Enhancers on the edge - how the nuclear envelope controls gene regulatory elements.

Precise temporal and sequential control of gene expression during development and in response to environmental stimuli requires tight regulation of the physical contact between gene regulatory elements and promoters. Current models describing how the genome folds in 3D space to establish these interactions often ignore the role of the most stable structural nuclear feature - the nuclear envelope. While contributions of 3D folding within/between topologically associated domains (TADs) have been extensively described, mechanical contributions from the nuclear envelope can impact enhancer-promoter interactions both directly and indirectly through influencing intra/inter-TAD interactions. Importantly, these nuclear envelope contributions clearly link this mechanism to development and, when defective, to human disease. Here, we discuss evidence for nuclear envelope regulation of tissue-specific enhancer-promoter pairings, potential mechanisms for this regulation, exciting recent findings that other regulatory elements such as microRNAs and long noncoding RNAs are under nuclear envelope regulation, the possible involvement of condensates, and how disruption of this regulation can lead to disease.

Metabolic, fibrotic and splicing pathways are all altered in Emery-Dreifuss muscular dystrophy spectrum patients to differing degrees.

Emery-Dreifuss muscular dystrophy (EDMD) is a genetically and clinically variable disorder. Previous attempts to use gene expression changes to find its pathomechanism were unavailing, so we engaged a functional pathway analysis. RNA-Seq was performed on cells from 10 patients diagnosed with an EDMD spectrum disease with different mutations in seven genes. Upon comparing to controls, the pathway analysis revealed that multiple genes involved in fibrosis, metabolism, myogenic signaling and splicing were affected in all patients. Splice variant analysis revealed alterations of muscle-specific variants for several important muscle genes. Deeper analysis of metabolic pathways revealed a reduction in glycolytic and oxidative metabolism and reduced numbers of mitochondria across a larger set of 14 EDMD spectrum patients and 7 controls. Intriguingly, the gene expression signatures segregated the patients into three subgroups whose distinctions could potentially relate to differences in clinical presentation. Finally, differential expression analysis of miRNAs changing in the patients similarly highlighted fibrosis, metabolism and myogenic signaling pathways. This pathway approach revealed a transcriptome profile that can both be used as a template for establishing a biomarker panel for EDMD and direct further investigation into its pathomechanism. Furthermore, the segregation of specific gene changes into distinct groups that appear to correlate with clinical presentation may template development of prognostic biomarkers, though this will first require their testing in a wider set of patients with more clinical information.

Reduction in Nuclear Size by DHRS7 in Prostate Cancer Cells and by Estradiol Propionate in DHRS7-Depleted Cells.

Increased nuclear size correlates with lower survival rates and higher grades for prostate cancer. The short-chain dehydrogenase/reductase (SDR) family member DHRS7 was suggested as a biomarker for use in prostate cancer grading because it is largely lost in higher-grade tumors. Here, we found that reduction in DHRS7 from the LNCaP prostate cancer cell line with normally high levels of DHRS7 increases nuclear size, potentially explaining the nuclear size increase observed in higher-grade prostate tumors where it is lost. An exogenous expression of DHRS7 in the PC3 prostate cancer cell line with normally low DHRS7 levels correspondingly decreases nuclear size. We separately tested 80 compounds from the Microsource Spectrum library for their ability to restore normal smaller nuclear size to PC3 cells, finding that estradiol propionate had the same effect as the re-expression of DHRS7 in PC3 cells. However, the drug had no effect on LNCaP cells or PC3 cells re-expressing DHRS7. We speculate that separately reported beneficial effects of estrogens in androgen-independent prostate cancer may only occur with the loss of DHRS7/ increased nuclear size, and thus propose DHRS7 levels and nuclear size as potential biomarkers for the likely effectiveness of estrogen-based treatments.

Nuclear size rectification: A potential new therapeutic approach to reduce metastasis in cancer.

Research on metastasis has recently regained considerable interest with the hope that single cell technologies might reveal the most critical changes that support tumor spread. However, it is possible that part of the answer has been visible through the microscope for close to 200 years. Changes in nuclear size characteristically occur in many cancer types when the cells metastasize. This was initially discarded as contributing to the metastatic spread because, depending on tumor types, both increases and decreases in nuclear size could correlate with increased metastasis. However, recent work on nuclear mechanics and the connectivity between chromatin, the nucleoskeleton, and the cytoskeleton indicate that changes in this connectivity can have profound impacts on cell mobility and invasiveness. Critically, a recent study found that reversing tumor type-dependent nuclear size changes correlated with reduced cell migration and invasion. Accordingly, it seems appropriate to now revisit possible contributory roles of nuclear size changes to metastasis.

Chemical Interrogation of Nuclear Size Identifies Compounds with Cancer Cell Line-Specific Effects on Migration and Invasion.

Background: Lower survival rates for many cancer types correlate with changes in nuclear size/scaling in a tumor-type/tissue-specific manner. Hypothesizing that such changes might confer an advantage to tumor cells, we aimed at the identification of commercially available compounds to guide further mechanistic studies. We therefore screened for Food and Drug Administration (FDA)/European Medicines Agency (EMA)-approved compounds that reverse the direction of characteristic tumor nuclear size changes in PC3, HCT116, and H1299 cell lines reflecting, respectively, prostate adenocarcinoma, colonic adenocarcinoma, and small-cell squamous lung cancer. Results: We found distinct, largely nonoverlapping sets of compounds that rectify nuclear size changes for each tumor cell line. Several classes of compounds including, e.g., serotonin uptake inhibitors, cyclo-oxygenase inhibitors, ß-adrenergic receptor agonists, and Na+/K+ ATPase inhibitors, displayed coherent nuclear size phenotypes focused on a particular cell line or across cell lines and treatment conditions. Several compounds from classes far afield from current chemotherapy regimens were also identified. Seven nuclear size-rectifying compounds selected for further investigation all inhibited cell migration and/or invasion. Conclusions: Our study provides (a) proof of concept that nuclear size might be a valuable target to reduce cell migration/invasion in cancer treatment and (b) the most thorough collection of tool compounds to date reversing nuclear size changes specific to individual cancer-type cell lines. Although these compounds still need to be tested in primary cancer cells, the cell line-specific nuclear size and migration/invasion responses to particular drug classes suggest that cancer type-specific nuclear size rectifiers may help reduce metastatic spread.

Genomic loci mispositioning in Tmem120a knockout mice yields latent lipodystrophy.

Little is known about how the observed fat-specific pattern of 3D-spatial genome organisation is established. Here we report that adipocyte-specific knockout of the gene encoding nuclear envelope transmembrane protein Tmem120a disrupts fat genome organisation, thus causing a lipodystrophy syndrome. Tmem120a deficiency broadly suppresses lipid metabolism pathway gene expression and induces myogenic gene expression by repositioning genes, enhancers and miRNA-encoding loci between the nuclear periphery and interior. Tmem120a-/- mice, particularly females, exhibit a lipodystrophy syndrome similar to human familial partial lipodystrophy FPLD2, with profound insulin resistance and metabolic defects that manifest upon exposure to an obesogenic diet. Interestingly, similar genome organisation defects occurred in cells from FPLD2 patients that harbour nuclear envelope protein encoding LMNA mutations. Our data indicate TMEM120A genome organisation functions affect many adipose functions and its loss may yield adiposity spectrum disorders, including a miRNA-based mechanism that could explain muscle hypertrophy in human lipodystrophy.

STING nuclear partners contribute to innate immune signaling responses.

STimulator of INterferon Genes (STING) is an adaptor for cytoplasmic DNA sensing by cGAMP/cGAS that helps trigger innate immune responses (IIRs). Although STING is mostly localized in the ER, we find a separate inner nuclear membrane pool of STING that increases mobility and redistributes to the outer nuclear membrane upon IIR stimulation by transfected dsDNA or dsRNA mimic poly(I:C). Immunoprecipitation of STING from isolated nuclear envelopes coupled with mass spectrometry revealed a distinct nuclear envelope-STING proteome consisting of known nuclear membrane proteins and enriched in DNA- and RNA-binding proteins. Seventeen of these nuclear envelope STING partners are known to bind direct interactors of IRF3/7 transcription factors, and testing a subset of these revealed STING partners SYNCRIP, MEN1, DDX5, snRNP70, RPS27a, and AATF as novel modulators of dsDNA-triggered IIRs. Moreover, we find that SYNCRIP is a novel antagonist of the RNA virus, influenza A, potentially shedding light on reports of STING inhibition of RNA viruses.

Tm7sf2 Disruption Alters Radial Gene Positioning in Mouse Liver Leading to Metabolic Defects and Diabetes Characteristics.

Tissue-specific patterns of radial genome organization contribute to genome regulation and can be established by nuclear envelope proteins. Studies in this area often use cancer cell lines, and it is unclear how well such systems recapitulate genome organization of primary cells or animal tissues; so, we sought to investigate radial genome organization in primary liver tissue hepatocytes. Here, we have used a NET47/Tm7sf2-/- liver model to show that manipulating one of these nuclear membrane proteins is sufficient to alter tissue-specific gene positioning and expression. Dam-LaminB1 global profiling in primary liver cells shows that nearly all the genes under such positional regulation are related to/important for liver function. Interestingly, Tm7sf2 is a paralog of the HP1-binding nuclear membrane protein LBR that, like Tm7sf2, also has an enzymatic function in sterol reduction. Fmo3 gene/locus radial mislocalization could be rescued with human wild-type, but not TM7SF2 mutants lacking the sterol reductase function. One central pathway affected is the cholesterol synthesis pathway. Within this pathway, both Cyp51 and Msmo1 are under Tm7sf2 positional and expression regulation. Other consequences of the loss of Tm7sf2 included weight gain, insulin sensitivity, and reduced levels of active Akt kinase indicating additional pathways under its regulation, several of which are highlighted by mispositioning genes. This study emphasizes the importance for tissue-specific radial genome organization in tissue function and the value of studying genome organization in animal tissues and primary cells over cell lines.

The NEMP family supports metazoan fertility and nuclear envelope stiffness.

Human genome-wide association studies have linked single-nucleotide polymorphisms (SNPs) in NEMP1 (nuclear envelope membrane protein 1) with early menopause; however, it is unclear whether NEMP1 has any role in fertility. We show that whole-animal loss of NEMP1 homologs in Drosophila, Caenorhabditis elegans, zebrafish, and mice leads to sterility or early loss of fertility. Loss of Nemp leads to nuclear shaping defects, most prominently in the germ line. Biochemical, biophysical, and genetic studies reveal that NEMP proteins support the mechanical stiffness of the germline nuclear envelope via formation of a NEMP-EMERIN complex. These data indicate that the germline nuclear envelope has specialized mechanical properties and that NEMP proteins play essential and conserved roles in fertility.

Nucleoplasmic signals promote directed transmembrane protein import simultaneously via multiple channels of nuclear pores.

Roughly 10% of eukaryotic transmembrane proteins are found on the nuclear membrane, yet how such proteins target and translocate to the nucleus remains in dispute. Most models propose transport through the nuclear pore complexes, but a central outstanding question is whether transit occurs through their central or peripheral channels. Using live-cell high-speed super-resolution single-molecule microscopy we could distinguish protein translocation through the central and peripheral channels, finding that most inner nuclear membrane proteins use only the peripheral channels, but some apparently extend intrinsically disordered domains containing nuclear localization signals into the central channel for directed nuclear transport. These nucleoplasmic signals are critical for central channel transport as their mutation blocks use of the central channels; however, the mutated proteins can still complete their translocation using only the peripheral channels, albeit at a reduced rate. Such proteins can still translocate using only the peripheral channels when central channel is blocked, but blocking the peripheral channels blocks translocation through both channels. This suggests that peripheral channel transport is the default mechanism that was adapted in evolution to include aspects of receptor-mediated central channel transport for directed trafficking of certain membrane proteins.

A multistage sequencing strategy pinpoints novel candidate alleles for Emery-Dreifuss muscular dystrophy and supports gene misregulation as its pathomechanism.

BACKGROUND: As genome-wide approaches prove difficult with genetically heterogeneous orphan diseases, we developed a new approach to identify candidate genes. We applied this to Emery-Dreifuss muscular dystrophy (EDMD), characterised by early onset contractures, slowly progressive muscular wasting, and life-threatening heart conduction disturbances with wide intra- and inter-familial clinical variability. Roughly half of EDMD patients are linked to six genes encoding nuclear envelope proteins, but the disease mechanism remains unclear because the affected proteins function in both cell mechanics and genome regulation. METHODS: A primer library was generated to test for mutations in 301 genes from four categories: (I) all known EDMD-linked genes; (II) genes mutated in related muscular dystrophies; (III) candidates generated by exome sequencing in five families; (IV) functional candidates - other muscle nuclear envelope proteins functioning in mechanical/genome processes affected in EDMD. This was used to sequence 56 unlinked patients with EDMD-like phenotype. FINDINGS: Twenty-one patients could be clearly assigned: 18 with mutations in genes of similar muscular dystrophies; 3 with previously missed mutations in EDMD-linked genes. The other categories yielded novel candidate genes, most encoding nuclear envelope proteins with functions in gene regulation. INTERPRETATION: Our multi-pronged approach identified new disease alleles and many new candidate EDMD genes. Their known functions strongly argue the EDMD pathomechanism is from altered gene regulation and mechanotransduction due to connectivity of candidates from the nuclear envelope to the plasma membrane. This approach highlights the value of testing for related diseases using primer libraries and may be applied for other genetically heterogeneous orphan diseases. FUNDING: The Wellcome Trust, Muscular Dystrophy UK, Medical Research Council, European Community's Seventh Framework Programme "Integrated European -omics research project for diagnosis and therapy in rare neuromuscular and neurodegenerative diseases (NEUROMICS)".

Casting a Wider Net: Differentiating between Inner Nuclear Envelope and Outer Nuclear Envelope Transmembrane Proteins.

The nuclear envelope (NE) surrounds the nucleus with a double membrane in eukaryotic cells. The double membranes are embedded with proteins that are synthesized on the endoplasmic reticulum and often destined specifically for either the outer nuclear membrane (ONM) or the inner nuclear membrane (INM). These nuclear envelope transmembrane proteins (NETs) play important roles in cellular function and participate in transcription, epigenetics, splicing, DNA replication, genome architecture, nuclear structure, nuclear stability, nuclear organization, and nuclear positioning. These vital functions are dependent upon both the correct localization and relative concentrations of NETs on the appropriate membrane of the NE. It is, therefore, important to understand the distribution and abundance of NETs on the NE. This review will evaluate the current tools and methodologies available to address this important topic.

Lamin A molecular compression and sliding as mechanisms behind nucleoskeleton elasticity.

Lamin A is a nuclear intermediate filament protein critical for nuclear architecture and mechanics and mutated in a wide range of human diseases. Yet little is known about the molecular architecture of lamins and mechanisms of their assembly. Here we use SILAC cross-linking mass spectrometry to determine interactions within lamin dimers and between dimers in higher-order polymers. We find evidence for a compression mechanism where coiled coils in the lamin A rod can slide onto each other to contract rod length, likely driven by a wide range of electrostatic interactions with the flexible linkers between coiled coils. Similar interactions occur with unstructured regions flanking the rod domain during oligomeric assembly. Mutations linked to human disease block these interactions, suggesting that this spring-like contraction can explain in part the dynamic mechanical stretch and flexibility properties of the lamin polymer and other intermediate filament networks.

The cell-wide web coordinates cellular processes by directing site-specific Ca2+ flux across cytoplasmic nanocourses.

Ca2+ coordinates diverse cellular processes, yet how function-specific signals arise is enigmatic. We describe a cell-wide network of distinct cytoplasmic nanocourses with the nucleus at its centre, demarcated by sarcoplasmic reticulum (SR) junctions (≤400 nm across) that restrict Ca2+ diffusion and by nanocourse-specific Ca2+-pumps that facilitate signal segregation. Ryanodine receptor subtype 1 (RyR1) supports relaxation of arterial myocytes by unloading Ca2+ into peripheral nanocourses delimited by plasmalemma-SR junctions, fed by sarco/endoplasmic reticulum Ca2+ ATPase 2b (SERCA2b). Conversely, stimulus-specified increases in Ca2+ flux through RyR2/3 clusters selects for rapid propagation of Ca2+ signals throughout deeper extraperinuclear nanocourses and thus myocyte contraction. Nuclear envelope invaginations incorporating SERCA1 in their outer nuclear membranes demarcate further diverse networks of cytoplasmic nanocourses that receive Ca2+ signals through discrete RyR1 clusters, impacting gene expression through epigenetic marks segregated by their associated invaginations. Critically, this circuit is not hardwired and remodels for different outputs during cell proliferation.

Spatial Genome Organization: From Development to Disease.

Every living organism, from bacteria to humans, contains DNA encoding anything from a few hundred genes in intracellular parasites such as Mycoplasma, up to several tens of thousands in many higher organisms. The first observations indicating that the nucleus had some kind of organization were made over a hundred years ago. Understanding of its significance is both limited and aided by the development of techniques, in particular electron microscopy, fluorescence in situ hybridization, DamID and most recently HiC. As our knowledge about genome organization grows, it becomes apparent that the mechanisms are conserved in evolution, even if the individual players may vary. These mechanisms involve DNA binding proteins such as histones, and a number of architectural proteins, some of which are very much conserved, with some others having diversified and multiplied, acquiring specific regulatory functions. In this review we will look at the principles of genome organization in a hierarchical manner, from DNA packaging to higher order genome associations such as TADs, and the significance of radial positioning of genomic loci. We will then elaborate on the dynamics of genome organization during development, and how genome architecture plays an important role in cell fate determination. Finally, we will discuss how misregulation can be a factor in human disease.

Host Vesicle Fusion Protein VAPB Contributes to the Nuclear Egress Stage of Herpes Simplex Virus Type-1 (HSV-1) Replication.

The primary envelopment/de-envelopment of Herpes viruses during nuclear exit is poorly understood. In Herpes simplex virus type-1 (HSV-1), proteins pUL31 and pUL34 are critical, while pUS3 and some others contribute; however, efficient membrane fusion may require additional host proteins. We postulated that vesicle fusion proteins present in the nuclear envelope might facilitate primary envelopment and/or de-envelopment fusion with the outer nuclear membrane. Indeed, a subpopulation of vesicle-associated membrane protein-associated protein B (VAPB), a known vesicle trafficking protein, was present in the nuclear membrane co-locating with pUL34. VAPB knockdown significantly reduced both cell-associated and supernatant virus titers. Moreover, VAPB depletion reduced cytoplasmic accumulation of virus particles and increased levels of nuclear encapsidated viral DNA. These results suggest that VAPB is an important player in the exit of primary enveloped HSV-1 virions from the nucleus. Importantly, VAPB knockdown did not alter pUL34, calnexin or GM-130 localization during infection, arguing against an indirect effect of VAPB on cellular vesicles and trafficking. Immunogold-labelling electron microscopy confirmed VAPB presence in nuclear membranes and moreover associated with primary enveloped HSV-1 particles. These data suggest that VAPB could be a cellular component of a complex that facilitates UL31/UL34/US3-mediated HSV-1 nuclear egress.

Telomere elongation through hTERT immortalization leads to chromosome repositioning in control cells and genomic instability in Hutchinson-Gilford progeria syndrome fibroblasts, expressing a novel SUN1 isoform.

Immortalizing primary cells with human telomerase reverse transcriptase (hTERT) has been common practice to enable primary cells to be of extended use in the laboratory because they avoid replicative senescence. Studying exogenously expressed hTERT in cells also affords scientists models of early carcinogenesis and telomere behavior. Control and the premature ageing disease-Hutchinson-Gilford progeria syndrome (HGPS) primary dermal fibroblasts, with and without the classical G608G mutation have been immortalized with exogenous hTERT. However, hTERT immortalization surprisingly elicits genome reorganization not only in disease cells but also in the normal control cells, such that whole chromosome territories normally located at the nuclear periphery in proliferating fibroblasts become mislocalized in the nuclear interior. This includes chromosome 18 in the control fibroblasts and both chromosomes 18 and X in HGPS cells, which physically express an isoform of the LINC complex protein SUN1 that has previously only been theoretical. Additionally, this HGPS cell line has also become genomically unstable and has a tetraploid karyotype, which could be due to the novel SUN1 isoform. Long-term treatment with the hTERT inhibitor BIBR1532 enabled the reduction of telomere length in the immortalized cells and resulted that these mislocalized internal chromosomes to be located at the nuclear periphery, as assessed in actively proliferating cells. Taken together, these findings reveal that elongated telomeres lead to dramatic chromosome mislocalization, which can be restored with a drug treatment that results in telomere reshortening and that a novel SUN1 isoform combined with elongated telomeres leads to genomic instability. Thus, care should be taken when interpreting data from genomic studies in hTERT-immortalized cell lines.