Journal: bioRxiv

Article Title: RNA polymerase loss by nuclear rupture drives LMNA cardiomyopathy

doi: 10.64898/2026.04.03.716433

Figure Lengend Snippet: A) Immunofluorescence for bromouridine (BrU) after cardiomyocytes were cultured with BrU for 2 hr to label nascent RNAs. One binucleated cardiomyocyte per image, with nuclei zoomed in. Texts and icons below images indicate nuclear states. Scale bar: 5 μm. All data in , except , are from cardiomyocytes freshly isolated from mice at day 14 post tamoxifen. B) Nuclear BrU intensity by nuclear states after 1-hr BrU incubation. Density and box plots: BrU intensity distribution and interquartile range. Circles: mean intensity within biological replicates (color coded). Asterisks: P < 0.05 from t -tests on linear regression-estimated means with mouse-clustered standard errors. Underlying data: 244 intact nuclei from 4 WT mice, 275 intact, 111 ruptured, 104 resealed nuclei from 3 Lmna CKO mice. C) Relationship between nuclear BrU intensity and nuclear NLS–tdTomato intensity in all types of nuclei in Lmna CKO cardiomyocytes (519 nuclei from 3 mice). Line: simple linear regression fit with 95% confidence interval. R: Pearson correlation coefficient. P: t -test p -value on linear regression-estimated means with mouse-clustered standard errors. See for individual replicates. D) Immunofluorescence for RNA polymerase II (Pol II) in cardiomyocytes. Scale bar: 5 μm. E) Nuclear Pol II intensity by nuclear states. Underlying data: 285 intact nuclei from 3 WT mice, 180 intact, 133 ruptured, 136 resealed nuclei from 3 Lmna CKO mice. Graph annotations and statistics as in (B) . F) Relationship between Pol II intensity and NLS–tdTomato intensity in all types of nuclei in Lmna CKO cardiomyocytes (495 nuclei from 3 mice). Graph annotations and statistics as in (C) . G) Pol II ChIP-seq and input read coverage in WT and Lmna CKO cardiomyocytes (3 mice per genotype). ChIP-seq signals are normalized to internal spike-in controls. H) Average Pol II ChIP-seq signals across 21,177 protein-coding genes. X-axis: 100 equally-spaced bins in gene bodies, 5 bins for 1 kb-upstream regions, and 10 bins for 2 kb-downstream regions. I) Statistical comparison of gene-body Pol II signals in Lmna CKO versus WT cardiomyocytes for 11,942 Pol II-bound genes. Pol II-lost or gained genes are defined at limma p -value < 0.05. J) Ten most enriched Gene Ontology terms among the 1,759 Pol II-lost genes in Lmna CKO cardiomyocytes, with three representative genes for each term. P: Metascape p -value. K) Gene expression state of Pol II-lost, Pol II-gained, and all other genes in Lmna CKO (n=5) versus WT (n=7) hearts. P, DESeq2 p -value. RNA-seq data from En et al. 2024. L) Summary of . Nuclear rupture causes transcriptional deficiency due to RNA Pol II loss.

Article Snippet: Lysates were incubated with rabbit anti-Pol II NTD antibody (Cell Signaling Technology, #14958) overnight at 4 °C.

Techniques: Immunofluorescence, Cell Culture, Isolation, Incubation, ChIP-sequencing, Comparison, Gene Expression, RNA Sequencing

Journal: bioRxiv

Article Title: RNA polymerase loss by nuclear rupture drives LMNA cardiomyopathy

doi: 10.64898/2026.04.03.716433

Figure Lengend Snippet: A) Relationship between nuclear BrU intensity and nuclear NLS–tdTomato intensity in cardiomyocytes for each of 3 Lmna CKO mice. Line: simple linear regression fit with 95% confidence interval. R: Pearson’s correlation coefficient. All data in are derived from mice at day 14 post tamoxifen. B) Relationship between nuclear RNA Pol II intensity and nuclear NLS-tdTomato intensity in cardiomyocytes for each of 3 Lmna CKO mice. Other details as in (A) . C) Immunofluorescence for Pol II CTD phospho-Ser5 (Pol II Ser5p) in isolated cardiomyocytes expressing NLS-tdTomato and GFP-icGAS. Scale bar: 5 μm. D) Nuclear Pol II Ser5p intensity by nuclear states. Density and box plots (interquartile range): signal distribution of all nuclei. Circles: mean intensity within individual biological replicates (color coded). Asterisks: P < 0.05 from t-tests on linear regression-estimated means with mouse-clustered standard errors. Underlying data: 218 intact nuclei from 3 WT mice, 187 intact, 111 ruptured, 91 resealed nuclei from 3 Lmna CKO mice. E, F) Relationship between Pol II Ser5p intensity and NLS–tdTomato intensity in nuclei of Lmna CKO cardiomyocytes. (E) 424 nuclei from three biological replicates (mice). (F) Analysis within individual biological replicates. Other details as in (A) . G) Immunofluorescence for Pol II CTD phospho-Ser2 (Pol II Ser2p) in isolated cardiomyocytes expressing NLS-tdTomato and GFP-icGAS. Scale bar: 5 μm. H) Nuclear Pol II Ser2p intensity by nuclear states. Underlying data: 198 intact nuclei from 3 WT mice, 233 intact, 101 ruptured, 131 resealed nuclei from 3 Lmna CKO mice. N.S.: not significant. Other details as in (D) . I, J) Relationship between Pol II Ser2p intensity and NLS-tdTomato intensity in nuclei of Lmna CKO cardiomyocytes. (I) 517 nuclei from three biological replicates (mice). (J) Analysis within individual biological replicates. Other details as in (A) .

Article Snippet: Lysates were incubated with rabbit anti-Pol II NTD antibody (Cell Signaling Technology, #14958) overnight at 4 °C.

Techniques: Derivative Assay, Immunofluorescence, Isolation, Expressing

Journal: bioRxiv

Article Title: RNA polymerase loss by nuclear rupture drives LMNA cardiomyopathy

doi: 10.64898/2026.04.03.716433

Figure Lengend Snippet: A) Number of Pol II ChIP-seq and input sequencing reads aligned to the mouse genome (experimental) or the human genome (spike-in control). Scale factors are computed by spike-in control reads and sequencing depths and used to normalize Pol II ChIP-seq signals. All data in are derived from mice at 2 weeks post tamoxifen. B) Pol II ChIP-seq read coverage in all mouse genes stratified by gene-body Pol II coverage. Genes with Pol II coverage greater than or equal to 100 (2 in Log 10 ) were considered Pol II-bound (11,942 genes). C) Pol II ChIP-seq read coverage in all human genes, derived from the spike-in control chromatin. D) Gene-body Pol II ChIP-seq read coverage between every pair of biological replicates. E) Principal Component Analysis (PCA) of gene-body Pol II coverage in 11,942 Pol II-bound protein-coding genes. F) Cumulative fraction of 1,759 Pol II-lost genes and all other genes (y-axis) along the scale of differential gene expression between Lmna CKO hearts and wild-type hearts (x-axis). P, Kolmogorov-Smirnov test p -value comparing log 2 fold change of gene expression between Pol II-lost genes and all other genes. G) Gene expression state of Pol II-lost genes, Pol II-gained genes, and all other genes in the cardiomyocyte population in Lmna CKO (n=3) versus WT (n=3) hearts derived from single-nucleus RNA-seq in En et al. 2024. P, DESeq2 p -value. H) Same as F, but along the scale of differential gene expression between Lmna CKO and wild-type pseudo-bulk cardiomyocytes from the single-nucleus RNA-seq. P, Kolmogorov-Smirnov test p -value comparing log 2 fold change of gene expression between Pol II-lost genes and all other genes.

Article Snippet: Lysates were incubated with rabbit anti-Pol II NTD antibody (Cell Signaling Technology, #14958) overnight at 4 °C.

Techniques: ChIP-sequencing, Sequencing, Control, Derivative Assay, Gene Expression, RNA Sequencing

Journal: bioRxiv

Article Title: RNA polymerase loss by nuclear rupture drives LMNA cardiomyopathy

doi: 10.64898/2026.04.03.716433

Figure Lengend Snippet: A) Immunofluorescence for BANF1 and Desmin in human hearts. Top: heart biopsy from an individual with a LMNA p.R541C heterozygous mutation. Middle: autopsy heart from an individual with a pathogenic ACTC1 mutation. Bottom: apparently healthy autopsy heart. Inset: magnified areas enclosed by white squares. Scale bar: 20 μm. B) Three-dimensional reconstruction of BANF1 (green) and DNA (red) of nuclei (#1-4) in the LMNA p.R541C heart indicated in ( A ). Scale bar: 5 μm. C) Nuclei positive for nuclear-tip BANF1 per specimen field (normal heart, n=4 fields; ACTC1 heart, n=5 fields, LMNA -R541C heart, n=5 fields). Box: interquartile range. Asterisk: p -value <0.05 in Kruskal–Wallis test with Holm-corrected Wilcoxon tests. D) Model. Nuclear rupture drives LMNA -cardiomyopathy due to Pol II loss. Ruptured nuclei accumulate in mutant hearts due to frequent re-rupture of resealed nuclei.

Article Snippet: Lysates were incubated with rabbit anti-Pol II NTD antibody (Cell Signaling Technology, #14958) overnight at 4 °C.

Techniques: Immunofluorescence, Mutagenesis