Sedentary Behavior Impacts on the Epigenome and Transcriptome: Lessons from Muscle Inactivation in Drosophila Larvae.

The biological mechanisms linking sedentary lifestyles and metabolic derangements are incompletely understood. In this study, temporal muscle inactivation in Drosophila larvae carrying a temperature-sensitive mutation in the shibire (shi1) gene was induced to mimic sedentary behavior during early life and study its transcriptional outcome. Our findings indicated a significant change in the epigenetic profile, as well as the genomic profile, of RNA Pol II binding in the inactive muscles relative to control, within a relatively short time period. Whole-genome analysis of RNA-Pol II binding to DNA by muscle-specific targeted DamID (TaDa) protocol revealed that muscle inactivity altered Pol II binding in 121 out of 2010 genes (6%), with a three-fold enrichment of genes coding for lncRNAs. The suppressed protein-coding genes included genes associated with longevity, DNA repair, muscle function, and ubiquitin-dependent proteostasis. Moreover, inducing muscle inactivation exerted a multi-level impact upon chromatin modifications, triggering an altered epigenetic balance of active versus inactive marks. The downregulated genes in the inactive muscles included genes essential for muscle structure and function, carbohydrate metabolism, longevity, and others. Given the multiple analogous genes in Drosophila for many human genes, extrapolating our findings to humans may hold promise for establishing a molecular link between sedentary behavior and metabolic diseases.

Regulation of chromatin microphase separation by binding of protein complexes.

We show evidence of the association of RNA polymerase II (RNAP) with chromatin in a core-shell organization, reminiscent of microphase separation where the cores comprise dense chromatin and the shell, RNAP and chromatin with low density. These observations motivate our physical model for the regulation of core-shell chromatin organization. Here, we model chromatin as a multiblock copolymer, comprising active and inactive regions (blocks) that are both in poor solvent and tend to be condensed in the absence of binding proteins. However, we show that the solvent quality for the active regions of chromatin can be regulated by the binding of protein complexes (e.g., RNAP and transcription factors). Using the theory of polymer brushes, we find that such binding leads to swelling of the active chromatin regions which in turn modifies the spatial organization of the inactive regions. In addition, we use simulations to study spherical chromatin micelles, whose cores comprise inactive regions and shells comprise active regions and bound protein complexes. In spherical micelles the swelling increases the number of inactive cores and controls their size. Thus, genetic modifications affecting the binding strength of chromatin-binding protein complexes may modulate the solvent quality experienced by chromatin and regulate the physical organization of the genome.

The LINC Complex Inhibits Excessive Chromatin Repression.

The Linker of Nucleoskeleton and Cytoskeleton (LINC) complex transduces nuclear mechanical inputs suggested to control chromatin organization and gene expression; however, the underlying mechanism is currently unclear. We show here that the LINC complex is needed to minimize chromatin repression in muscle tissue, where the nuclei are exposed to significant mechanical inputs during muscle contraction. To this end, the genomic binding profiles of Polycomb, Heterochromatin Protein1 (HP1a) repressors, and of RNA-Pol II were studied in Drosophila larval muscles lacking functional LINC complex. A significant increase in the binding of Polycomb and parallel reduction of RNA-Pol-II binding to a set of muscle genes was observed. Consistently, enhanced tri-methylated H3K9 and H3K27 repressive modifications and reduced chromatin activation by H3K9 acetylation were found. Furthermore, larger tri-methylated H3K27me3 repressive clusters, and chromatin redistribution from the nuclear periphery towards nuclear center, were detected in live LINC mutant larval muscles. Computer simulation indicated that the observed dissociation of the chromatin from the nuclear envelope promotes growth of tri-methylated H3K27 repressive clusters. Thus, we suggest that by promoting chromatin-nuclear envelope binding, the LINC complex restricts the size of repressive H3K27 tri-methylated clusters, thereby limiting the binding of Polycomb transcription repressor, directing robust transcription in muscle fibers.

Common genetic basis of ALS patients and soccer players may contribute to disease risk.

OBJECTIVE: The aim of the present study was to determine the prevalence of the ACSL A/G single nucleotide polymorphism among athletes and patients with amyotrophic lateral sclerosis (ALS). ALS is a progressive neurodegenerative disorder of motor neurons that leads to paralysis and death usually within 3-5 years from onset. Previous epidemiological studies reported a higher risk of ALS among soccer players. The ACSL (long-chain-fatty-acid-CoA ligase 1) gene codes the long-chain fatty-acid-coenzyme A ligase family that plays a key role in lipid biosynthesis and fatty acid oxidation. The ACSL A/G polymorphism is associated with endurance trainability. METHODS: One hundred and seventy-eight ALS patients, 172 athletes (60 soccer players, 112 middle- and long-distance runners), and 111 nonathletic controls participated in the study. Genomic DNA was extracted from blood or buccal cells according to the salting-out procedure. Genotypes were determined using the TaqMan allelic discrimination assay. RESULTS: The prevalence of the ACSL AA genotype was significantly higher among soccer players (35.0%) and ALS patients (39.3%) compared to runners (16.1%) and controls (18.0%). However, ALS GG carriers had a higher mortality rate. CONCLUSION: We postulate that soccer players and ALS patients carry a common genetic predisposition that is related to impaired fatty acid utilization. Moreover, while the A allele might be associated with a genetic predisposition toward ALS, especially among soccer players, the G allele might be associated with disease severity. Further research is needed in order to explore the role of the ACSL rs6552828 polymorphism in ALS.

Evaluation of chromatin mesoscale organization.

Chromatin organization in the nucleus represents an important aspect of transcription regulation. Most of the studies so far focused on the chromatin structure in cultured cells or in fixed tissue preparations. Here, we discuss the various approaches for deciphering chromatin 3D organization with an emphasis on the advantages of live imaging approaches.

A Comparison Between Pressure Wire and Microcatheter Measurements for Evaluating the Cerebral Venous Pressure Gradient.

Introduction: A pressure gradient of over 8 mm Hg across the stenosis (usually located in the transverse-sigmoid junction) is one of the criteria for cerebral venous stenting in idiopathic intracranial hypertension (IIH) patients. The possible inaccuracy of the traditional microcatheter-based pressure measurements has been discussed in previous studies. In the cardiology field, a dual-sensor pressure wire is routinely used for the evaluation of stenotic lesions. Using a pressure wire for cerebral vasculature was previously discussed in a small case series and case reports. In this study, we compared venous pressure measurements obtained using both a microcatheter and a pressure wire in patients who were candidates for stenting. Methods: A retrospective study was conducted, comparing the two methods of pressure measurements in 26 patients with venous stenosis. Altogether, 120 measurements were performed using both methods. Demographic characteristics, medical history, procedural details, medications, indications for the procedure, and complications were collected from the patient charts. Results: Based on an 8-mm Hg pressure gradient cutoff indication, 19 patients were found eligible to go through unilateral venous stenting based on catheter measurements alone. The wire results corroborated the catheter results in detecting all cases indicated for a stent. This finding implies a sensitivity equal to 100% for the wire measurements. There were no wire-related complications, demonstrating its safety. Conclusions: We conclude that the pressure wire is as safe as the microcatheter and can identify cases requiring intervention. A larger-scale study is needed to assess the measurement accuracy of the pressure wire in brain vasculature.

Live imaging of chromatin distribution reveals novel principles of nuclear architecture and chromatin compartmentalization.

The three-dimensional organization of chromatin contributes to transcriptional control, but information about native chromatin distribution is limited. Imaging chromatin in live Drosophila larvae, with preserved nuclear volume, revealed that active and repressed chromatin separates from the nuclear interior and forms a peripheral layer underneath the nuclear lamina. This is in contrast to the current view that chromatin distributes throughout the nucleus. Furthermore, peripheral chromatin organization was observed in distinct Drosophila tissues, as well as in live human effector T lymphocytes and neutrophils. Lamin A/C up-regulation resulted in chromatin collapse toward the nuclear center and correlated with a significant reduction in the levels of active chromatin. Physical modeling suggests that binding of lamina-associated domains combined with chromatin self-attractive interactions recapitulate the experimental chromatin distribution profiles. Together, our findings reveal a novel mode of mesoscale organization of peripheral chromatin sensitive to lamina composition, which is evolutionary conserved.

Mesoscale phase separation of chromatin in the nucleus.

Intact-organism imaging of Drosophila larvae reveals and quantifies chromatin-aqueous phase separation. The chromatin can be organized near the lamina layer of the nuclear envelope, conventionally fill the nucleus, be organized centrally, or as a wetting droplet. These transitions are controlled by changes in nuclear volume and the interaction of chromatin with the lamina (part of the nuclear envelope) at the nuclear periphery. Using a simple polymeric model that includes the key features of chromatin self-attraction and its binding to the lamina, we demonstrate theoretically that it is the competition of these two effects that determines the mode of chromatin distribution. The qualitative trends as well as the composition profiles obtained in our simulations compare well with the observed intact-organism imaging and quantification. Since the simulations contain only a small number of physical variables we can identify the generic mechanisms underlying the changes in the observed phase separations.

A minimal constraint device for imaging nuclei in live Drosophila contractile larval muscles reveals novel nuclear mechanical dynamics.

Muscle contractions produce reiterated cytoplasmic mechanical variations, which potentially influence nuclear mechanotransduction, however information regarding the dynamics of muscle nuclei (myonuclei) in the course of muscle contraction is still missing. Towards that end, a minimal constraint device was designed in which intact live Drosophila larva is imaged, while its muscles still contract. The device is placed under spinning disc confocal microscope enabling imaging of fluorescently labeled sarcomeres and nuclei during muscle contraction, without any external stimulation. As a proof of principle we studied myonuclei dynamics in wild-type, as well as in Nesprin/klar mutant larvae lacking proper nuclear-cytoskeletal connections. Myonuclei in control larvae exhibited comparable dynamics in the course of multiple contractile events, independent of their position along the muscle fiber. In contrast, myonuclei of mutant larvae displayed differential dynamics at distinct positions along individual myofibers. Moreover, we identified a linear link between myonuclear volume and its acceleration values during muscle contraction which, in Nesprin/klar mutants exhibited an opposite tendency relative to control. Estimation of the drag force applied on individual myonuclei revealed that force fluctuations in time, but not the average force, differed significantly between control and Nesprin/klar mutant, and were considerably higher in the mutant myonuclei. Taken together these results imply significant alterations in the mechanical dynamics of individual myonuclei in the Nesprin/klar myonuclei relative to control. Such differences provide novel mechanical insight into Nesprin function in contractile muscles, and might reveal the mechanical basis underlying Nesprin-related human diseases.

Ma2/d promotes myonuclear positioning and association with the sarcoplasmic reticulum.

The cytoplasm of striated myofibers contains a large number of membrane organelles, including sarcoplasmic reticulum (SR), T-tubules and the nuclear membrane. These organelles maintain a characteristic juxtaposition that appears to be essential for efficient inter-membranous exchange of RNA, proteins and ions. We found that the membrane-associated Muscle-specific α2/δ (Ma2/d) subunit of the Ca2+ channel complex localizes to the SR and T-tubules, and accumulates at the myonuclear surfaces. Furthermore, Ma2/d mutant larval muscles exhibit nuclear positioning defects, disruption of the nuclear-SR juxtapositioning, as well as impaired larval locomotion. Ma2/d localization at the nuclear membrane depends on the proper function of the nesprin ortholog Msp300 and the BAR domain protein Amphiphysin (Amph). Importantly, live imaging of muscle contraction in intact Drosophila larvae indicated altered distribution of Sarco/Endoplamic Reticulum Ca2+-ATPase (SERCA) around the myonuclei of Ma2/d mutant larvae. Co-immunoprecipitation analysis supports association between Ma2/d and Amph, and indirectly with Msp300. We therefore suggest that Ma2/d, in association with Msp300 and Amph, mediates interactions between the SR and the nuclear membrane.

The efficacy of an immunoisolating membrane system for islet xenotransplantation in minipigs.

Developing a device that protects xenogeneic islets to allow treatment and potentially cure of diabetes in large mammals has been a major challenge in the past decade. Using xenogeneic islets for transplantation is required in light of donor shortage and the large number of diabetic patients that qualify for islet transplantation. Until now, however, host immunoreactivity against the xenogeneic graft has been a major drawback for the use of porcine islets. Our study demonstrates the applicability of a novel immunoprotective membrane that allows successful xenotransplantation of rat islets in diabetic minipigs without immunosuppressive therapy. Rat pancreatic islets were encapsulated in highly purified alginate and integrated into a plastic macrochamber covered by a poly-membrane for subcutaneous transplantation. Diabetic Sinclair pigs were transplanted and followed for up to 90 days. We demonstrated a persistent graft function and restoration of normoglycemia without the need for immunosuppressive therapy. This concept could potentially offer an attractive strategy for a more widespread islet replacement therapy that would restore endogenous insulin secretion in diabetic patients without the need for immunosuppressive drugs and may even open up an avenue for safe utilization of xenogeneic islet donors.