Huntington\'s disease (HD) is caused by a polyglutamine expansion of the huntingtin protein, resulting in the formation of polyglutamine aggregates. The mechanisms of toxicity that result in the complex HD pathology remain only partially understood. Here, we show that nuclear polyglutamine aggregates induce nuclear envelope (NE) blebbing and ruptures that are often repaired incompletely. These ruptures coincide with disruptions of the nuclear lamina and lead to lamina scar formation. Expansion microscopy enabled resolving the ultrastructure of nuclear aggregates and revealed polyglutamine fibrils sticking into the cytosol at rupture sites, suggesting a mechanism for incomplete repair. Furthermore, we found that NE repair factors often accumulated near nuclear aggregates, consistent with stalled repair. These findings implicate nuclear polyQ aggregate-induced loss of NE integrity as a potential contributing factor to Huntington\'s disease and other polyglutamine diseases.

The Spalt transcriptional regulators participate in a variety of cell fate specification processes during development, regulating transcription through interactions with DNA AT-rich regions. Spalt proteins also bind to heterochromatic regions, and some of their effects require interactions with the NuRD chromatin remodeling and deacetylase complex. Most of the biological roles of Spalt proteins have been characterized in diploid cells engaged in cell proliferation. Here, we address the function of Drosophila Spalt genes in the development of a larval tissue formed by polyploid cells, the prothoracic gland, the cells of which undergo several rounds of DNA replication without mitosis during larval development. We show that prothoracic glands depleted of Spalt expression display severe changes in the size of the nucleolus, the morphology of the nuclear envelope and the disposition of the chromatin within the nucleus, leading to a failure in the synthesis of ecdysone. We propose that loss of ecdysone production in the prothoracic gland of Spalt mutants is primarily caused by defects in nuclear pore complex function that occur as a consequence of faulty interactions between heterochromatic regions and the nuclear envelope.

Stem cell loss in aging and disease is associated with nuclear deformation. Yet, how nuclear shape influences stem cell homeostasis is poorly understood. We investigated this connection using Drosophila germline stem cells, as survival of these stem cells is compromised by dysfunction of the nuclear lamina, the extensive protein network that lines the inner nuclear membrane and gives shape to the nucleus. To induce nuclear distortion in germline stem cells, we used the GAL4-UAS system to increase expression of the permanently farnesylated nuclear lamina protein, Kugelkern, a rate limiting factor for nuclear growth. We show that elevated Kugelkern levels cause severe nuclear distortion in germline stem cells, including extensive thickening and lobulation of the nuclear envelope and nuclear lamina, as well as alteration of internal nuclear compartments. Despite these changes, germline stem cell number, proliferation, and female fertility are preserved, even as females age. Collectively, these data demonstrate that disruption of nuclear architecture does not cause a failure of germline stem cell survival or homeostasis, revealing that nuclear deformation does not invariably promote stem cell loss.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic condition characterized by features of accelerated aging, and individuals with HGPS seldom live beyond their mid-teens. The syndrome is commonly caused by a point mutation in the LMNA gene which codes for lamin A and its splice variant lamin C, components of the nuclear lamina. The mutation causing HGPS leads to production of a truncated, farnesylated form of lamin A referred to as \"progerin.\" Progerin is also expressed at low levels in healthy individuals and appears to play a role in normal aging. HGPS is associated with an accumulation of genomic DNA double-strand breaks (DSBs) and alterations in the nature of DSB repair. The source of DSBs in HGPS is often attributed to stalling and subsequent collapse of replication forks in conjunction with faulty recruitment of repair factors to damage sites. In this work, we used a model system involving immortalized human cell lines to investigate progerin-induced genomic damage. Using an immunofluorescence approach to visualize phosphorylated histone H2AX foci which mark sites of genomic damage, we report that cells engineered to express progerin displayed a significant elevation of endogenous damage in the absence of any change in the cell cycle profile or doubling time of cells. Genomic damage was enhanced and persistent in progerin-expressing cells treated with hydroxyurea. Overexpression of wild-type lamin A did not elicit the outcomes associated with progerin expression. Our results show that DNA damage caused by progerin can occur independently from global changes in replication or cell proliferation.

Progerin, the protein that causes Hutchinson-Gilford progeria syndrome, triggers nuclear membrane (NM) ruptures and blebs, but the mechanisms are unclear. We suspected that the expression of progerin changes the overall structure of the nuclear lamina. High-resolution microscopy of smooth muscle cells (SMCs) revealed that lamin A and lamin B1 form independent meshworks with uniformly spaced openings (~0.085 µm2). The expression of progerin in SMCs resulted in the formation of an irregular meshwork with clusters of large openings (up to 1.4 µm2). The expression of progerin acted in a dominant-negative fashion to disrupt the morphology of the endogenous lamin B1 meshwork, triggering irregularities and large openings that closely resembled the irregularities and openings in the progerin meshwork. These abnormal meshworks were strongly associated with NM ruptures and blebs. Of note, the progerin meshwork was markedly abnormal in nuclear blebs that were deficient in lamin B1 (~50% of all blebs). That observation suggested that higher levels of lamin B1 expression might normalize the progerin meshwork and prevent NM ruptures and blebs. Indeed, increased lamin B1 expression reversed the morphological abnormalities in the progerin meshwork and markedly reduced the frequency of NM ruptures and blebs. Thus, progerin expression disrupts the overall structure of the nuclear lamina, but that effect-along with NM ruptures and blebs-can be abrogated by increased lamin B1 expression.

Heterochromatin is a nuclear area that contains highly condensed and transcriptionally inactive chromatin. Alterations in the organization of heterochromatin are correlated with changes in gene expression and genome stability, which affect various aspects of plant life. Thus, studies of the molecular mechanisms that regulate heterochromatin organization are important for understanding the regulation of plant physiology. Microscopically, heterochromatin can be characterized as chromocenters that are intensely stained with DNA-binding fluorescent dyes. Arabidopsis thaliana exhibits distinctive chromocenters in interphase nuclei, and genetic studies combined with cytological analyses have identified a number of factors that are involved in heterochromatin assembly and organization. In this review, I will summarize the factors involved in the regulation of heterochromatin organization in plants.

DNA replication in differentiated cells follows a defined program, but when and how it is established during mammalian development is not known. Here we show using single-cell sequencing, that late replicating regions are established in association with the B compartment and the nuclear lamina from the first cell cycle after fertilization on both maternal and paternal genomes. Late replicating regions contain a relative paucity of active origins and few but long genes and low G/C content. In both bovine and mouse embryos, replication timing patterns are established prior to embryonic genome activation. Chromosome breaks, which form spontaneously in bovine embryos at sites concordant with human embryos, preferentially locate to late replicating regions. In mice, late replicating regions show enhanced fragility due to a sparsity of dormant origins that can be activated under conditions of replication stress. This pattern predisposes regions with long neuronal genes to fragility and genetic change prior to separation of soma and germ cell lineages. Our studies show that the segregation of early and late replicating regions is among the first layers of genome organization established after fertilization.

Cell fate changes require rewiring of transcriptional programs to generate functionally specialized cell states. Reconfiguration of transcriptional networks requires overcoming epigenetic barriers imposed by silenced heterochromatin in order to activate lineage-specific genes. Further, cell fate decisions are made in a tissue-specific context, where cells are physically linked to each other as well as to the connective tissue environment. Here, cells are continuously exposed to a multitude of mechanical forces emanating from cellular dynamics in their local microenvironments, for example through cell movements, cell divisions, tissue contractions, or fluid flow. Through their ability to deform cellular structures and activate receptors, mechanical forces can be sensed at the plasma membrane, but also at the nuclear periphery through direct or cytoskeleton-mediated deformation of the nuclear envelope. This deformation and the associated signaling is capable of triggering changes in the mechanical state of the nuclear membranes, the organization and rigidity of the underlying nuclear lamina, compaction state of chromatin, and ultimately transcription. This review focuses on the role of nuclear architecture, particularly the nuclear lamina-chromatin interface, and its mechanical regulation in cell fate decisions as well as its physiological role in development and cellular reprogramming.

Epigenetics is the study of heritable changes to the genome and gene expression patterns that are not caused by direct changes to the DNA sequence. Examples of these changes include posttranslational modifications to DNA-bound histone proteins, DNA methylation, and remodeling of nuclear architecture. Collectively, epigenetic changes provide a layer of regulation that affects transcriptional activity of genes while leaving DNA sequences unaltered. Sequence variants or mutations affecting enzymes responsible for modifying or sensing epigenetic marks have been identified in patients with congenital heart disease (CHD), and small-molecule inhibitors of epigenetic complexes have shown promise as therapies for adult heart diseases. Additionally, transgenic mice harboring mutations or deletions of genes encoding epigenetic enzymes recapitulate aspects of human cardiac disease. Taken together, these findings suggest that the evolving field of epigenetics will inform our understanding of congenital and adult cardiac disease and offer new therapeutic opportunities.

Nuclear envelope (NE) ruptures are emerging observations in Lamin-related dilated cardiomyopathy, an adult-onset disease caused by loss-of-function mutations in Lamin A/C, a nuclear lamina component. Here, we test a prevailing hypothesis that NE ruptures trigger the pathological cGAS-STING cytosolic DNA-sensing pathway using a mouse model of Lamin cardiomyopathy. The reduction of Lamin A/C in cardio-myocyte of adult mice causes pervasive NE ruptures in cardiomyocytes, preceding inflammatory transcription, fibrosis, and fatal dilated cardiomyopathy. NE ruptures are followed by DNA damage accumulation without causing immediate cardiomyocyte death. However, cGAS-STING-dependent inflammatory signaling remains inactive. Deleting cGas or Sting does not rescue cardiomyopathy in the mouse model. The lack of cGAS-STING activation is likely due to the near absence of cGAS expression in adult cardiomyocytes at baseline. Instead, extracellular matrix (ECM) signaling is activated and predicted to initiate pro-inflammatory communication from Lamin-reduced cardiomyocytes to fibroblasts. Our work nominates ECM signaling, not cGAS-STING, as a potential inflammatory contributor in Lamin cardiomyopathy.