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.

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)".

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.

Analysis of RNA-Seq datasets reveals enrichment of tissue-specific splice variants for nuclear envelope proteins.

Laminopathies yield tissue-specific pathologies, yet arise from mutation of ubiquitously-expressed genes. A little investigated hypothesis to explain this is that the mutated proteins or their partners have tissue-specific splice variants. To test this, we analyzed RNA-Seq datasets, finding novel isoforms or isoform tissue-specificity for: Lap2, linked to cardiomyopathy; Nesprin 2, linked to Emery-Dreifuss muscular dystrophy and Lmo7, that regulates the Emery-Dreifuss muscular dystrophy linked emerin gene. Interestingly, the muscle-specific Lmo7 exon is rich in serine phosphorylation motifs, suggesting regulatory function. Muscle-specific splice variants in non-nuclear envelope proteins linked to other muscular dystrophies were also found. Nucleoporins tissue-specific variants were found for Nup54, Nup133, Nup153 and Nup358/RanBP2. RT-PCR confirmed novel Lmo7 and RanBP2 variants and specific knockdown of the Lmo7 variantreduced myogenic index. Nuclear envelope proteins were enriched for tissue-specific splice variants compared to the rest of the genome, suggesting that splice variants contribute to its tissue-specific functions.

Microinjection of Antibodies Targeting the Lamin A/C Histone-Binding Site Blocks Mitotic Entry and Reveals Separate Chromatin Interactions with HP1, CenpB and PML.

Lamins form a scaffold lining the nucleus that binds chromatin and contributes to spatial genome organization; however, due to the many other functions of lamins, studies knocking out or altering the lamin polymer cannot clearly distinguish between direct and indirect effects. To overcome this obstacle, we specifically targeted the mapped histone-binding site of A/C lamins by microinjecting antibodies specific to this region predicting that this would make the genome more mobile. No increase in chromatin mobility was observed; however, interestingly, injected cells failed to go through mitosis, while control antibody-injected cells did. This effect was not due to crosslinking of the lamin polymer, as Fab fragments also blocked mitosis. The lack of genome mobility suggested other lamin-chromatin interactions. To determine what these might be, mini-lamin A constructs were expressed with or without the histone-binding site that assembled into independent intranuclear structures. HP1, CenpB and PML proteins accumulated at these structures for both constructs, indicating that other sites supporting chromatin interactions exist on lamin A. Together, these results indicate that lamin A-chromatin interactions are highly redundant and more diverse than generally acknowledged and highlight the importance of trying to experimentally separate their individual functions.

The predicted influence of climate change on lesser prairie-chicken reproductive parameters.

The Southern High Plains is anticipated to experience significant changes in temperature and precipitation due to climate change. These changes may influence the lesser prairie-chicken (Tympanuchus pallidicinctus) in positive or negative ways. We assessed the potential changes in clutch size, incubation start date, and nest survival for lesser prairie-chickens for the years 2050 and 2080 based on modeled predictions of climate change and reproductive data for lesser prairie-chickens from 2001-2011 on the Southern High Plains of Texas and New Mexico. We developed 9 a priori models to assess the relationship between reproductive parameters and biologically relevant weather conditions. We selected weather variable(s) with the most model support and then obtained future predicted values from climatewizard.org. We conducted 1,000 simulations using each reproductive parameter's linear equation obtained from regression calculations, and the future predicted value for each weather variable to predict future reproductive parameter values for lesser prairie-chickens. There was a high degree of model uncertainty for each reproductive value. Winter temperature had the greatest effect size for all three parameters, suggesting a negative relationship between above-average winter temperature and reproductive output. The above-average winter temperatures are correlated to La Niña events, which negatively affect lesser prairie-chickens through resulting drought conditions. By 2050 and 2080, nest survival was predicted to be below levels considered viable for population persistence; however, our assessment did not consider annual survival of adults, chick survival, or the positive benefit of habitat management and conservation, which may ultimately offset the potentially negative effect of drought on nest survival.