Developmental analysis of Spalt function in the Drosophila ring gland.

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, whose cells 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 envelop.

Progerin forms an abnormal meshwork and has a dominant-negative effect on the nuclear lamina.

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.

Lamin B1 curtails early human papillomavirus infection by safeguarding nuclear compartmentalization and autophagic capacity.

Human papillomavirus (HPV) infection is a primary cause of cervical and head-and-neck cancers. The HPV genome enters the nucleus during mitosis when the nuclear envelope disassembles. Given that lamins maintain nuclear integrity during interphase, we asked to what extent their loss would affect early HPV infection. To address this question, we infected human cervical cancer cells and keratinocytes lacking the major lamins with a HPV16 pseudovirus (HP-PsV) encoding an EGFP reporter. We found that a sustained reduction or complete loss of lamin B1 significantly increased HP-PsV infection rate. A corresponding greater nuclear HP-PsV load in LMNB1 knockout cells was directly related to their prolonged mitotic window and extensive nuclear rupture propensity. Despite the increased HP-PsV presence, EGFP transcript levels remained virtually unchanged, indicating an additional defect in protein turnover. Further investigation revealed that LMNB1 knockout led to a substantial decrease in autophagic capacity, possibly linked to the persistent activation of cGAS by cytoplasmic chromatin exposure. Thus, the attrition of lamin B1 increases nuclear perviousness and attenuates autophagic capacity, creating an environment conducive to unrestrained accumulation of HPV capsids. Our identification of lower lamin B1 levels and nuclear BAF foci in the basal epithelial layer of several human cervix samples suggests that this pathway may contribute to an increased individual susceptibility to HPV infection.

CRWN nuclear lamina components maintain the H3K27me3 landscape and promote successful reproduction in Arabidopsis.

Arabidopsis lamin analogs CROWDED NUCLEIs (CRWNs) are necessary to maintain nuclear structure, genome function, and proper plant growth. However, whether and how CRWNs impact reproduction and genome-wide epigenetic modifications is unknown. Here, we investigate the role of CRWNs during the development of gametophytes, seeds, and endosperm, using genomic and epigenomic profiling methods. We observed defects in crwn mutant seeds including seed abortion and reduced germination rate. Quadruple crwn null genotypes were rarely transmitted through gametophytes. Because defects in seeds often stem from abnormal endosperm development, we focused on crwn1 crwn2 (crwn1/2) endosperm. These mutant seeds exhibited enlarged chalazal endosperm cysts and increased expression of stress-related genes and the MADS-box transcription factor PHERES1 and its targets. Previously, it was shown that PHERES1 expression is regulated by H3K27me3 and that CRWN1 interacts with the PRC2 interactor PWO1. Thus, we tested whether crwn1/2 alters H3K27me3 patterns. We observed a mild loss of H3K27me3 at several hundred loci, which differed between endosperm and leaves. These data indicate that CRWNs are necessary to maintain the H3K27me3 landscape, with tissue-specific chromatin and transcriptional consequences.

Interaction with B-type lamin reveals the function of Drosophila Keap1 xenobiotic response factor in nuclear architecture.

BACKGROUND: The Keap1-Nrf2 pathway serves as a central regulator that mediates transcriptional responses to xenobiotic and oxidative stimuli. Recent studies have shown that Keap1 and Nrf2 can regulate transcripts beyond antioxidant and detoxifying genes, yet the underlying mechanisms remain unclear. Our research has uncovered that Drosophila Keap1 (dKeap1) and Nrf2 (CncC) proteins can control high-order chromatin structure, including heterochromatin. METHODS AND RESULTS: In this study, we identified the molecular interaction between dKeap1 and lamin Dm0, the Drosophila B-type lamin responsible for the architecture of nuclear lamina and chromatin. Ectopic expression of dKeap1 led to an ectopic localization of lamin to the intra-nuclear area, corelated with the spreading of the heterochromatin marker H3K9me2 into euchromatin regions. Additionally, mis-regulated dKeap1 disrupted the morphology of the nuclear lamina. Knocking down of dKeap1 partially rescued the lethality induced by lamin overexpression, suggesting their genetic interaction during development. CONCLUSIONS: The discovered dKeap1-lamin interaction suggests a novel role for the Keap1 oxidative/xenobiotic response factor in regulating chromatin architecture.

Regulation of heterochromatin organization in plants.

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.

Epigenetics.

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.

A lineage-specific protein network at the trypanosome nuclear envelope.

The nuclear envelope (NE) separates translation and transcription and is the location of multiple functions, including chromatin organization and nucleocytoplasmic transport. The molecular basis for many of these functions have diverged between eukaryotic lineages. Trypanosoma brucei, a member of the early branching eukaryotic lineage Discoba, highlights many of these, including a distinct lamina and kinetochore composition. Here, we describe a cohort of proteins interacting with both the lamina and NPC, which we term lamina-associated proteins (LAPs). LAPs represent a diverse group of proteins, including two candidate NPC-anchoring pore membrane proteins (POMs) with architecture conserved with S. cerevisiae and H. sapiens, and additional peripheral components of the NPC. While many of the LAPs are Kinetoplastid specific, we also identified broadly conserved proteins, indicating an amalgam of divergence and conservation within the trypanosome NE proteome, highlighting the diversity of nuclear biology across the eukaryotes, increasing our understanding of eukaryotic and NPC evolution.

The nexus of nuclear envelope dynamics, circular economy and cancer cell pathophysiology.

The nuclear envelope (NE) is a critical component in maintaining the function and structure of the eukaryotic nucleus. The NE and lamina are disassembled during each cell cycle to enable an open mitosis. Nuclear architecture construction and deconstruction is a prime example of a circular economy, as it fulfills a highly efficient recycling program bound to continuous assessment of the quality and functionality of the building blocks. Alterations in the nuclear dynamics and lamina structure have emerged as important contributors to both oncogenic transformation and cancer progression. However, the knowledge of the NE breakdown and reassembly is still limited to a fraction of participating proteins and complexes. As cancer cells contain highly diverse nuclei in terms of DNA content, but also in terms of nuclear number, size, and shape, it is of great interest to understand the intricate relationship between these nuclear features in cancer cell pathophysiology. In this review, we provide insights into how those NE dynamics are regulated, and how lamina destabilization processes may alter the NE circular economy. Moreover, we expand the knowledge of the lamina-associated domain region by using strategic algorithms, including Artificial Intelligence, to infer protein associations, assess their function and location, and predict cancer-type specificity with implications for the future of cancer diagnosis, prognosis and treatment. Using this approach we identified NUP98 and MECP2 as potential proteins that exhibit upregulation in Acute Myeloid Leukemia (LAML) patients with implications for early diagnosis.

Progerin Can Induce DNA Damage in the Absence of Global Changes in Replication or Cell Proliferation.

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.

Lamin A/C and PI(4,5)P2-A Novel Complex in the Cell Nucleus.

Lamins, the nuclear intermediate filaments, are important regulators of nuclear structural integrity as well as nuclear functional processes such as DNA transcription, replication and repair, and epigenetic regulations. A portion of phosphorylated lamin A/C localizes to the nuclear interior in interphase, forming a lamin A/C pool with specific properties and distinct functions. Nucleoplasmic lamin A/C molecular functions are mainly dependent on its binding partners; therefore, revealing new interactions could give us new clues on the lamin A/C mechanism of action. In the present study, we show that lamin A/C interacts with nuclear phosphoinositides (PIPs), and with nuclear myosin I (NM1). Both NM1 and nuclear PIPs have been previously reported as important regulators of gene expression and DNA damage/repair. Furthermore, phosphorylated lamin A/C forms a complex with NM1 in a phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2)-dependent manner in the nuclear interior. Taken together, our study reveals a previously unidentified interaction between phosphorylated lamin A/C, NM1, and PI(4,5)P2 and suggests new possible ways of nucleoplasmic lamin A/C regulation, function, and importance for the formation of functional nuclear microdomains.

Pervasive nuclear envelope ruptures precede ECM signaling and disease onset without activating cGAS-STING in Lamin-cardiomyopathy mice.

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.

Depletion of lamins B1 and B2 promotes chromatin mobility and induces differential gene expression by a mesoscale-motion-dependent mechanism.

BACKGROUND: B-type lamins are critical nuclear envelope proteins that interact with the three-dimensional genomic architecture. However, identifying the direct roles of B-lamins on dynamic genome organization has been challenging as their joint depletion severely impacts cell viability. To overcome this, we engineered mammalian cells to rapidly and completely degrade endogenous B-type lamins using Auxin-inducible degron technology. RESULTS: Using live-cell Dual Partial Wave Spectroscopic (Dual-PWS) microscopy, Stochastic Optical Reconstruction Microscopy (STORM), in situ Hi-C, CRISPR-Sirius, and fluorescence in situ hybridization (FISH), we demonstrate that lamin B1 and lamin B2 are critical structural components of the nuclear periphery that create a repressive compartment for peripheral-associated genes. Lamin B1 and lamin B2 depletion minimally alters higher-order chromatin folding but disrupts cell morphology, significantly increases chromatin mobility, redistributes both constitutive and facultative heterochromatin, and induces differential gene expression both within and near lamin-associated domain (LAD) boundaries. Critically, we demonstrate that chromatin territories expand as upregulated genes within LADs radially shift inwards. Our results indicate that the mechanism of action of B-type lamins comes from their role in constraining chromatin motion and spatial positioning of gene-specific loci, heterochromatin, and chromatin domains. CONCLUSIONS: Our findings suggest that, while B-type lamin degradation does not significantly change genome topology, it has major implications for three-dimensional chromatin conformation at the single-cell level both at the lamina-associated periphery and the non-LAD-associated nuclear interior with concomitant genome-wide transcriptional changes. This raises intriguing questions about the individual and overlapping roles of lamin B1 and lamin B2 in cellular function and disease.

LMNA R644C associates with hepatic steatosis in a large cohort and increases cellular lipid droplet accumulation in vitro.

The R644C variant of lamin A is controversial, as it has been linked to multiple phenotypes in familial studies, but has also been identified in apparently healthy volunteers. Here we present data from a large midwestern US cohort showing that this variant associates genetically with hepatic steatosis, and with related traits in additional publicly available datasets, while in vitro testing demonstrated that this variant increased cellular lipid droplet accumulation. Taken together, these data support this LMNA variant's potential pathogenicity in lipodystrophy and metabolic liver disease.

Development and characterization of polyclonal antibodies against the linker region of the telomere-binding protein TRF2

Background: TRF2 (telomeric repeat binding factor 2) is an essential component of the telomere-binding protein complex shelterin. TRF2 induces the formation of a special structure of telomeric DNA and counteracts activation of DNA damage-response pathways telomeres. TRF2 has a poorly characterized linker region (udTRF2) between its homodimerization and DNA-binding domains. Some lines of evidence have shown that this region could be involved in TRF2 interaction with nuclear lamina. Results: In this study, the fragment of the TERF2 gene encoding udTRF2 domain of telomere-binding protein TRF2 was produced by PCR and cloned into the pET32a vector. The resulting plasmid pET32a-udTRF2 was used for the expression of the recombinant udTRF2 in E. coli RosettaBlue (DE3). The protein was isolated and purified using ammonium sulfate precipitation followed by ion-exchange chromatography. The purified recombinant protein udTRF2 was injected into guinea pigs to generate polyclonal antibodies. The ability of anti-udTRF2 antibodies to bind endogenous TRF2 in human skin fibroblasts was tested by western blotting and immunofluorescent staining. Conclusions: In this study, the recombinant protein udTRF2 and antibodies to it were generated. Both protein and antibodies will provide a useful tool for investigation of the functions of the udTRF2 domain and its role in the interaction between TRF2 and nuclear lamina.