LMNA encodes nuclear lamin A/C that tethers lamina-associated heterochromatin domains (LADs) to the nuclear periphery. Point mutations in LMNA cause degenerative disorders including the premature aging disorder Hutchinson-Gilford progeria, but the mechanisms are unknown. We report that Ser22phosphorylated Lamin A/C (pS22-Lamin A/C) was localized to the interior of the nucleus in human fibroblasts throughout the cell cycle. pS22-Lamin A/C interacted with a specific subset of putative active enhancers, not LADs, primarily at locations co-bound by the transcriptional activator c-Jun. In progeriapatient fibroblasts, a subset of pS22-Lamin A/C-binding sites were lost whereas new pS22-Lamin A/Cbinding sites emerged in normally quiescent loci. These new pS22-Lamin A/C-binding sites displayed increased histone acetylation and c-Jun binding, implying increased enhancer activity. The genes near these new binding sites, implicated in clinical components of progeria including carotid artery diseases, hypertension, and cardiomegaly, were upregulated in progeria. These results suggest that Lamin A/C regulates gene expression by direct enhancer binding in the nuclear interior. Disruption of the gene regulatory rather than LAD function of Lamin A/C presents a novel mechanism for disorders caused by LMNA mutations including progeria. HIGHLIGHTS• pS22-Lamin A/C is present in the nuclear interior throughout interphase.• pS22-Lamin A/C associates with active enhancers, not lamina-associated domains.• pS22-Lamin A/C-genomic binding sites are co-bound by the transcriptional activator c-Jun.• New pS22-Lamin A/C binding in progeria accompanies upregulation of disease-related genes.

20LMNA encodes nuclear lamin A/C that tethers lamina-associated domains (LADs) to the nuclear lamina.Hutchinson-Gilford progeria is a premature aging disorder caused by heterozygous LMNA point mutations, however, the mechanism by which LMNA mutations cause progeria is unclear. We report that Ser22-phosphorylated LMNA (pS22-LMNA) was localized to the interior of the nucleus in human fibroblasts throughout the cell cycle. pS22-LMNA interacted with a specific subset of putative active 25 enhancers, not LADs, primarily at locations co-bound by the transcriptional activator c-Jun. In progeriapatient fibroblasts, some pS22-LMNA-binding sites were lost whereas new pS22-LMNA-binding sites emerged at abnormal locations. New pS22-LMNA-binding in progeria cells was accompanied by increased H3K27 acetylation, increased c-Jun binding, and upregulated expression of genes implicated in coronary artery diseases, hypertension, and cardiomegaly, clinical components of progeria. Thus, 30 pS22-LMNA bound to enhancers and progeria mutations affected pS22-LMNA-bound enhancer function.These observations expand the genomic role of LMNA to include direct enhancer binding and modulation, and introduce a novel molecular mechanism whereby LMNA mutation may contribute to disease distinct from LMNA's role at the nuclear lamina. 35interior was soluble and highly mobile (Broers et al., 1999;Shimi et al., 2008) thus present as a nonpolymerized form and not constituting a scaffold structure. The specific function of LMNA in the nuclear interior has been difficult to ascertain, mainly due to a lack of understanding about how LMNA is directed 65 to the nuclear interior, and a lack of laboratory approaches to isolate nuclear-interior LMNA.Polymerization and depolymerization of nuclear lamins, required for nuclear envelope breakdown and the cell cycle, are regulated by phosphorylation of specific serine residues (Gerace and Blobel, 1980;Heald and McKeon, 1990;Peter et al., 1990;Ward and Kirschner, 1990). Ser22 (S22) and Ser392 (S392) of LMNA are well characterized and are known as "mitotic sites" because they are phosphorylated during 70 mitosis, leading to LMNA depolymerization (Heald and McKeon, 1990;Peter et al., 1990;Ward and Kirschner, 1990). Early studies reported that phosphorylation of S22 and S392 begins at late G2 stage and is mediated by CDK1/Cyclin B, a kinase complex that promotes cell-cycle progression from G2 to mitosis (Georgatos et al., 1997;Heald and McKeon, 1990;Ward and Kirschner, 1990). More recently, S22 and S392 phosphorylation have been reported in the nuclear interior of interphase cells (Kochin et 75 al., 2014), suggesting that S22/S392-phosphorylated, non-polymerized LMNA may represent a nuclearinterior pool of LMNA in interphase cells (Torvaldson et al., 2015). Separate studies proposed that S22 and S392 phosphorylation are increased upon changes in the mechanical environment of the cell and promote LMNA disassembly and degradation (Buxboim et al., 2014;Swift et al., 2013). Therefore, LMNA S22/S392 phosphorylation has b...

Cardiomyopathies caused by mutations in LMNA, encoding nuclear Lamin A/C, are highly malignant and prevalent. How LMNA mutations cause cardiomyopathies remains unknown. We characterized cellular, molecular, and pathological evolution of mouse models of LMNA -related cardiomyopathy and provide evidence for a model in which nuclear rupture generates nuclear-localized proinflammatory signaling as a candidate molecular mechanism underlying disease pathogenesis. We observed that cardiomyocyte-specific, tamoxifen-inducible deletion of Lmna in adult mice ( Lmna CMKO ) caused a gradual reduction of Lamin A/C protein at the nuclear lamina, reflecting the slow turnover of Lamin A/C. A modest reduction of Lamin A/C in Lmna CMKO was sufficient to cause extensive fibrosis, reduced ejection fraction, and chamber dilation by 3 weeks after Lmna gene deletion. Lmna CMKO cardiomyocytes exhibited localized rupture of the nuclear envelope 2 weeks prior to the development of fibrosis and reduction of ejection fraction. Nuclear rupture in Lmna CMKO was immediately followed by an extensive upregulation of pro-inflammatory gene expression programs. We hypothesized that nuclear rupture might expose nuclear DNA to the cytoplasm thereby activating the pro-inflammatory cGas-STING cytosolic DNA sensing pathway. However, we did not observe localization of the cytosolic DNA sensor cGas to cytoplasmic DNA protruded from the ruptured nuclei in Lmna CMKO cardiomyocytes. Instead, we found that HMGB1, a potent proinflammatory protein normally sequestered in the nucleus, was released from the ruptured nuclei in Lmna CMKO cardiomyocytes. Mass spectrometry identified a strong interaction between Lamin A/C and HMGB1 in normal human fibroblast cells. Our data suggested that Lamin A/C tethers HMGB1 to the nuclear periphery by direct interaction and that reduction of Lamin A/C unleashes HMGB1 to the cytoplasm upon nuclear rupture. Future work will examine the hypothesis that cytoplasmic HMGB1 triggers pathogenic sterile inflammation leading to dilated cardiomyopathies in Lmna CMKO mice. In conclusion, we identified the nuclear rupture-induced cytoplasmic release of HMGB1 as a candidate mechanism underlying LMNA -related cardiomyopathies.

The segregation of heterochromatin domains (LADs) at the nuclear periphery by the nuclear lamina, composed by polymerized nuclear Lamin A/C, provides a longstanding paradigm for the control of gene expression and for the mechanisms underlying Lamin-A/C-associated disorders, including progeria and cardiomyopathy. Here, we provide evidence supporting a novel paradigm that Lamin A/C functions as a transcription factor in the nuclear interior. We discovered that Ser22-phosphorylated Lamin A/C (pS22-Lamin A/C), required for lamin depolymerization during mitosis, populated the nuclear interior throughout the cell cycle. pS22-Lamin A/C ChIP-deq demonstrated localization at a large subset of putative active enhancers, not LADs. pS22-Lamin A/C-binding sites were co-occupied by the transcriptional activator c-Jun. In progeria patient-derived fibroblasts, a subset of pS22-Lamin A/C-binding sites were lost whereas new pS22-Lamin A/C-binding sites emerged. New pS22-Lamin A/C binding was accompanied by increased histone acetylation and increased c-Jun binding, whereas loss of pS22-Lamin A/C-binding was accompanied by loss of histone acetylation and c-Jun binding. New pS22-Lamin A/C enhancer binding in progeria was associated with upregulated expression of genes implicated in progeria pathophysiology, including cardiovascular disease. In contrast, alteration of LADs in progeria-patient cells could not explain the observed gene expression changes. These results suggest that Lamin A/C regulates gene expression by enhancer binding in the nuclear interior, independent of its function at the nuclear lamina, providing a new paradigm for the pathogenesis of lamin-associated disorders. pS22-Lamin A/C was also present in the nuclear interior of adult mouse cardiomyocytes. Cardiomyocyte-specific deletion of Lmna encoding Lamin A/C in adult mice caused extensive transcriptional changes in the heart and dilated cardiomyopathy, without apparent reduction of nuclear peripheral Lamin A/C. Disruption of the gene regulatory rather than LAD tethering function of Lamin A/C may underlie the pathogenesis of disorders caused by LMNA mutations, including cardiomyopathy.