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.

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.

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.

Recruitment of BAF to the nuclear envelope couples the LINC complex to endoreplication.

DNA endoreplication has been implicated as a cell strategy for cell growth and in tissue injury. Here, we demonstrate that barrier-to-autointegration factor (BAF) represses endoreplication in Drosophila myofibers. We show that BAF localization at the nuclear envelope is eliminated in flies with mutations of the linker of nucleoskeleton and cytoskeleton (LINC) complex in which the LEM-domain protein Otefin is excluded, or after disruption of the nucleus-sarcomere connections. Furthermore, BAF localization at the nuclear envelope requires the activity of the BAF kinase VRK1/Ball, and, consistently, non-phosphorylatable BAF-GFP is excluded from the nuclear envelope. Importantly, removal of BAF from the nuclear envelope correlates with increased DNA content in the myonuclei. E2F1, a key regulator of endoreplication, overlaps BAF localization at the myonuclear envelope, and BAF removal from the nuclear envelope results in increased E2F1 levels in the nucleoplasm and subsequent elevated DNA content. We suggest that LINC-dependent and phosphosensitive attachment of BAF to the nuclear envelope, through its binding to Otefin, tethers E2F1 to the nuclear envelope thus inhibiting its accumulation in the nucleoplasm.

Pituicyte Cues Regulate the Development of Permeable Neuro-Vascular Interfaces.

The hypothalamo-neurohypophyseal system (HNS) regulates homeostasis through the passage of neurohormones and blood-borne proteins via permeable blood capillaries that lack the blood-brain barrier (BBB). Why neurohypophyseal capillaries become permeable while the neighboring vasculature of the brain forms BBB remains unclear. We show that pituicytes, the resident astroglial cells of the neurohypophysis, express genes that are associated with BBB breakdown during neuroinflammation. Pituicyte-enriched factors provide a local microenvironment that instructs a permeable neurovascular conduit. Thus, genetic and pharmacological perturbations of Vegfa and Tgfß3 affected HNS vascular morphogenesis and permeability and impaired the expression of the fenestral marker plvap. The anti-inflammatory agent dexamethasone decreased HNS permeability and downregulated the pituicyte-specific cyp26b gene, encoding a retinoic acid catabolic enzyme. Inhibition of Cyp26b activity led to upregulation of tight junction protein Claudin-5 and decreased permeability. We conclude that pituicyte-derived factors regulate the "decision" of endothelial cells to adopt a permeable endothelial fate instead of forming a BBB.

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.

Mechanotransduction via the LINC complex regulates DNA replication in myonuclei.

Nuclear mechanotransduction has been implicated in the control of chromatin organization; however, its impact on functional contractile myofibers is unclear. We found that deleting components of the linker of nucleoskeleton and cytoskeleton (LINC) complex in Drosophila melanogaster larval muscles abolishes the controlled and synchronized DNA endoreplication, typical of nuclei across myofibers, resulting in increased and variable DNA content in myonuclei of individual myofibers. Moreover, perturbation of LINC-independent mechanical input after knockdown of ß-Integrin in larval muscles similarly led to increased DNA content in myonuclei. Genome-wide RNA-polymerase II occupancy analysis in myofibers of the LINC mutant klar indicated an altered binding profile, including a significant decrease in the chromatin regulator barrier-to-autointegration factor (BAF) and the contractile regulator Troponin C. Importantly, muscle-specific knockdown of BAF led to increased DNA content in myonuclei, phenocopying the LINC mutant phenotype. We propose that mechanical stimuli transmitted via the LINC complex act via BAF to regulate synchronized cell-cycle progression of myonuclei across single myofibers.

Nesprin provides elastic properties to muscle nuclei by cooperating with spectraplakin and EB1.

Muscle nuclei are exposed to variable cytoplasmic strain produced by muscle contraction and relaxation, but their morphology remains stable. Still, the mechanism responsible for maintaining myonuclear architecture, and its importance, is currently elusive. Herein, we uncovered a unique myonuclear scaffold in Drosophila melanogaster larval muscles, exhibiting both elastic features contributed by the stretching capacity of MSP300 (nesprin) and rigidity provided by a perinuclear network of microtubules stabilized by Shot (spectraplakin) and EB1. Together, they form a flexible perinuclear shield that protects myonuclei from intrinsic or extrinsic forces. The loss of this scaffold resulted in significantly aberrant nuclear morphology and subsequently reduced levels of essential nuclear factors such as lamin A/C, lamin B, and HP1. Overall, we propose a novel mechanism for maintaining myonuclear morphology and reveal its critical link to correct levels of nuclear factors in differentiated muscle fibers. These findings may shed light on the underlying mechanism of various muscular dystrophies.

Homeodomain protein otp and activity-dependent splicing modulate neuronal adaptation to stress.

Regulation of corticotropin-releasing hormone (CRH) activity is critical for the animal's adaptation to stressful challenges, and its dysregulation is associated with psychiatric disorders in humans. However, the molecular mechanism underlying this transcriptional response to stress is not well understood. Using various stress paradigms in mouse and zebrafish, we show that the hypothalamic transcription factor Orthopedia modulates the expression of CRH as well as the splicing factor Ataxin 2-Binding Protein-1 (A2BP1/Rbfox-1). We further show that the G protein coupled receptor PAC1, which is a known A2BP1/Rbfox-1 splicing target and an important mediator of CRH activity, is alternatively spliced in response to a stressful challenge. The generation of PAC1-hop messenger RNA isoform by alternative splicing is required for termination of CRH transcription, normal activation of the hypothalamic-pituitary-adrenal axis and adaptive anxiety-like behavior. Our study identifies an evolutionarily conserved biochemical pathway that modulates the neuronal adaptation to stress through transcriptional activation and alternative splicing.

Enhanced sensitivity of midline glial cells to apoptosis is achieved by HOW(L)-dependent repression of Diap1.

The selective sensitivity of cells to programmed cell death (PCD) depends on the positive and negative death-inducing signals that converge into the apoptotic pathway. In Drosophila, the midline glial (MG) cells undergo selective death during development. Here, we show that the long isoform of the RNA-binding protein Held Out Wing (HOW(L)) is essential for enhancing the sensitivity of the MG cells to PCD. In how mutant embryos, the number of MG cells was elevated. This phenotype could be rescued by midline expression of the HOW(L) repressor isoform. In how mutant embryos, the levels of the caspase inhibitor of apoptosis, Diap1 were elevated, in parallel to reduction in the levels of activated caspase. Similarly, reducing the levels of HOW in S2 cells led to elevation of Diap1, whereas over expression of HOW(L) promoted reduction of Diap1 protein as well as mRNA levels. Importantly, deletion of the two HOW binding sites from diap1 3'UTR abrogated HOW-dependent repression of Diap1, suggesting that HOW represses diap1 by binding to its 3'UTR. These results suggest that HOW(L) enhances the sensitivity of MG cells to apoptotic signals by reducing the levels of diap1 in these cells in, demonstrating a novel mode of regulation of PCD at the mRNA level.

The splicing factor crooked neck associates with the RNA-binding protein HOW to control glial cell maturation in Drosophila.

In both vertebrates and invertebrates, glial cells wrap axonal processes to ensure electrical conductance. Here we report that Crooked neck (Crn), the Drosophila homolog of the yeast Clf1p splicing factor, is directing peripheral glial cell maturation. We show that crooked neck is expressed and required in glial cells to control migration and axonal wrapping. Within the cytoplasm, Crn interacts with the RNA-binding protein HOW and then translocates to the nucleus where the Crn/HOW complex controls glial differentiation by facilitating splicing of specific target genes. By using a GFP-exon trap approach, we identified some of the in vivo target genes that encode proteins localized in autocellular septate junctions. In conclusion, here we show that glial cell differentiation is controlled by a cytoplasmic assembly of splicing components, which upon translocation to the nucleus promote the splicing of genes involved in the assembly of cellular junctions.