Journal: EMBO Molecular Medicine

Article Title: Trans‐generational epigenetic regulation associated with the amelioration of Duchenne Muscular Dystrophy

doi: 10.15252/emmm.202012063

Figure Lengend Snippet: A GSΔ44 myoblasts were transfected with an empty vector (CTRL) or with a CELF2a overexpressing construct (OE CELF2A) (Martone et al , ). Cells were collected after 10 days in differentiation medium (DM), and the RNA was analysed by RT–PCR for CELF2a expression and DMD exon 45 skipping. GAPDH was used as loading control. The arrow indicates the DMD exon 45‐skipped band. Representative results are shown ( n = 3). B Representative immunofluorescence for myosin heavy chain (MHC in red) in combination with DAPI staining (in blue) of iPSCs obtained from WT (WT#1), UPΔ44 (UPΔ44#3) and Δ44 edited clones (UPΔ44#3.1 and UPΔ44#3.5) differentiated for 9 days into myocytes by MYOD/BAF60c overexpression. Scale bar 100 μm. C Histogram represents fusion index quantification. At least 7 randomly chosen microscope fields of two independent biological samples were analysed ( n = 2). Data are presented as mean ± SD of the biological replicates. D RNA extracted from myocytes obtained by differentiation of WT (WT#1), UPΔ44 (UPΔ44#3) and edited UPΔ44 iPSC (UPΔ44#3.1 ΔCELF2a and UPΔ44#3.5 ΔCELF2a) clones was analysed by RT–PCR for CELF2a, CELF2b, CELF2c and DMD exon 45 skipping. GAPDH was used as loading control. The arrow indicates the DMD exon 45‐skipped band. Representative results are shown ( n = 3). E Western blot on proteins (40 μg) extracted from myocytes obtained by differentiation of WT (WT#1), UPΔ44 (UPΔ44#3) and edited UPΔ44 iPSC (UPΔ44#3.1 ΔCELF2a and UPΔ44#3.5 ΔCELF2a) clones probed with antibodies against dystrophin (DMD). Actinin (ACTN) was used as a loading control. For the WT 5% sample, 2 μg of proteins was diluted in 38 μg of proteins belonging to UPΔ44#3 to reach a total amount of 40 μg. Representative results are shown ( n = 3). Source data are available online for this figure.

Article Snippet: Three top ranking guides for the region containing the first exon of Celf2a (Chr10:11047177‐11047403, GRCh37/hg19 assembly) were selected and cloned into the CRISPR/Cas9 plasmid pX330 (Li et al , ; Addgene#42230).

Techniques: Transfection, Plasmid Preparation, Construct, Reverse Transcription Polymerase Chain Reaction, Expressing, Control, Immunofluorescence, Staining, Clone Assay, Over Expression, Microscopy, Western Blot

Journal: EMBO Molecular Medicine

Article Title: Trans‐generational epigenetic regulation associated with the amelioration of Duchenne Muscular Dystrophy

doi: 10.15252/emmm.202012063

Figure Lengend Snippet: A Representative phase contrast image of UPΔ44#3 pluripotent stem cell morphology. Scale bar 100 μm. B qPCR analyses of pluripotency‐associated genes (NANOG, OCT4, SOX2 and REX1) and of the direct inhibitor of OCT4 (NR2F2) expression in the patient‐specific iPSC clone (UPΔ44#3) relative to the somatic cell control (UPΔ44 fibroblasts). PCRs were normalized against an internal control (GAPDH) and plotted relative to the expression level in the individual parent fibroblast cell lines set to a value of 1. The mean ± SEM of triplicates from one representative experiment is shown ( n = 3). C RT–PCR for the exogenous OCT4 mRNA in RNA extracted from UPΔ44 fibroblasts (UPΔ44 Fibro), fibroblasts collected 3 days after lentiviral infection with hSTEMCCA vector (Inf Fibro) and the patient‐specific iPSC clone#3 (UPΔ44#3). GAPDH was used as control. Lane (−) indicates the negative control. Representative results are shown ( n = 3). D Normal karyotype of WT#1 and UPΔ44#3 iPSC clones (46, XY). E Genetic identity with the parental cell line was assessed by PCR on gDNA. PCR was performed on gDNA from control and UPΔ44 fibroblasts cells and iPSCs amplifying the indicated DMD exons. Lane (−) indicates the negative control. F Schematic representation of the gene‐editing strategy. Vectors encoding Cas9 and sgRNA targeting the first exon of CELF2a gene, together with a donor plasmid containing a selection cassette flanked by Lox P sites and two homologous sequences (˜800 bp each) to upstream and downstream regions of the first CELF2a exon, were co‐transfected in UPΔ44 iPSCs. The edited clones were selected based on puromycin resistance, whose insertion was validated by PCR (F1‐R1). Homozygous clones were selected based on the absence of the CELF2a genomic region assessed by RT–PCR using the indicated oligonucleotides (F2‐R2). G qPCR analysis of the indicated muscle differentiation markers in iPSCs prior (iPSC) and after the induction of muscle differentiation (Myo) in the indicated clones. Relative levels of mRNAs were calculated with the delta delta C t method. qPCRs were normalized against an internal control (GAPDH) and plotted relative to the expression level in iPSCs (0) set to a value of 1. The mean ± SEM of triplicates from one representative experiment is shown ( n = 3). H Sanger sequencing data confirming the exon 45 skipping in the UPΔ44 CRISPR/Cas9 edited clones depleted for CELF2a (UPΔ44#3.1 and UPΔ44#3.5). Source data are available online for this figure.

Article Snippet: Three top ranking guides for the region containing the first exon of Celf2a (Chr10:11047177‐11047403, GRCh37/hg19 assembly) were selected and cloned into the CRISPR/Cas9 plasmid pX330 (Li et al , ; Addgene#42230).

Techniques: Expressing, Control, Reverse Transcription Polymerase Chain Reaction, Infection, Plasmid Preparation, Negative Control, Clone Assay, Selection, Transfection, Sequencing, CRISPR

Journal: EMBO Molecular Medicine

Article Title: Trans‐generational epigenetic regulation associated with the amelioration of Duchenne Muscular Dystrophy

doi: 10.15252/emmm.202012063

Figure Lengend Snippet: A Representative phase‐contrast images of GSΔ44 (#2, #8) and GSM (#1) pluripotent stem cell clones’ morphology. Scale bar 100 μm. B qPCR analyses of pluripotency‐associated genes (NANOG, OCT4, SOX2 and REX1) and of the direct inhibitor of OCT4 (NR2F2) in indicated patient‐specific iPSC clones relative to the somatic cells control (fibroblasts). PCRs were normalized against an internal control (GAPDH) and plotted relative to the expression level in the individual parent fibroblast cell lines set to a value of 1. The mean ± SEM of triplicates from one representative experiment is shown ( n = 3). C RT–PCR for the exogenous OCT4 mRNA in RNA extracted from GSΔ44 fibroblasts (GSΔ44 Fibro), fibroblasts collected 3 days after lentiviral infection with hSTEMCCA vector (Inf Fibro), patient‐specific iPSC clones #2 and 8 (GSΔ44#2 and GSΔ44#8) and the mother‐specific iPSC clone #1 (GSM#1). GAPDH was used as control. Lane (−) indicates the negative control. Representative results are shown ( n = 3). D Normal karyotype of GSΔ44#2 and GSΔ44#8 iPSC clones (46, XY). E Left panel: Schematic representation of GSΔ44‐specific gDNA deletion on the DMD gene showing the location of the oligonucleotides used to amplify deletion boundaries. Middle and right panel: Genetic identity with the parental cell line was assessed by PCR on gDNA. PCR was performed on gDNA from control fibroblasts (middle panel) and iPSCs (right panel) amplifying the indicated DMD exons and the DMD deletion boundary characteristic of GSΔ44 and GSM individuals. Lane (−) indicates the negative control. F qPCR analysis of the indicated muscle differentiation markers in iPSCs prior (iPSC) and after the induction of muscle differentiation (Myo) in the indicated clones. Relative levels of mRNAs were calculated with the delta delta C t method. qPCRs were normalized against an internal control (GAPDH) and plotted relative to the expression level in iPSCs (iPSC) set to a value of 1. The mean ± SEM of triplicates from one representative experiment is shown ( n = 3). G RT–PCR analyses of CELF2a, MyoG, MyoD, Baf60c and Mef2c in control (WT1 and WT2) and in GSΔ44 myoblasts transfected with a plasmid for the overexpression of MyoD alone (epB‐MyoD) or in combination with Baf60c (epB‐Baf60c) collected before treatment (Un) or at the indicated time points. GAPDH was used as control. Representative results are shown ( n = 3). H Selection of GSΔ44‐edited clones based on puromycin resistance, which was validated by PCR (F1‐R1). Homozygous clones were selected based on the absence of the CELF2a genomic region assessed by RT–PCR using the indicated oligonucleotides (F2‐R2) according to the schematic representation of the gene‐editing strategy shown in (Fig F). I Sanger sequencing data confirming the exon 45 skipping in the GSΔ44#8.2 CRISPR/Cas9‐edited clone depleted for CELF2a. Source data are available online for this figure.

Article Snippet: Three top ranking guides for the region containing the first exon of Celf2a (Chr10:11047177‐11047403, GRCh37/hg19 assembly) were selected and cloned into the CRISPR/Cas9 plasmid pX330 (Li et al , ; Addgene#42230).

Techniques: Clone Assay, Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Infection, Plasmid Preparation, Negative Control, Transfection, Over Expression, Selection, Sequencing, CRISPR

Journal: EMBO Molecular Medicine

Article Title: Trans‐generational epigenetic regulation associated with the amelioration of Duchenne Muscular Dystrophy

doi: 10.15252/emmm.202012063

Figure Lengend Snippet: A iPSCs obtained from a control (WT#1; Lenzi et al , ), GSΔ44 (GSΔ44#2, GSΔ44#8) and GSM (GSM#1) were differentiated into myocytes by MYOD/BAF60c overexpression. Cells were collected before the induction (iPSC) and after 9 days in DM (Myo), and RNA was analysed by RT–PCR for CELF2a, CELF2b, CELF2c and DMD expression. GAPDH was used as loading control. Representative results are shown ( n = 3). B Representative immunofluorescence for myosin heavy chain (MHC in red) in combination with DAPI staining (in blue) of iPSCs obtained from WT#1, GSΔ44 (GSΔ44#8) and GSΔ44 edited clones (GSΔ44#8.2) differentiated for 9 days into myocytes by MYOD/BAF60c overexpression. Scale bar 100 μm. C Histogram represents fusion index quantification. At least 7 randomly chosen microscope fields of two independent biological samples were analysed ( n = 2). Data are presented as mean ± SD of the biological replicates. D RNA extracted from myocytes obtained by differentiation of WT#1, GSΔ44 (GSΔ44#8) and GSΔ44‐edited clones (GSΔ44#8.2 ΔCELF2a) was analysed by RT–PCR for CELF2a, CELF2b, CELF2c and DMD exon 45 skipping. GAPDH was used as loading control. The arrow indicates the DMD exon 45‐skipped band. Representative results are shown ( n = 3). E Western blot on proteins (40 μg) extracted from myocytes obtained by differentiation of WT (WT#1), GSΔ44 (GSΔ44#8) and edited GSΔ44 (GSΔ44#8.2 ΔCELF2a) iPSC clones probed with antibodies against dystrophin (DMD). Actinin (ACTN) was used as a loading control. For the WT 5% sample, 2 μg of proteins was diluted in 38 μg of proteins belonging from GSΔ44#8 to reach the total amount of 40 μg. Representative results are shown ( n = 3). Source data are available online for this figure.

Article Snippet: Three top ranking guides for the region containing the first exon of Celf2a (Chr10:11047177‐11047403, GRCh37/hg19 assembly) were selected and cloned into the CRISPR/Cas9 plasmid pX330 (Li et al , ; Addgene#42230).

Techniques: Control, Over Expression, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunofluorescence, Staining, Clone Assay, Microscopy, Western Blot

Journal: EMBO Molecular Medicine

Article Title: Trans‐generational epigenetic regulation associated with the amelioration of Duchenne Muscular Dystrophy

doi: 10.15252/emmm.202012063

Figure Lengend Snippet: A Scatterplots of ATAC‐seq peak intensities between two conditions. B ATAC‐seq fragment sizes generated from WT and GSΔ44. C RT–PCR analysis for CELF2a, CELF2b, CELF2c expression in WT and GSΔ44 myoblasts. GAPDH was used as the loading control. Lane (−) indicates the negative control. Representative results are shown ( n = 3). D DNA accessibility (ATAC‐seq) and chromatin marks (ChIP‐seq) signal in the first exon region of CELF2a and b isoforms using the UCSC genome browser. ATAC‐seq peaks called in the two conditions are reported below each track. The differential peaks specific for one of the two conditions are reported in the tracks ATACseq_specific peaks_WT and ATACseq_specific peaks_GSΔ44. ChIP‐seq signals for H3K4me3, H3K27ac and H3K27me3 in WT and GSΔ44 myoblasts are reported (WT myoblasts in light orange; GSΔ44‐derived myoblasts in light blue). ChIP‐seq for MyoD in control cells was obtained from ref. MacQuarrie et al . Blue boxes highlight MyoD binding site peaks with differential DNA accessibility between WT final vs GSΔ44 (Peak 1 and 2).

Article Snippet: Three top ranking guides for the region containing the first exon of Celf2a (Chr10:11047177‐11047403, GRCh37/hg19 assembly) were selected and cloned into the CRISPR/Cas9 plasmid pX330 (Li et al , ; Addgene#42230).

Techniques: Generated, Reverse Transcription Polymerase Chain Reaction, Expressing, Control, Negative Control, ChIP-sequencing, Derivative Assay, Binding Assay

Journal: EMBO Molecular Medicine

Article Title: Trans‐generational epigenetic regulation associated with the amelioration of Duchenne Muscular Dystrophy

doi: 10.15252/emmm.202012063

Figure Lengend Snippet: A Schematic representation of the exon/intron organization of Celf2 genomic locus. Arrows indicate TSS sites. B RT–PCR analyses of indicated RNAs in control (WT), Δ44 and in GSΔ44 myocytes collected after 9 days upon the induction of differentiation. GAPDH was used as control. Representative results are shown ( n = 3). C RT–PCR analyses for CELF2a and DUXAP8 expression in WT and GSΔ44 myoblasts (GM) and myocytes (DM). GAPDH was used as control. Representative results are shown ( n = 3). D RT–PCR analysis of DUXAP8 expression in RNA extracted from GSΔ44, GSΔ44's mother (GSM) and GSΔ44's father (F) fibroblasts. GAPDH was used as control. Lane (−) indicates the negative control. Representative results are shown ( n = 3). E RT–PCR analysis for DUXAP8 expression in RNA extracted from WT and GSΔ44 myocytes and from WT (WT#1), GSΔ44 (GSΔ44#2, GSΔ44#8) and GSM (GSM#1) iPSCs induced to differentiation for 9 days. GAPDH was used as control. Lane (−) indicates the negative control. Representative results are shown ( n = 3). F RT–PCR for the indicated RNAs on extracts from GSΔ44 myoblasts treated with either control siRNAs (siSCR) or siRNAs against DUXAP8 (siDUXAP8) and collected 2 days after transfection. GAPDH was used as control. Lane (−) indicates the negative control. Representative results are shown ( n = 3). G qPCR analysis of DUXAP8 and CELF2a expression in control myoblasts transfected with a plasmid for the overexpression of DUXAP8 (OE DUXAP8) or an empty vector (Empty), and collected after 96 h. Relative mRNA levels were calculated with the delta delta Ct method. qPCRs were normalized against an internal control (GAPDH) and plotted relative to the expression level in the sample treated with the empty vector which was set to a value of 1. The mean ± SEM of triplicates from one representative experiment is shown ( n = 3). Source data are available online for this figure.

Article Snippet: Three top ranking guides for the region containing the first exon of Celf2a (Chr10:11047177‐11047403, GRCh37/hg19 assembly) were selected and cloned into the CRISPR/Cas9 plasmid pX330 (Li et al , ; Addgene#42230).

Techniques: Reverse Transcription Polymerase Chain Reaction, Control, Expressing, Negative Control, Transfection, Plasmid Preparation, Over Expression

Journal: EMBO Molecular Medicine

Article Title: Trans‐generational epigenetic regulation associated with the amelioration of Duchenne Muscular Dystrophy

doi: 10.15252/emmm.202012063

Figure Lengend Snippet: A Screenshot of UCSC Genome Browser displaying annotated lncRNAs in the Celf2 locus (chr10 in GRCh37/hg19 assembly). B RT–PCR for the indicated lncRNAs on extracts from WT, Δ44 and GSΔ44 myocytes. GAPDH was used as control. Representative results are shown ( n = 3). C RT–PCR of CELF2a and SFTA1P on cytoplasm (C) and nuclear (N) RNA obtained from GSΔ44 myoblasts transfected with a plasmid for SFTA1P overexpression (OE SFTA1P) or an empty vector (Empty) and incubated 48 h. GAPDH mRNA and pre‐mRNA (pre‐GAPDH) were used, respectively, as cytoplasm and nuclear controls. Lane (−) indicates the negative control. Representative results are shown ( n = 3). D RT–PCR for CELF2a and SFTA1P expression in RNA extracted from WT myoblasts treated with either a control gapmer (Gapmer SCR) or a gapmer against SFTA1P (Gapmer SFTA1P) and collected 2 days after transfection. GAPDH was used as control. Representative results are shown ( n = 3). E RT–PCR for SFTA1P and CELF2a expression in RNA extracted from GSΔ44 myoblasts transfected with an empty vector (Empty) or a plasmid for the overexpression of CELF2a (OE CELF2a) and collected after 48 h. GAPDH was used as control. Lane (−) indicates the negative control. Representative results are shown ( n = 3). F iPSCs’ RNA analysed in Fig A was further analysed by RT–PCR for SFTA1P expression. GAPDH has been shown twice to make the image easier to understand. Representative results are shown ( n = 3). G Venn diagram showing the number of differentially expressed lncRNAs between WT and Δ44 (blue) and WT and GSΔ44 (orange) samples. H RT–PCR on cytoplasm (C) nucleoplasm (N) and chromatin (Chr) GSΔ44 myoblast extracts showing the subcellular localization of DUXAP8. GAPDH mRNA and pre‐mRNA (pre‐GAPDH) were used as cytoplasm, nucleoplasm and chromatin controls. Lane (−) indicates the negative control. Representative results are shown ( n = 3). I RT–PCR on cytoplasm (C) nucleoplasm (N) and chromatin (Chr) WT extracts showing the subcellular localization of DUXAP8. WT myoblasts were transfected with a plasmid for the overexpression of DUXAP8 and collected after 96 h. GAPDH mRNA and pre‐mRNA (pre‐GAPDH) were used as cytoplasm, nucleoplasm and chromatin controls. Lane (−) indicates the negative control. Representative results are shown ( n = 3). Source data are available online for this figure.

Article Snippet: Three top ranking guides for the region containing the first exon of Celf2a (Chr10:11047177‐11047403, GRCh37/hg19 assembly) were selected and cloned into the CRISPR/Cas9 plasmid pX330 (Li et al , ; Addgene#42230).

Techniques: Reverse Transcription Polymerase Chain Reaction, Control, Transfection, Plasmid Preparation, Over Expression, Incubation, Negative Control, Expressing

Journal: EMBO Molecular Medicine

Article Title: Trans‐generational epigenetic regulation associated with the amelioration of Duchenne Muscular Dystrophy

doi: 10.15252/emmm.202012063

Figure Lengend Snippet: First panel: In WT condition, the lncRNA DUXAP8 is not expressed and the chromatin of Celf2a regulatory region is characterized by a permissive signature (H3K27ac and H3K4me3) that allows the expression of this factor. In the presence of Celf2a protein, the ex45 is normally included in the mature form of DMD mRNA. Second panel: In GSΔ44 cells, the lncRNA DUXAP8 is aberrantly expressed. The chromatin of Celf2a regulatory region is marked by the repressive H3K27me3, Celf2a is not expressed, and the ex45 is not included in the mature form of DMD mRNA allowing the production of an in‐frame DMD transcript (Δ44–Δ45). Third panel: in Δ44 condition DUXAP8 is absent, Celf2a is expressed and ex45 is included in the mature form of DMD mRNA producing an out of frame transcript (Δ44). Fourth panel: In Δ44 cells, the depletion of Celf2a isoform is paralleled by a recovery of dystrophin protein (5%) obtained by the exclusion of ex45 from the mature DMD transcript (Δ44–Δ45).

Article Snippet: Three top ranking guides for the region containing the first exon of Celf2a (Chr10:11047177‐11047403, GRCh37/hg19 assembly) were selected and cloned into the CRISPR/Cas9 plasmid pX330 (Li et al , ; Addgene#42230).

Techniques: Expressing