dataset EV1 and EV2). Our profiling identified 7,379 and 6,945 RNA species with m6A modifications in progenitor cells and differentiated cells, respectively. Among them, 260 RNAs have markedly reduced m6A modification upon differentiation (fold change > 2), whereas 1,474 RNAs have elevated levels of m6A modification. GO term enrichment analysis of RNAs with altered m6A modification revealed a variety of enriched clusters following keratinocyte differentiation (Appendix Fig S3), including annotations pertaining to RNA processing and metabolisms. LncRNAs have emerged as an important class of regulators involved in many processes, including skin development and homeostasis (Hombach & Kretz, 2013; Wan & Wang, 2014). Our analysis revealed substantially altered m6A modifications of different lncRNAs during skin differentiation, including Gas5 (growth arrest-specific 5), Neat1 (nuclear enriched abundant transcript 1), Malat1 (metastasis associated in lung adenocarcinoma transcript 1), Snhg18 (small nucleolar RNA host gene 18) and Pvt1 (Fig 3A). Among these, Pvt1, a potential oncogenic lncRNA associated with MYC, displayed significantly reduced m6A modification in differentiated cells. We verified this change by RT–PCR from differentiated and undifferentiated cells. Pvt1 contains five potential m6A modification sites that match the Pu[G>A]m6AC[A/C/U] consensus sequence (Harper et al, 1990). The five potential sites are distributed as two clusters (A282, A294, and A303 in cluster 1, and A446 and A452 in cluster 2) at Exon 2. To examine their potential modification, we prepared different mutants of Pvt1 and then determined their m6A methylation level by α-m6A immunoprecipitation followed with RT–PCR. When each cluster was mutated (A–G), we saw ~50% reduction in the m6A modification (Fig 3B). When both clusters were mutated (A5G), methylation of Pvt1 was nearly abolished (Fig 3B). Figure 3. Pvt1 regulates epidermal stemness and tissue homeostasis Profiling of RNA methylome by m6A-seq shows significant changes of lncRNA modifications upon epidermal differentiation in vitro. Mutations in the predicted m6A modification sites can significantly reduce Pvt1 methylation. n = 3. Error bar represents s.d. Deletion of endogenous Pvt1 by an inducible Cas9 (iCas9) system can significantly reduce Pvt1 RNA level (quantification from RT–PCR, left panel) and MYC protein level (quantification from immunoblot, right panel) expression. n = 3, P < 0.01 (Student’s t-test). Error bar represents s.d. CFE of WT, Pvt1-inducible KO, and Pvt1-inducible KO cells rescued with WT or mutant Pvt1 was quantified and presented as bar graph. n = 3, **P < 0.01 (Student’s t-test). Error bar represents s.d. Skin organoids derived from WT or Pvt1-inducible KO cells were grafted to nude mice. The regenerated skin was analyzed by H/E staining. Epidermal thickness of WT and Pvt1 KO skin grafts was quantified and presented as box and whisker plots. The plot indicates the mean (open circle within the box), 25th percentile (bottom line of the box), median (middle line of the box), 75th percentile (top line of the box), 5th and 95th percentile (whiskers), 1st and 99th percentile (solid triangles), and minimum and maximum measurements (solid squares). n = 6 (biological repeats), P < 0.05 (Student’s t-test). n = 18, P < 0.01 (Student’s t-test). Number of p63-positive cells in WT and cKO skin was quantified and shown as bar graph. n = 5, P < 0.01 (Student’s t-test). Error bar represents s.d. Fluorescence microscopy demonstrates different survival capability of WT and Pvt1-inducible KO cells with or without Doxycycline (Dox) treatment. Ratio of WT and Pvt1-inducible KO cells in the co-culture model" @default.
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- W3136409253 title "N <sup>6</sup> ‐methyladenosine modification of lncRNA <i>Pvt1</i> governs epidermal stemness" @default.
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