The long noncoding RNA Charme supervises cardiomyocyte maturation by controlling cell differentiation programs in the developing heart
- Department of Biology and Biotechnologies “Charles Darwin”, Sapienza University of Rome, Italy
- Center for Life Nano- and Neuro-Science, Istituto Italiano di Tecnologia (IIT), Italy
- Department of Medical Surgical Sciences and Biotechnologies, Sapienza University of Rome, Italy
- Center for Human Technologies, Istituto Italiano di Tecnologia, Italy
- Epigenetics and Neurobiology Unit, EMBL-Rome, Italy
- DAHFMO-Unit of Histology and Medical Embryology, Sapienza University of Rome, Italy
Figures
Figure 1 with 1 supplement
Charme locus expression in developing mouse embryos and in the heart.
(A) Transcriptional start site (TSS) usage analysis from FANTOM5 CAGE phase1 and 2 datasets (skeletal muscle is not included) on the last update of Zenbu browser (https://fantom.gsc.riken.jp/zenbu/; …
Figure 1—figure supplement 1
Study of Charme locus expression by WISH and scRNA-seq.
(A) In situ hybridization (ISH) performed on E13.5 embryonal cryo-sections using digoxigenin-labeled RNA antisense (Charme, left panel) or sense (control, right panel) probes against Charme. T, …
Figure 2 with 1 supplement
Genome-wide profiling of cardiac CharmeWT and CharmeKO transcriptomes.
(A) Heatmap visualization from RNA-seq analysis of CharmeWT and CharmeKO neonatal (PN2) hearts. Plot was produced by heatmap3 (Zhao, 2021). Expression values were calculated as FPKM, were log2-transf…
Figure 2—figure supplement 1
Transcriptomic and phenotypic characterization of CharmeWT and CharmeKO hearts.
(A) Schematic overview of the workflow to identifying differentially expressed genes (DEGs) from CharmeWT and CharmeKO transcriptomes (left panel). Multidimensional scaling plot of leading fold …
Figure 3 with 1 supplement
In fetal cardiomyocytes pCharme promotes MATR3 nuclear condensation.
(A) RNA-FISH for pCharme (red) and DAPI staining (blue) in CharmeWT cardiac and spinal cord from E15.5 tissue sections. Whole heart (white dashed lines), spinal cord (yellow dashed line). A, atria; …
Figure 3—figure supplement 1
pCharme and MATR3 nuclear localization analyses.
(A) Quantification of the subcellular distribution of pCharme and mCharme in cardiac tissues from neonatal mice. Histogram shows the quantification by RT-qPCR of the RNA abundance (%) in cytoplasmic …
Figure 4 with 1 supplement
MATR3/pCharme nuclear condensates contain key regulators of heart development.
(A) Schematic representation of MATR3 CLIP-seq workflow from fetal (E15.5) CharmeWT and CharmeKO hearts. See ‘Materials and methods’ for details. (B) MATR3 CLIP-seq from fetal hearts. Upper panel: a …
Figure 4—figure supplement 1
Experimental workflow, data output and validation of MATR3 CLIP-seq.
(A) Schematic representation of MATR3-CLIP assay as performed from fetal hearts (E15.5) in CharmeWT and CharmeKO conditions. MATR3 Western blot analysis on the retrieved protein fractions is shown. …
Figure 5 with 1 supplement
The pCharme/MATR3 interaction in cardiomyocytes sustains developmental genes expression.
(A) Pie charts showing the percentage of MATR3 targets in CharmeKO downregulated, invariant, or upregulated differentially expressed genes (DEGs). Significance of enrichment or depletion was …
Figure 5—figure supplement 1
Study of MATR3 binding properties from MATR3 CLIP-seq CharmeWT and CharmeKO datasets.
(A) Empirical cumulative distribution functions (ECDF) showing the RNA abundance of MATR3 targets in CharmeKO condition compared to the other expressed genes. Significance was determined using a …
Figure 6
Proposed model for pCharme functions during heart development.
At developmental stages (CharmeWT), pCharme is required for the expression of genes involved in cardiomyocyte maturation. This activity is accompanied by the formation of nuclear condensates, …
Author response image 1
Correlation of Tbx5 and pCharme expression in cardiomyocytes.
(A) COTAN heatmap obtained using the whole scRNA-seq dataset showing Charme positive correlation with Tbx5, both expressed in cardiomyocytes, and its negative correlation with Klf2, Pecam1, Sox9 and …
Author response image 2
Expression analysis of compact layer marker genes.
(A) Left panel: RT-qPCR quantification of Hey2 RNA levels in E15. 5 CharmeWT vs CharmeKO cardiac extract. RT-qPCR data were normalized to GAPDH mRNA and represent means ± SEM of WT and KO (n=3) …
Author response image 3
Expression analysis of cardiac trabeculae marker genes in neonatal hearts.
Average expression from neonatal (PN) RNA-seq (FPKM) in E15.5 CharmeWT vs CharmeKO cardiac extract of trabeculae marker genes.
Author response image 4
Expression analysis of proliferation marker genes in primary cardiac cells.
RT-qPCR quantification of cell cycle genes mRNA expression in primary cardiac cells treated with si-SCR or si-MATR3. Data were normalized to GAPDH mRNA and represent mean ± SEM of 4 independent …
Author response image 5
Expression analysis of Charme neighbouring genes.
(A) Contact map depicting Hi-C data of left ventricular mice heart retrived from GEO accession ID GSM2544836. Data related to 1 Mb region around Charme locus were visualized using Juicebox Web App …
Author response image 6
Quantification (FPKM) of Charme neighbouring genes expression from C2C12 RNA-seq data.
Average expression from RNA-seq (FPKM) quantification of Charme neighbouring genes in C2C12 differentiated myotubes treated with Gap-scr vs Gap-Charme. Values for Gap-Charme represent the average …
Author response image 7
Expression analysis of Gata4, Nkx2-5 and Tbx5 transcripts at embryonal stages.
RT-qPCR quantification Gata4, Nkx2-5 and Tbx5 in CharmeWT and CharmeKO cardiac extract at E12.5, E15.5 and E18.5 days of embryonal development. Data were normalized to GAPDH mRNA and represent means …
Author response image 8
Charme and Malat-1 lncRNAs expression in neonatal hearts.
Charme (left) and Malat-1 (right) in situ hybridization performed on whole (PN) sections. Black squares outline the magnified areas. Magnification shows that the signal is restricted to the nucleus. …
Additional files
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Supplementary file 1
Charme Tss usage data collected from Zenbu genome browser.
- https://cdn.elifesciences.org/articles/81360/elife-81360-supp1-v2.xlsx
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Supplementary file 2
RNA-seq in CharmeWT and CharmeKO neonatal hearts.
- https://cdn.elifesciences.org/articles/81360/elife-81360-supp2-v2.xls
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Supplementary file 3
Echocardiography measurement for CharmeWT and CharmeKO animals.
- https://cdn.elifesciences.org/articles/81360/elife-81360-supp3-v2.xlsx
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Supplementary file 4
MATR3 CLIP-seq in CharmeWT and CharmeKO fetal hearts.
- https://cdn.elifesciences.org/articles/81360/elife-81360-supp4-v2.xlsx
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Supplementary file 5
List and sequences of the oligonucleotides, siRNAs, antibodies, and imaging probes used.
- https://cdn.elifesciences.org/articles/81360/elife-81360-supp5-v2.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/81360/elife-81360-mdarchecklist1-v2.pdf
