Fig 1: Transcriptional activation and epigenetic priming in mESCs upon lamin A/C loss results in cardiac programming.a Volcano plot showing the distribution of DEG between Lmna+/+ and Lmna-/- mESCs (log2 fold change =-0.58, =0.58; p-value <0.05, determined by DESeq2 algorithm). b Heat-map representation of RNA-Seq analysis of control and Lmna-/- mESCs. Representative genes and enriched GO terms in upregulated (red) and downregulated (blue) genes are presented. c Confocal images of immunostaining for GATA4, MYL4 (red), and DAPI (blue) of Lmna+/+ and Lmna-/- mESCs. Scale bars, 20 µm. d Overlap of genes within lamin A LADs, lamin B1 LADs and genes upregulated in Lmna-/- mESCs. e Hierarchical cluster analysis of genes within lamin A LADs and upregulated either in Lmna-/- mESCs, Lmna-/- CPs, or Lmna-/- CMs. f Genome tracks of ATAC-Seq reads in Lmna+/+ and Lmna-/- ESCs and normalized RNA-Seq reads in control (black) and Lmna-/- (red) ESCs, CPs and CMs at Chr. 14. Red arrows indicate large-scale chromatin opening. g Normalized ATAC-Seq signal intensity at the TSS ± 5 kb of all genes (left) as well as genes upregulated (middle) and downregulated (right) upon lamin A/C LOF in Lmna+/+ and Lmna-/- ESCs. h Genome tracks of merged ATAC-Seq and RNA-Seq reads of Lmna+/+ and Lmna-/- ESCs at example genes. i Hierarchical cluster analysis of genes showing increased chromatin accessibility in ESCs and upregulation either in Lmna-/- ESCs, Lmna-/- CPs or Lmna-/- CMs. j, m Overlap between genes showing increased ATAC-Seq signal and genes upregulated upon Lmna LOF in ESCs (j) or in CPs and CMs (m). k, n GO analysis of the overlapping genes in (j and m, respectively). l, o Enrichment of known TF motifs in ATAC-Seq peaks of genes showing increased chromatin accessibility and upregulation in Lmna-/- ESCs (l) or upregulation in both Lmna-/- CPs and CMs (o). a-o: ATAC-Seq and RNA-Seq datasets from control and Lmna-/- ESCs, CPs, and CMs are from n = 3 biologically independent samples. b, k, n P-values are from GO pathway analysis using DAVID Bioinformatics Resources 6.8. l, o P-values were determined using Homer motif enrichment analysis.
Fig 2: MYL4+mosaicism by gender and disease. (A) Male subjects had more MYL4+ cells, independent of disease state (p = 8.66e-15). (B) In left ventricular segments, MYL4+ cells were increased in HCM compared to control tissues (TMAs 1&5). There were no control septal tissues to compare to the HCM cases on TMA4. The MYL4 residual is of a regression of sqrt(%MYL4+) cells adjusted for multiple measures per individual and effects of different TMAs, age, and sex.
Fig 3: Representative images of mosaic MYL4 staining patterns in various cardiac diseases. (A) Female, sudden cardiac death, 1% MYL4+ (B) Female, non-cardiac cause of death, 13.8% MYL4+ (C) Male, ACM, 32.3% MYL4+ (D) Male, non-cardiac cause of death 48.2% MYL4+ (E) Male, hypertrophic cardiomyopathy, 55.9% MYL4+ (F) Male, non-cardiac cause of death 78.7% MYL4+. All images taken from the same TMA.
Fig 4: Key function of lamin A/C in naïve pluripotent stem cells for cardiac development and disease.In naïve pluripotent stem cells, lamin A/C tethers cardiac-specific genes and genes involved in stem cell differentiation to the repressive nuclear periphery. Lamin A/C loss leads to their dissociation from the nuclear lamina accompanied by major chromatin reorganization resulting in either ectopic expression in mESCs (e.g., Gata4, Myl4) or epigenetic priming for activation later in development. These changes lead to precocious activation of a gene expression program promoting CM versus endothelial cell fate, accompanied by premature cardiomyocyte differentiation, cell cycle withdrawal, and abnormal contractility, which is dependent on Gata4. The figure was designed with BioRender.
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