A transposable element into the human long noncoding RNA CARMEN is a switch for cardiac precursor cell specification

Figures

• 

  Figure 1. CARMEN-201 controls SMC specification via its second exon. (A and B) Representative images and quantification of ACTN2-positive TNNI-positive CMs and SMMHC-positive SMCs in differentiating fetal and adult CPC cultures. Scale bar: 50 µm. (C) Annotated CARMEN isoforms. The CARMEN-201second exon is highlighted. (D) Absolute quantification of CARMEN-201 (C-201), CARMEN-205 (C-205) and CARMEN-217 (C-217) in differentiating fetal and adult CPCs. (E) Nuclear and cytoplasmic levels of CARMEN-201, CARMEN-205, CARMEN-217, ACTB, and NEAT. (F) Expression of CARMEN-201, CARMEN-205, CARMEN-217, SMC markers (MYH11; CNN1; TAGLN), and CM markers (MYH6; MYH7) in adult WT and Δ201Ex2 CPC clones lacking CARMEN-201 Exon 2. (G and H) Representative images and quantification of SMMHC-positive CNN1-positive TAGLN-positive SMCs in cultures of differentiating adult WT or Δ201Ex2 CPC clones. Scale bar: 50 µm. Data represent mean values ± SEM; *P < 0.05 as compared with fetal CPCs in expansion; §P < 0.05 compared with the indicated conditions (n = 3–6). ANOVA with post hoc Tukey. See also see Supplementary material online, Figure S1–S3.

• 

  Figure 2. The CARMEN-201 Exon 2 contains a functional transposable element implicated in SMC specification. (A) Expression of CARMEN isoforms and (B) SMC markers (MYH11; CNN1; TAGLN; CALD1) in differentiating fetal cells either untransfected (None), transfected with the SAM system in the absence of gRNA (SAM) or with the SAM system with a gRNA targeting sequences upstream the CARMEN TSS (SAM/gRNA). (C) Representative images and quantification of SMCs in cultures of differentiating fetal CPCs transfected as in (A). Scale bar: 50 µm. (D) Position of the MIRc transposable element in the CARMEN-201second exon, and sequence conservation. (E) Expression of CARMEN-201 using either a primer pair specific for the endogenous transcript (P1) or the exogenous Exon 2 (P3), and (F) SMC markers (MYH11; CNN1; TAGLN; CALD1) in differentiating fetal CPCs either not transduced (None), transduced with a lentiviral vector encoding CARMEN(C)-201 Ex2 or transduced with a lentiviral vector encoding a mutated C-201 Ex2 (C-201 mutEx2). (G) Representative images and quantification of SMMHC-positive CNN1-positive SMCs in cultures of differentiating fetal CPCs transfected as in (F). Scale bar: 50 µm. Data represent…

• 

  Figure 3. Transcriptomic analysis and identification of upstream regulators of CM and SMC specification in fetal CPCs with or without C-201 Exon 2 overexpression. (A) Principal Component Analysis (PCA) visualizing transcriptomic data in a two-dimensional space. (B) Expression heatmap of regulators and markers of the CM and the SMC fate. The heatmaps show scaled TPM values. (C and D) TRIAGE transformation of input RNA Seq data predicts regulatory genes controlling cell differentiation based on TRIAGE rank order (left) compared with ranking observed using simple gene expression (right). Control differentiation (None) vs. differentiation following C-201 Exon 2 overexpression (C-201 Ex2) 1 (d1) or 7 days (d7) after transduction. Regulators implicated in cardiovascular differentiation are highlighted in red. (E) Human heart single-cell data analysis reveals genes positively and negatively correlated with CARMEN expression during development. (F) UMAP plots showing epicardium-, pericyte-, and SMC-specific expression of CARMEN in the human heart 6.5 weeks post-conception. See also Supplementary material online, Figure S4 and S5.

• 

  Figure 4. Identification of CARMEN-201 protein partners. (A) Quantification of REST, (B) NSUN6 and (C) RPA1 by Western blotting in a protein pulldown assay using biotinylated sense or antisense CARMEN-201 transcript in adult CPC lysates. Graphs show mean values ± SEM *P < 0.05 as compared with input; §P < 0.05 comparing sense and antisense probe (n = 3). (D–F) Quantification of CARMEN-201 enrichment after RNA immunoprecipitation using a control immunoglobulin G (IgG) or IgG directed against REST (anti-REST IgG), NSUN6 (anti-NSUN6 IgG) and RPA1 (anti-RPA1 IgG). Graphs show mean values ± SEM; *P < 0.05 as compared with control (n = 3). (G) Expression of REST, CARMEN-201 and SMC markers (MYH11; CNN1; TAGLN) in differentiating fetal CPCs either not transduced (None), transduced with a lentiviral vector encoding C-201 Exon 2 (C-201 Ex2), and treated with either a scrambled siRNA (Scr siRNA) or a siRNA directed against REST (Anti-REST siRNA). (H) Representative images and quantification of SMMHC-positive CNN1-positive SMCs and ACTN2-psoitive CMs in cultures of differentiating fetal CPCs transfected as in (G). Scale bars: 50 µm. Data represent mean values ± SEM; *P < 0.05 as compared with fetal…

• 

  Figure 5. Identification of IRX1, IRX5, SFRP1 and ISL1 as target genes of C-201 action. (A) Significant DNA-binding domains (DBDs) identified in the mature sequence of C-201 and C-205 when analyzed against the differentially downregulated genes on Day 1 and Day 7 following induction of differentiation. Graph shows the number of DNA-binding sites (DBS) for each DBD. (b) Venn diagram illustrating the overlap of promoters predicted to form triple helices with C-201 and C-205. Functional enrichment analysis of the isoform-specific bound promoters. Graph shows the negative logarithm of the P value. (C) Venn diagram illustrating the identification of IRX1, IRX5, ISL1, and SFRP1 as common to the indicated lists of genes. Hypergeometric tests were performed to explore the significance of the overlap (D) REST occupancy at the promoters of IRX1, IRX5, SFRP1, and ISL1 in adult CPCs with or without GapmeR-mediated C-201 silencing as determined by ChIP-qPCR. Occupancy at the GAPDH and the SYN1 promoters was used as negative and positive controls, respectively. (E) Expression of IRX1, IRX5, ISL1, and SFRP1 in differentiating fetal cells either untransfected (None), transfected with the SAM system in the…

• 

  Figure 6. Validation of candidate cardiogenic factor downregulation. (A–D) Expression of epicardial genes (WT1, TCF21, TBX18) in differentiating fetal CPCs following IRX1, IRX5, ISL1, and SFRP1 silencing. (E) Epicardial (WT1, TCF21, TBX18) SMC (MYH11, TAGLN, CNN1) and CM marker (MYH6) expression in differentiating fetal CPCs overexpressing C-201 Ex2, and in fetal CPCs treated with siRNAs directed against the indicated factors in combination. (F) Representative images and quantification of ACTN2-positive CMs and SMMHC-positive CNN1-positive SMCs in cultures of differentiating fetal CPCs transfected as in (E). Scale bars: 50 µm. Expression of CARMEN-201 (C-201), CARMEN-205 (C-205) and CARMEN-217 (C-217) in differentiating fetal CPCs treated as in (E). Data represent mean values ± SEM; *P < 0.05 as compared with fetal CPCs in expansion; §P < 0.05 as compared with fetal CPCs in differentiation (n = 6–12). ANOVA with post hoc Tukey. See also see Supplementary material online, Figure S8.

• 

  Figure 7. CARMEN-201 expression is increased in response to myocardial infarction in humans. (A) Association of CARMEN with cardiovascular traits using CTG-VIEW. (B–E) Detection of C-201 in the failing human heart by RNA in situ hybridization assay (BaseScope). (B and C) Representative images of sections of an explanted heart obtained from a heart failure patient. (B). Top: Hematoxylin/Eosin; scale bar: 500 μm; Bottom: Masson Trichrome staining; scale bar: 500 μm. (C) Left: Masson Trichrome staining; scale bar: 100 µm; Right: Hematoxylin/Eosin; scale bar: 20 µm. Red dots: Positive C-201 BaseScope signals. (D and E) Quantification of C-201 expression in CMs and mural cells. Data represent mean values ± SEM; *P < 0.05; ANOVA with post hoc Tukey. Two patients; 5 sections per patient; 5 to 10 different areas per section. LV: Left ventricle; RV: Right ventricle; LA: Left atria; RA: Right atria. (F) Immunostaining detection of VIMENTIN-positive cells (endothelial cells and fibroblasts) and SMMHC-positive cells (SMCs) in adjacent sections of that used in b and c; scale bar: 25 µm. (G) Time course of blood sample collection, and expression of C-201 in plasma of STEMI and NSTEMI patients (Data…