Deficiency in the nuclear long noncoding RNA Charme causes myogenic defects and heart remodeling in mice
2018; Springer Nature; Volume: 37; Issue: 18 Linguagem: Inglês
10.15252/embj.201899697
ISSN1460-2075
AutoresMonica Ballarino, Andrea Cipriano, Rossella Tita, Tiziana Santini, Fabio Desideri, Mariangela Morlando, Alessio Colantoni, Claudia Carrieri, Carmine Nicoletti, Antonio Musarò, Dònal O’ Carroll, Irene Bozzoni,
Tópico(s)RNA Research and Splicing
ResumoArticle3 September 2018Open Access Source DataTransparent process Deficiency in the nuclear long noncoding RNA Charme causes myogenic defects and heart remodeling in mice Monica Ballarino Monica Ballarino Department of Biology and Biotechnology Charles Darwin, Sapienza University of Rome, Rome, Italy Search for more papers by this author Andrea Cipriano Andrea Cipriano Department of Biology and Biotechnology Charles Darwin, Sapienza University of Rome, Rome, Italy Search for more papers by this author Rossella Tita Rossella Tita Department of Biology and Biotechnology Charles Darwin, Sapienza University of Rome, Rome, Italy Search for more papers by this author Tiziana Santini Tiziana Santini Center for Life Nano [email protected], Istituto Italiano di Tecnologia, Rome, Italy Search for more papers by this author Fabio Desideri Fabio Desideri Department of Biology and Biotechnology Charles Darwin, Sapienza University of Rome, Rome, Italy Search for more papers by this author Mariangela Morlando Mariangela Morlando Department of Biology and Biotechnology Charles Darwin, Sapienza University of Rome, Rome, Italy Search for more papers by this author Alessio Colantoni Alessio Colantoni Department of Biology and Biotechnology Charles Darwin, Sapienza University of Rome, Rome, Italy Search for more papers by this author Claudia Carrieri Claudia Carrieri MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Carmine Nicoletti Carmine Nicoletti DAHFMO-Unit of Histology and Medical Embryology, Sapienza University of Rome, Rome, Italy Search for more papers by this author Antonio Musarò Antonio Musarò Center for Life Nano [email protected], Istituto Italiano di Tecnologia, Rome, Italy DAHFMO-Unit of Histology and Medical Embryology, Sapienza University of Rome, Rome, Italy Search for more papers by this author Dònal O' Carroll Dònal O' Carroll orcid.org/0000-0002-8626-2217 MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Irene Bozzoni Corresponding Author Irene Bozzoni [email protected] orcid.org/0000-0002-3485-8537 Department of Biology and Biotechnology Charles Darwin, Sapienza University of Rome, Rome, Italy Center for Life Nano [email protected], Istituto Italiano di Tecnologia, Rome, Italy Institute Pasteur Fondazione Cenci-Bolognetti, Sapienza University of Rome, Rome, Italy Institute of Molecular Biology and Pathology, CNR, Sapienza University of Rome, Rome, Italy Search for more papers by this author Monica Ballarino Monica Ballarino Department of Biology and Biotechnology Charles Darwin, Sapienza University of Rome, Rome, Italy Search for more papers by this author Andrea Cipriano Andrea Cipriano Department of Biology and Biotechnology Charles Darwin, Sapienza University of Rome, Rome, Italy Search for more papers by this author Rossella Tita Rossella Tita Department of Biology and Biotechnology Charles Darwin, Sapienza University of Rome, Rome, Italy Search for more papers by this author Tiziana Santini Tiziana Santini Center for Life Nano [email protected], Istituto Italiano di Tecnologia, Rome, Italy Search for more papers by this author Fabio Desideri Fabio Desideri Department of Biology and Biotechnology Charles Darwin, Sapienza University of Rome, Rome, Italy Search for more papers by this author Mariangela Morlando Mariangela Morlando Department of Biology and Biotechnology Charles Darwin, Sapienza University of Rome, Rome, Italy Search for more papers by this author Alessio Colantoni Alessio Colantoni Department of Biology and Biotechnology Charles Darwin, Sapienza University of Rome, Rome, Italy Search for more papers by this author Claudia Carrieri Claudia Carrieri MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Carmine Nicoletti Carmine Nicoletti DAHFMO-Unit of Histology and Medical Embryology, Sapienza University of Rome, Rome, Italy Search for more papers by this author Antonio Musarò Antonio Musarò Center for Life Nano [email protected], Istituto Italiano di Tecnologia, Rome, Italy DAHFMO-Unit of Histology and Medical Embryology, Sapienza University of Rome, Rome, Italy Search for more papers by this author Dònal O' Carroll Dònal O' Carroll orcid.org/0000-0002-8626-2217 MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Irene Bozzoni Corresponding Author Irene Bozzoni [email protected] orcid.org/0000-0002-3485-8537 Department of Biology and Biotechnology Charles Darwin, Sapienza University of Rome, Rome, Italy Center for Life Nano [email protected], Istituto Italiano di Tecnologia, Rome, Italy Institute Pasteur Fondazione Cenci-Bolognetti, Sapienza University of Rome, Rome, Italy Institute of Molecular Biology and Pathology, CNR, Sapienza University of Rome, Rome, Italy Search for more papers by this author Author Information Monica Ballarino1,‡, Andrea Cipriano1,‡, Rossella Tita1,‡, Tiziana Santini2, Fabio Desideri1, Mariangela Morlando1, Alessio Colantoni1, Claudia Carrieri3, Carmine Nicoletti4, Antonio Musarò2,4, Dònal O' Carroll3 and Irene Bozzoni *,1,2,5,6 1Department of Biology and Biotechnology Charles Darwin, Sapienza University of Rome, Rome, Italy 2Center for Life Nano [email protected], Istituto Italiano di Tecnologia, Rome, Italy 3MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK 4DAHFMO-Unit of Histology and Medical Embryology, Sapienza University of Rome, Rome, Italy 5Institute Pasteur Fondazione Cenci-Bolognetti, Sapienza University of Rome, Rome, Italy 6Institute of Molecular Biology and Pathology, CNR, Sapienza University of Rome, Rome, Italy ‡These authors contributed equally to this work *Corresponding author. Tel: +39 06 4991 2202; Fax: +39 06 4991 2500; E-mail: [email protected] The EMBO Journal (2018)37:e99697https://doi.org/10.15252/embj.201899697 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 Myogenesis is a highly regulated process that involves the conversion of progenitor cells into multinucleated myofibers. Besides proteins and miRNAs, long noncoding RNAs (lncRNAs) have been shown to participate in myogenic regulatory circuitries. Here, we characterize a murine chromatin-associated muscle-specific lncRNA, Charme, which contributes to the robustness of the myogenic program in vitro and in vivo. In myocytes, Charme depletion triggers the disassembly of a specific chromosomal domain and the downregulation of myogenic genes contained therein. Notably, several Charme-sensitive genes are associated with human cardiomyopathies and Charme depletion in mice results in a peculiar cardiac remodeling phenotype with changes in size, structure, and shape of the heart. Moreover, the existence of an orthologous transcript in human, regulating the same subset of target genes, suggests an important and evolutionarily conserved function for Charme. Altogether, these data describe a new example of a chromatin-associated lncRNA regulating the robustness of skeletal and cardiac myogenesis. Synopsis The study characterises the chromatin-associated long noncoding RNA (lncRNA) Charme, a novel murine muscle-specific lncRNA conserved in human. Depletion studies in vitro and in vivo show that Charme regulates myogenesis by stabilising long-range chromosomal interactions required to control the expression of pro-myogenic loci. Charme is a novel chromatin-associated muscle-specific lncRNA. Charme regulates the robustness of skeletal and cardiac myogenesis by controlling the chromosomal architecture of pro-myogenic genomic loci. Functional depletion of Charme in mice results in a severe cardiac phenotype and leads to lifespan reduction. Introduction Myogenesis is a highly regulated multistep process, in which the conversion of progenitor cells into multinucleated and functional myofibers can be easily reproduced in vitro. Besides the well-characterized protein factors and miRNAs (Buckingham & Rigby, 2014), also long noncoding RNAs (lncRNAs) have been shown to participate in myogenic regulatory circuitries (Rinn & Chang, 2012; Fatica & Bozzoni, 2014; Ballarino et al, 2015). lncRNAs, through their ability to interact with nucleic acids as well as with proteins (Guttman & Rinn, 2012; Rinn & Chang, 2012; Batista & Chang, 2013; Cipriano & Ballarino, 2018), act as scaffolds for the formation of specific functional complexes where different proteins and RNA and even DNA molecules can be tethered together (Wutz et al, 2002; Clemson et al, 2009; Tsai et al, 2010; Ariel et al, 2014; Hacisuleyman et al, 2014; Ribeiro et al, 2017). Nuclear lncRNAs have been described to control the transcriptional myogenic program through diverse modes of action (Ballarino et al, 2016): as enhancer-associated transcripts (Mousavi et al, 2014; Mueller et al, 2015; Ounzain et al, 2015), as guides for the recruitment of PRCs and MLL epigenetic regulators or DNA methyltransferases (DNMT; Wang et al, 2015, 2016), and also as allosteric inhibitors (Han et al, 2014; Wang et al, 2016). Important functions have also been ascribed to cytoplasmic lncRNAs through their ability to control mRNA stability and translation (Wang et al, 2013), to modulate miRNA function acting as competing endogenous RNA (Cesana et al, 2011; Salmena et al, 2011; Han et al, 2015), or to encode for micropeptides (Anderson et al, 2015; Nelson et al, 2016). Interestingly, de-regulation of these molecules was often associated with skeletal (Ballarino et al, 2015) and cardiac (Han et al, 2014; Ounzain & Pedrazzini, 2015; Uchida & Dimmeler, 2015; Wang et al, 2016) diseases. We recently identified a novel nuclear lncRNA, Charme (Chromatin architect of muscle expression), conserved in human and with restricted expression in skeletal and cardiac muscles (Ballarino et al, 2015). Here, we show that the depletion of Charme produces the downregulation of myogenic genes, many of which involved in human cardiomyopathies (Becker et al, 2011; Harvey & Leinwand, 2011). Through a combination of ChIRP and FISH approaches, we identified a Charme/chromatin interaction that is required for the correct expression of a subset of relevant myogenic genes. Notably, the functional knockout of Charme in mice produced morphological alterations of both skeletal and cardiac muscles and resulted in a shorter life span. The structural and functional conservation of Charme in human further corroborates its relevance in the control of proper muscle differentiation and homeostasis. Results Charme is a chromatin-associated long noncoding RNA Previous transcriptome analysis from murine C2C12 myoblasts and myotubes discovered novel lncRNAs with muscle restricted expression (Ballarino et al, 2015). Among them, lnc-405, here named Charme (Chromatin architect of muscle expression; Figs 1A and EV1A), is a novel isoform of the 5430431A17Rik transcript of particular interest for (i) its specific expression in both skeletal and cardiac muscles, (ii) its predominant nuclear localization, and (iii) the presence of an orthologue transcript in human. Figure 1. Charme is a novel long noncoding transcript associated with myogenesis Genomic structure of the Charme locus. The position of PCR primers, LNA GAPmers (GAP-2, GAP-2/3), and in situ probes (green—intronic; or red—exonic) used in this study are shown together with the produced Charme isoforms. Semiquantitative RT–PCR (sqRT–PCR) quantification of pCharme and mCharme in cytoplasmic (Cyt), nuclear (Nu), nucleoplasmic (Np), or chromatin (Chr) fractions from 2-day differentiated myotubes. The quality of fractionation was tested with mature (GAPDH) and precursor (pre-GAPDH) RNAs. Co-staining of MHC protein (green) and Charme RNA (red) in fully differentiated myotubes. DAPI, 4′,6-diamidino-2-phenylindole. MHC, myosin heavy chain. Scale bar = 10 μm. sqRT–PCR quantification of Charme amplicons in growth (GM) and differentiated (DM) conditions. GAPDH mRNA serves as control. −, RT-minus control. Real-time RT–PCR (qRT–PCR) quantification of mCharme and pCharme, MCK, and MHC in differentiated myotubes treated with GAP-2, GAP-2/3, or GAP-scr as negative control. Data were normalized to GAPDH mRNA and represent mean ± SD of triplicates. Immunofluorescence staining for MHC on C2C12 cells treated with GAP-scr, GAP-2, or GAP-2/3 (right) and merged with the DAPI staining (left). Scale bar = 100 μm. Quantification of myotubes formation (F.I., Nu/MT, MonoMHC+) on cells treated with GAP-2, GAP-2/3, or GAP-scr. Bars represent mean ± SD of triplicates of randomly chosen microscope fields. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, paired Student's t-test. Source data are available online for this figure. Source Data for Figure 1 [embj201899697-sup-0007-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Genomic features and expression analysis of the murine Charme locus UCSC visualization showing the chromosome position and the genomic coordinates of Charme (highlighted in yellow) in the mm9 mouse genome. Red box indicates the genomic position of the previously annotated 5430431A17Rik transcript. Left: 5′-RACE analyses of Charme in proliferating (GM) and differentiating (DM) conditions. The position of the Racer primer is indicated. RNA treated to the same experimental regime, but with tobacco acid pyrophosphatase digestion omitted (−TAP), did not generate a product. The PCR products were sequenced, and the identified Charme transcription start site is reported (+1). Middle: Northern blot analysis of Charme on total RNA from proliferating (GM), differentiating (DM, GAP-scr), and Charme-depleted (DM, GAP-2) myotubes. The reduced level of the signal upon Charme depletion confirms the specificity of the observed band. 18S rRNA and 28S rRNA serve as a loading control. Right: RT–PCR quantification of Charme in cytoplasmic (Cyt), nuclear (Nu), nucleoplasmic (Np), or chromatin (Chr) fractions from differentiated myotubes. The quality of fractionation is shown in Fig 1B. Three biological replicates were analyzed, and a representative experiment is shown. −, RT-minus control. RNA-seq coverage visualization of Charme locus during the time-course of C2C12 differentiation. A quantification of the intronic reads is represented in the histogram aside. Normalized reads (NR) were obtained by dividing the total number of intronic reads for their respective lengths. RT–PCR validation of Charme intron 1 retention performed on RNA from cytoplasmic or nuclear samples. Assessment of Charme isoform stability. RNA half-lives were calculated upon 0, 3, and 6 h of actinomycin D (ActD) treatment; GAPDH and pre-GAPDH serve as controls. Data represent the means ± SD from three independent experiments. Source data are available online for this figure. Download figure Download PowerPoint The expression of Charme was studied by 5′-RACE, Northern blot, and RT–PCR analyses (Figs 1B and EV1B): It is predominantly localized in the nucleus, mainly associated with the chromatin, with a minor fraction being present in the cytoplasm. In C2C12 cells, RNA FISH showed a well-defined punctate localization of Charme in the nuclei of myofibers defined as multinucleated cells expressing the myogenic marker myosin heavy chain (MHC). Charme was instead absent in proliferating myoblasts, identified as mononucleated cells lacking MHC staining (Fig 1C). Charme expression starts at day 1 upon shift to differentiation medium (DM), reaching high levels at day 3 (Fig 1D). In agreement with RNA-seq data, RT–PCR analyses revealed the existence of an isoform (pCharme) still retaining intron 1 and devoid of intron 2 (Figs 1A and D, and EV1C and D). Notably, actinomycin experiments indicated that this isoform was as stable as the fully spliced Charme species (mCharme; Fig EV1E). RNA FISH with intron probes detected an average of three nuclear spots which co-localize with the exonic signals (Appendix Fig S1A and B). This pattern, whose specificity was tested by both RNaseA and RNAi sensitivity (Appendix Fig S1C), well correlates with the aneuploidy of C2C12 cells having three copies of chromosome 7, where Charme locus resides. The number, the intensity, and the focal plane distribution of the signals obtained with exonic vs. intronic probes allowed us to establish that the major fraction of Charme is retained at the sites of its own transcription as a precursor species. Charme depletion affects myogenesis To analyze the role of Charme in myogenesis, we treated C2C12 myotubes with two different LNA GAPmers (GAP-2 and GAP-2/3; Fig 1A), which provided 60–70% average reduction in both intron 1-harboring (pCharme) and mature (mCharme) Charme transcripts (Fig 1E). Upon knockdown, 50% decrease of the mRNAs for two diagnostic markers of differentiation, myosin creatine kinase (MCK) and the myosin heavy chain (MHC), was found (Fig 1E and Table EV1) indicating quite a clear impairment of myogenesis. Morphological parameters were also specifically affected: a consistent reduction in the caliber of differentiated MHC-positive myotubes (Fig 1F) was observed, together with a decrease of more than twofold of the fusion index (F.I.) and of the number of nuclei per fiber (Nu/MT). In parallel, the number of mononucleated (MonoMHC+) cells increased by a factor of 3 (Fig 1G). These results suggest that Charme inhibition mainly affects the ability of myogenic cells to fuse and to proceed to later stages of differentiation. RNA-seq analysis on cells treated with GAP-2 or GAP-2/3, in parallel with a scramble control (Appendix Fig S2A and B) led to the identification of a set of 826 genes differentially expressed upon Charme depletion with two different sets of LNA GAPmers (Fig 2A and Appendix Fig S2C). Of these, 302 were upregulated and 524 downregulated in comparison with the scramble condition (Fig 2A and Table EV1). Real-time quantitative PCR on a selected number of targets confirmed the sequencing data (Appendix Fig S2D). The Gene Ontology (GO) term enrichment study performed with the FIDEA web tool (D'Andrea et al, 2013) suggested that Charme may act by activating genes involved in muscle function and contraction processes (Fig 2B). To note, we did not observe significant enrichment of any cell-cycle- and/or proliferation-related categories. Figure 2. Charme regulates myogenesis by interacting with the nctc region RNA-seq analysis of transcriptome changes upon Charme depletion. Heatmap was drawn using the heatmap3 R package and represents hierarchical clustering of the common genes differentially expressed upon Charme depletion with both GAP-2 and GAP-2/3 GAPmers, compared to GAP-scr control. The expression levels correspond to mean-centered log2-transformed FPKM values. See also Table EV1. Gene Ontology (GO) term enrichment analyses performed by FIDEA (D'Andrea et al, 2013) on genes downregulated (top) or upregulated (bottom) upon Charme depletion. Bars indicate the top 10 categories of cellular components in decreasing order of significance. Threshold (P-value < 0.01) is indicated by the dashed black line. KEGG pathway enrichment analysis on genes downregulated by Charme depletion. Quantification of recovered RNA (left) and DNA (right) upon Charme ChIRP with the pool of biotinylated probes reported in Table EV4. RNA/DNA FISH for Charme RNA and nctc (left) or lnc-31 (right) loci in 2-day differentiated myotubes. Inserts show a magnification of the spots converted into binary images (I), 3D rendering (II), or rotated on Z-axis (III). Yellow arrows indicate signals overlapping. Right histogram reports the mean ± SD percentage of nuclei with paired or overlapped spots from two biological replicates. Scale bar = 5 μm. qRT–PCR quantification of Charme and Charme target genes in GAP-scr- vs. GAP-2-transfected (2-day differentiated) myotubes. PCR data were normalized to GAPDH and represent mean ± SD of triplicates. RNA Pol II (left) and H3K9ac (right) ChIP performed in GAP-scr- vs. GAP-2-transfected myotubes. The recovered chromatin was analyzed by qPCR in parallel with an intergenic region used to normalize the two (GAP-scr vs. GAP-2) conditions. Data were subtracted for background and are expressed as input percentage (% Input). Histograms represent the mean ± SEM of three biological replicates. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, unpaired Student's t-test. Source data are available online for this figure. Source Data for Figure 2 [embj201899697-sup-0008-SDataFig2.pdf] Download figure Download PowerPoint Interestingly, Charme depletion resulted in the downregulation of genes (i.e., Myh7 and Tnnt2; Appendix Fig S2E) implicated in several types of cardiomyopathies (Fig 2C), such as familial hypertrophic (HCM), dilated (DCM), and arrhythmogenic right ventricular (ARVC) cardiomyopathies (Harvey & Leinwand, 2011; Becker et al, 2011; Maron et al, 2012). pCharme is localized at specific chromosomal sites To elucidate Charme mode of action, we proceeded with the identification of the sites of chromatin interaction in C2C12 cells. Chromatin isolation by RNA purification (ChIRP) analysis was carried out with two different pools of four alternating antisense biotinylated oligonucleotides (even and odd), together with four oligonucleotides against lacZ sequences (Appendix Fig S2F). Figure 2D (panel RNA) shows that a specific pull-down of pCharme isoform was obtained when compared to the lacZ control and the GAPDH pre-mRNA, while no enrichment was obtained for mCharme isoform. The co-purified DNA was subjected to high-throughput sequencing (ChIRP-seq), and the reads from odd and even pull-downs were compared to those of the lacZ control (Table EV2). In agreement with the strong chromatin association, the major peak corresponded to the Charme locus. Several other genomic loci were identified (Table EV2); among them, the most prominent corresponded to a region (hereafter named as nctc) located on the same Charme chromosome (chr7:149,746,850–149,747,033) and containing several genes affected by Charme depletion (Appendix Fig S2G and Table EV1). The nctc peak was validated by PCR amplification on the initial ChIRP samples (Fig 2D, panel DNA) and reproduced in independent replicates. Interestingly, this region contains a shared core muscle enhancer (CME; Alzhanov et al, 2010) required for muscle-specific activation of Igf2 and H19 (Eun et al, 2013), which are both decreased upon Charme knockdown (Table EV1). Interestingly, the knockout of part of this enhancer was previously shown to produce the loss of Igf2 expression and defects in muscle differentiation (Kaffer et al, 2001). Therefore, for subsequent analysis we concentrated on this specific locus. The physical association of pCharme RNA with nctc (98 Mb far from Charme locus) was validated by FISH analysis with probes against Charme RNA (red) and BAC probes against the target loci (green). A detailed quantification of the signals indicated that in more than 60% of the nuclei Charme is in close proximity with the nctc region, with 2/3 of the signals showing partial overlapping (Fig 2E). No proximity was instead found when the unrelated lnc-31 locus (Ballarino et al, 2015), which was absent in the ChIRP-seq dataset, was used as negative control (Fig 2E). Notably, the surrounding 2.5 Mb of the ChIRP peak in the nctc region contains relevant myogenic genes, such as Igf2, Tnnt3, and Tnni2, that were downregulated upon Charme depletion in the RNA-seq dataset (Tables EV1 and EV2). qRT–PCR confirmed their downregulation upon Charme depletion both at the mRNA and pre-mRNA levels (Fig 2F). Along this line, ChIP analyses performed in conditions of Charme downregulation showed a strong decrease in the association of the RNA polymerase II (RNA Pol II) and in the acetylation of lysine K9 histone 3 (H3K9ac) of these genes (Fig 2G). Altogether, these data indicate that the effect of Charme on these target genes is exerted at the transcriptional level. Due to the strong accumulation of pCharme at the sites of its own transcription, we tested whether the Charme genomic locus itself could be in contact with nctc. To this purpose, FISH analyses were performed with DNA BAC probes flanking the Charme gene (red) or overlapping the nctc region (green; Figs 3 and EV2A–C). In GM conditions, when Charme is not expressed, the two districts are clearly distinguished as independent spots (Fig 3A, left panel, and Fig EV2C, GM). Notably, upon Charme expression, the two regions get close (Fig EV2C, DM1 and DM1.5, and Fig EV2D) with a consistent percentage of chromosomes showing overlapping or proximal signals at days 1 and 1.5 of differentiation (Fig 3A, right panels, and Fig EV2C). As control, no proximity was found with the unrelated lnc-31 locus (Figs 3A and EV2C, lower panels). Notably, the pCharme-mediated long-range interaction was significantly reduced when Charme was downregulated by 50% through GAP-2 treatment (Figs 3B and EV2E). Overall, these in vitro data suggest an important role of pCharme in a long-range physical interaction between two chromatin districts distantly located, but present on the same chromosome. Figure 3. pCharme contributes to the maintenance of chromatin contacts with the nctc region Double DNA FISH for Charme and nctc (top) or Charme and lnc-31 (bottom) loci in growth (GM) and differentiated (DM1) conditions. Histograms represent the percentages ± SD of chromosome 7 showing paired and overlapped signals in GM, DM1, and DM1.5 days of differentiation from two biological replicates. DNA/DNA FISH for Charme and nctc loci in GAP-scr- vs. GAP-2-transfected myotubes (DM1). Percentage of Charme locus ± SD from three biological replicates showing paired and overlapped signals with nctc is indicated in the histograms below. Data information: **P < 0.01, ***P < 0.001, unpaired Student's t-test. Scale bar = 5 μm. Source data are available online for this figure. Source Data for Figure 3 [embj201899697-sup-0009-SDataFig3.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Chromatin contacts and functional characterization of the murine Charme transcript Examples of proximal Charme/nctc DNA FISH spots on the same Z-section. Red and green signals correspond to Charme or nctc chromatin regions, respectively. White boxes show binarized images of single focal planes containing long-splitted, paired, or overlapped patterns. On top of the panels, the 3D interallelic distance interval (ND) for each category is shown. ND values were obtained by normalization to the diameter of the nuclei. The number of green/red DNA spots per nucleus (n = 3) reflects the C2C12 aneuploidy with three copies of chromosome 7. Scale bar = 5 μm. Plotting of Z-stack intensity distribution for each spot in the insets of (A). Gaussian fit curve (full line) for the data of fluorescence intensity (filled circles) in each channel is plotted along Z-planes (Z-step = 200 nm). The strong correspondence of green and red channel distribution indicates near co-localization and co-planarity of the signals. Full field view of the DNA FISH studies on the nuclear distribution of Charme and nctc (top) or Charme and lnc-31 (bottom) loci. Scale bar = 5 μm. Normalized 3D distances between Charme and nctc loci at the indicated time points (GM, DM 1–1.5 days). Interallelic distances were normalized to nuclei diameter. Normalized 3D distances between Charme and nctc loci in GAP-scr or GAP-2 treated cells at the indicated differentiation times (DM 1–1.5 days). Interallelic distances were normalized to nuclei diameter. Mean ± SD ND values are shown. **P < 0.01, ***P < 0.001, unpaired Student's t-test. Quantitative real-time RT–PCR analyses of mCharme, MHC, and MCK mRNAs in C2C12 cells transfected with GAP-scr or GAP-1 in combination with a mCharme mutant (mCharme-mut) in the GAP-1 targeting site. Transfections with the empty vector were used as negative control. PCR data were normalized to GAPDH mRNA. sqRT–PCR quantification of mCharme in cytoplasmic (Cyt) and nuclear (Nu) fractions from C2C12 cells transfected with mCharme-mut. The quality of fractionation was tested with mature (GAPDH) and precursor (pre-GAPDH) RNAs. −, RT-minus control. Source data are available online for this figure. Download figure Download PowerPoint A further evidence for Charme acting in the nucleus at the sites of its own transcription derives from experiments where we tried to rescue the phenotype of Charme depletion with a cDNA construct expressing a GAPmer-resistant mCharme. This construct failed to recover the expression of myogenic markers (Fig EV2F) and produced a transcript with a cytoplasmic-restricted localization (Fig EV2G). The lack of rescue with cytoplasmic RNA further supports the finding that the active Charme species is the nuclear one; moreover, these results are in line with the accepted notion that for chromatin-associated lncRNAs acting in cis, rescue phenotypes are not obtained with exogenous gene overexpression (Goff & Rinn, 2015; Wang et al, 2015). Charme functional knockout in mice affects the myogenic process In consideration of the interesting function of Charme observed in vitro, the study of its function in vivo was strongly demanded. Therefore, the CRIS
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