Knockdown of lamins and nesprins alters gene expression.

a) Schematic illustration of the regulation of gene expression by lamins and nesprins. The lamins and nesprins could affect gene expression in distal non-LADs through altering RNA synthesis, chromatin conformation change, and chromatin modification. NPC, nuclear pore complex; LADs, lamina-associated domains. b) Knockdown of lamin A, LMNA, and SYNE2 mRNA levels by targeting shRNAs (shLaminA, yellow; shLMNA, green; shSYNE2, red) was quantified by RT-qPCR and RNA-seq, respectively. In contrast, the relative gene levels of targeting genes (lamin A, LMNA, SYNE2) are unchanged in the U2OS cells with scramble shRNA construct (shScramble, blue). U2OS cells were treated or untreated with 100 ng ml-1 doxycycline for 48 h. The isoform log2 fold change of lamin A was calculated by IsoformSwitchAnalyzeR package. Data represent mean ± s.d. RT-qPCR, n = 7 per group, p-value < 0.05; RNA-seq, n = 3 per group, q-value < 0.05.

Impact of lamins and nesprins on RNA biosynthesis, chromatin conformation, and modification.

DEGs were identified using the DESeq2 package by comparing the doxycycline-treated group to the untreated group. Gene Ontology (GO) enrichment analysis of DEGs was conducted using the clusterProfiler package. Gene counts are displayed on the x-axis. The top pathways were selected based on a q-value cutoff of < 0.05 for shLaminA (a), shLMNA (b), and shSYNE2 (c), respectively. d) Volcano plots illustrating the distribution of DEGs located in lamina-associated domains (LADs) and non-LAD regions. DEGs were defined by an absolute log₂ fold change > 0.5 and p-value < 0.05.

Roles of lamins and nesprins in isoform switching.

Isoform switching analysis reveals differential expression of alternative transcript variants between conditions, highlighting a shift in predominant isoform usage. a, b, c) Volcano plots illustrating the dIF of differentially expressed isoform transcripts against the −log10 of the isoform switch q-values in shLaminA (a), shLMNA (b), and shSYNE2 (c). Isoform switching was detected using IsoformSwitchAnalyzeR (n = 3 per group, cutoff dIF > 0.1, q-value < 0.05). a) Lamin A knockdown leads to isoform switching in chromatin remodeling (JADE1) and alternative splicing regulation (RBM11). b) LMNA knockdown results in isoform switching of transcriptional repressors (SAFB2, LIN54) and transcription factors (TEAD2, AHR). c) SYNE2 knockdown significantly alters isoform ratios of RNA-binding proteins (HNRNPK, ILF3, HNRNPA1). d) UpSet plot displaying the number and overlap of differentially expressed isoforms in protein-coding mRNAs and lncRNAs, as well as the log2 fold change of isoform transcripts in shLaminA, shLMNA, and shSYNE2 conditions. Intersection sizes indicate the number of shared DEGs between knockdowns. Statistical analysis was performed using the non-parametric Kruskal-Wallis test to compare medians across multiple groups.

Regulation of gene expression in distal non-LAD regions by lamins and nesprins.

a) Venn diagram illustrating the number of DE mRNAs in shLaminA (yellow), shLMNA (green), and shSYNE2 (red), with minimal overlap with genes located in LADs (blue). LAD-associated genes were identified using a BED file derived from lamin A/C ChIP-seq data 54. DEGs were identified using thresholds of absolute log2 fold change > 0.5 and p-value < 0.05. b) GO enrichment analysis showing the genomic distribution of DEGs across chromosome arms. for shLaminA, shLMNA, and shSYNE2. The p-values (blue to red), and the counts of DE genes mapped to the reference chromosome coordinates are shown. Depletion of lamin A affects chr10q12, LMNA affects chr20q13, and SYNE2 affects chr9q34, chr1q36. c) DE genes from shLaminA, shLMNA, and shSYNE2 are spatially distant from LADs. GenometriCorr analysis (v1.1.24) was used to assess the spatial relationship between DEGs (query) and LADs (reference) 55. The x-axis shows the relative genomic distance between each DEG and the nearest LAD, scaled from –1 (far upstream) to 1 (far downstream), with 0 indicating closest proximity. The y-axis represents the density of DEGs at each distance bin. A color gradient indicates deviation from a randomized null distribution: red signifies enrichment (closer than expected), and blue signifies depletion. Statistical significance was determined using the Jaccard test (p < 0.05).

Modulation of lncRNA-mRNA Interactions by Lamins and Nesprins.

a) Expression distribution of lncRNAs versus mRNAs in log2 transcript per million (Log2TPM), highlighting differences in transcript abundance. b) DeepLncLoc predictions showing nuclear localization of identified lncRNAs. c) Cis-acting lncRNA-mRNA interaction network predicted based on highly correlated expression levels within 10 kb windows (Pearson correlation > 0.8, q-value < 0.05). d) Coexpression network analysis of lncRNA-mRNA interactions. Nodes represent transcripts; edges denote coexpression relationships (Pearson correlation > 0.8, q-value < 0.05).

Increased telomere dynamics following depletion of lamin A, LMNA, and SYNE2.

a) Representative fluorescence images of telomeres labeled with EGFP-dCas9 in U2OS cells. Cells expressing dCas9-EGFP, targeted to telomeric repeats, were imaged via TIRF microscopy. Scale bar = 5 µm. b) MSD analysis of telomere mobility over 30 s at 500 ms intervals in control and knockdown conditions. MSD curves represent n > 20 cells per group. c) Total telomere movement range tracked over a 30 s imaging window. Annotated numbers indicate the median values for each group. Statistical analysis was performed using the non-parametric Kruskal-Wallis test, with a significance threshold of *p-value < 0.05, **p-value < 0.001, and n.s. p-value > 0.05.