Inheritance of stress-induced, ATF-2-dependent epigenetic change.

Atf1, the fission yeast homolog of activation transcription factor-2 (ATF-2), contributes to heterochromatin formation. However, the role of ATF-2 in chromatin assembly in higher organisms remains unknown. This study reveals that Drosophila ATF-2 (dATF-2) is required for heterochromatin assembly, whereas the stress-induced phosphorylation of dATF-2, via Mekk1-p38, disrupts heterochromatin. The dATF-2 protein colocalized with HP1, not only on heterochromatin but also at specific loci in euchromatin. Heat shock or osmotic stress induced phosphorylation of dATF-2 and resulted in its release from heterochromatin. This heterochromatic disruption was an epigenetic event that was transmitted to the next generation in a non-Mendelian fashion. When embryos were exposed to heat stress over multiple generations, the defective chromatin state was maintained over multiple successive generations, though it gradually returned to the normal state. The results suggest a mechanism by which the effects of stress are inherited epigenetically via the regulation of a tight chromatin structure.

Identification of a novel role for Drosophila MESR4 in lipid metabolism.

Drosophila provides a powerful genetic model to analyze lipid metabolism. Drosophila has an adipose-like organ called the fat body, which plays a crucial role in energy homeostasis. Here, we conducted a fat body-specific misexpression screen to identify genes involved in lipid metabolism. We found that over-expression of a nuclear protein with nine C2 H2 type zinc-finger motifs and a PHD-finger, Misexpression suppressor of ras 4 (MESR4), reduces lipid accumulation in the fat body, whereas MESR4 knockdown increases it. We further show that MESR4 up-regulates the expression of major lipases, which may account for the reduction in lipid storage in the fat body and the release of free fatty acids (FFAs) in the body. These results suggest that MESR4 acts as an important upstream regulator of energy homeostasis.

The plant homeodomain finger protein MESR4 is essential for embryonic development in Drosophila.

Misexpression Suppressor of Ras 4 (MESR4), a plant homeodomain (PHD) finger protein with nine zinc-finger motifs has been implicated in various biological processes including the regulation of fat storage and innate immunity in Drosophila. However, the role of MESR4 in the context of development remains unclear. Here it is shown that MESR4 is a nuclear protein essential for embryonic development. Immunostaining of polytene chromosomes using anti-MESR4 antibody revealed that MESR4 binds to numerous bands along the chromosome arms. The most intense signal was detected at the 39E-F region, which is known to contain the histone gene cluster. P-element insertions in the MESR4 locus, which were homozygous lethal during embryogenesis with defects in ventral ectoderm formation and head encapsulation was identified. In the mutant embryos, expression of Fasciclin 3 (Fas3), an EGFR signal target gene was greatly reduced, and the level of EGFR signal-dependent double phosphorylated ERK (dp-ERK) remained low. However, in the context of wing vein formation, genetic interaction experiments suggested that MESR4 is involved in the EGFR signaling as a negative regulator. These results suggested that MESR4 is a novel chromatin-binding protein required for proper expression of genes including those regulated by the EGFR signaling pathway during development. genesis 53:701-708, 2015. © 2015 Wiley Periodicals, Inc.

Su(fu) switches Rdx functions to fine-tune hedgehog signaling in the Drosophila wing disk.

Hedgehog (Hh) signaling plays a central role in pattern formation by regulating transcription factor Cubitus interruptus (Ci). Previously, Roadkill (Rdx, also called HIB) was shown to inhibit Ci activity by two distinct mechanisms, depending on the Hh signal strength (Seong et al. 2010, PLoS One 5, e15365). In the anterior region abutting the anterior/posterior (A/P) boundary of the wing disk, where cells receive a strong Hh signal, Rdx blocks the nuclear entry of Ci-155. In contrast, in the region farther from the A/P boundary, where cells receive moderate levels of Hh, Rdx induces Ci-155 degradation in the nucleus. Here, we report that Suppressor of fused, Su(fu), causes the Rdx switch between mechanisms. A strong Hh signal induces rdx expression and suppresses su(fu) expression, whereas moderate levels of Hh induce moderate levels of rdx expression and high levels of su(fu) expression. Rdx blocks entry of Ci-155 into the nucleus in the absence of Su(fu) and Rdx induces the degradation of Ci-155 in the nucleus in the presence of a threshold level of Su(fu). Thus, the Su(fu)-induced switch between the dual actions of Rdx in response to the Hh signal strength plays a role in fine-tuning Hh signaling.

Inheritance and memory of stress-induced epigenome change: roles played by the ATF-2 family of transcription factors.

Data on the inheritance-of-stress effect have been accumulating and some mechanistic insights, such as epigenetic regulation, have also been suggested. In particular, the modern view of Lamarckian inheritance appears to be affected by the finding that stress-induced epigenetic changes can be inherited. This review summarizes the current data on the inheritance of stress effect and possible mechanisms involved in this process. In particular, we focus on the stress-induced epigenetic changes mediated by the ATF-2 family of transcription factors.

Road to sexual maturity: Behavioral event schedule from eclosion to first mating in each sex of Drosophila melanogaster.

Animals achieve their first mating through the process of sexual maturation. This study examined the precise and detailed timing of a series of behavioral events, including wing expansion, first feeding, first excretion, and courtship, during sexual maturation from eclosion to first mating in D. melanogaster. We found that the time of first mating is genetically invariant and is not affected by light/dark cycle or food intake after eclosion. We also found sexual dimorphism in locomotor activity after eclosion, with females increasing locomotor activity earlier than males. In addition, we found a time rapidly changing from extremely low to high sexual activity in males post eclosion (named "drastic male courtship arousal" or DMCA). These behavioral traits leading up to the first mating could serve as clear indicators of sexual maturation and establish precisely timed developmental landmarks to explore further the mechanisms underlying the integration of behavioral and physiological sexual maturation.

Noncanonical function of the Sex lethal gene controls the protogyny phenotype in Drosophila melanogaster.

Drosophila melanogaster females eclose on average 4 h faster than males owing to sexual differences in the pupal period, referred to as the protogyny phenotype. Here, to elucidate the mechanism underlying the protogyny phenotype, we used our newly developed Drosophila Individual Activity Monitoring and Detecting System (DIAMonDS) that detects the precise timing of both pupariation and eclosion in individual flies. Although sex transformation induced by tra-2, tra alteration, or msl-2 knockdown-mediated disruption of dosage compensation showed no effect on the protogyny phenotype, stage-specific whole-body knockdown and mutation of the Drosophila master sex switch gene, Sxl, was found to disrupt the protogyny phenotype. Thus, Sxl establishes the protogyny phenotype through a noncanonical pathway in D. melanogaster.

The Drosophila Individual Activity Monitoring and Detection System (DIAMonDS).

Here, we have developed DIAMonDS (Drosophila Individual Activity Monitoring and Detection System) comprising time-lapse imaging by a charge-coupled device (CCD) flatbed scanner and Sapphire, a novel algorithm and web application. DIAMonDS automatically and sequentially identified the transition time points of multiple life cycle events such as pupariation, eclosion, and death in individual flies at high temporal resolution and on a large scale. DIAMonDS performed simultaneous multiple scans to measure individual deaths (≤1152 flies per scanner) and pupariation and eclosion timings (≤288 flies per scanner) under various chemical exposures, environmental conditions, and genetic backgrounds. DIAMonDS correctly identified 74-85% of the pupariation and eclosion events and ~ 92% of the death events within ± 10 scanning frames. This system is a powerful tool for studying the influences of genetic and environmental factors on fruit flies and efficient, high-throughput genetic and chemical screening in drug discovery.

Publisher Correction: Paternal restraint stress affects offspring metabolism via ATF-2 dependent mechanisms in Drosophila melanogaster germ cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Paternal restraint stress affects offspring metabolism via ATF-2 dependent mechanisms in Drosophila melanogaster germ cells.

Paternal environmental factors can epigenetically influence gene expressions in offspring. We demonstrate that restraint stress, an experimental model for strong psychological stress, to fathers affects the epigenome, transcriptome, and metabolome of offspring in a MEKK1-dATF2 pathway-dependent manner in Drosophila melanogaster. Genes involved in amino acid metabolism are upregulated by paternal restraint stress, while genes involved in glycolysis and the tricarboxylic acid (TCA) cycle are downregulated. The effects of paternal restraint stress are also confirmed by metabolome analysis. dATF-2 is highly expressed in testicular germ cells, and restraint stress also induces p38 activation in the testes. Restraint stress induces Unpaired 3 (Upd3), a Drosophila homolog of Interleukin 6 (IL-6). Moreover, paternal overexpression of upd3 in somatic cells disrupts heterochromatin in offspring but not in offspring from dATF-2 mutant fathers. These results indicate that paternal restraint stress affects metabolism in offspring via inheritance of dATF-2-dependent epigenetic changes.

POSH, a scaffold protein for JNK signaling, binds to ALG-2 and ALIX in Drosophila.

Plenty of SH3s (POSH) functions as a scaffold protein for the Jun N-terminal kinase (JNK) signal transduction pathway, which leads to cell death in mammalian cultured cells and Drosophila. Here, we show that POSH forms a complex with Apoptosis-linked gene-2 (ALG-2) and ALG-2-interacting protein (ALIX/AIP1) in a calcium-dependent manner. Overexpression of ALG-2 or ALIX in developing imaginal eye discs resulted in roughened or melanized eyes, respectively. These phenotypes were enhanced by co-overexpression of POSH. We found that overexpression of either gene could induce ectopic JNK activation, suggesting that POSH/ALG-2/ALIX may function together in the regulation of the JNK pathway.

Longevity determination genes in Drosophila melanogaster.

Identification of longevity mutants is crucial for genetic approach to dissect the molecular mechanism of aging and longevity determination. In Drosophila melanogaster, several mutations have been shown to extend the longevity: methuselah encoding a putative G-protein coupled receptor, Indy encoding a sodium dicarboxylate cotransporter, chico encoding insulin receptor substrate, and InR encoding the insulin-like receptor. Extended longevity phenotypes were also observed in transgenic flies overexpressing antioxidant enzymes, Cu/Zn superoxide dismutase and Catalase, Cu/Zn SOD only, or a molecular chaperone, hsp70. Pleiotropism of mutations is a limitation associated with conventional mutagenesis for efficient detection of longevity determination genes. Using a conditional misexpression system, we identified Drosophila POSH (DPOSH), a scaffold protein containing RING finger and four SH3 domains, whose ubiquitous overexpression in adult stage extends the longevity. Neural-specific overexpression of DPOSH is sufficient to extend the longevity, whereas overexpression in non-neural tissues during development induces apoptosis through activation of JNK/SAPK pathway.

Inhibition of the nuclear import of cubitus interruptus by roadkill in the presence of strong hedgehog signal.

Hedgehog (Hh) signalling plays an important role in various developmental processes by activating the Cubitus interruptus (Ci)/Glioblastoma (Gli) family of transcription factors. In the process of proper pattern formation, Ci activity is regulated by multiple mechanisms, including processing, trafficking, and degradation. However, it remains elusive how Ci distinctly recognizes the strong and moderate Hh signals. Roadkill (Rdx) induces Ci degradation in the anterior region of the Drosophila wing disc. Here, we report that Rdx inhibited Ci activity by two different mechanisms. In the region abutting the anterior/posterior boundary, which receives strong Hh signal, Rdx inhibited the nuclear import of Ci by releasing importin α3 from Ci. In this region, Rdx negatively regulated the expression of transcription factor Knot/Collier. In farther anterior regions receiving moderate levels of Hh signal, Rdx induced Ci degradation, as reported previously. Thus, two different mechanisms by which Rdx negatively regulates Ci may play an important role in the fine-tuning of Hh responses.

Loss of Trx-2 enhances oxidative stress-dependent phenotypes in Drosophila.

Overexpression of thioredoxin (TRX) confers oxidative stress resistance and extends lifespan in mammals and insects. However, less is known about phenotypes associated with loss of TRX. We investigated loss-of-function phenotypes of Trx-2 in Drosophila, and found that the mutant flies are hyper-susceptible to paraquat, a free radical generator, but not to hydrogen peroxide. They contain a high amount of protein carbonyl, which dramatically increases with age. Trx-2 mutants express high levels of anti-oxidant genes, such as superoxide dismutase, catalase, and glutathione synthetase. This is the first demonstration of biochemical and physiological consequences caused by loss of Trx-2 in Drosophila.

Drosophila ATF-2 regulates sleep and locomotor activity in pacemaker neurons.

Stress-activated protein kinases such as p38 regulate the activity of transcription factor ATF-2. However, the physiological role of ATF-2, especially in the brain, is unknown. Here, we found that Drosophila melanogaster ATF-2 (dATF-2) is expressed in large ventral lateral neurons (l-LN(v)s) and also, to a much lesser extent, in small ventral lateral neurons, the pacemaker neurons. Only l-LN(v)s were stained with the antibody that specifically recognizes phosphorylated dATF-2, suggesting that dATF-2 is activated specifically in l-LN(v)s. The knockdown of dATF-2 in pacemaker neurons using RNA interference decreased sleep time, whereas the ectopic expression of dATF-2 increased sleep time. dATF-2 knockdown decreased the length of sleep bouts but not the number of bouts. The ATF-2 level also affected the sleep rebound after sleep deprivation and the arousal threshold. dATF-2 negatively regulated locomotor activity, although it did not affect the circadian locomotor rhythm. The degree of dATF-2 phosphorylation was greater in the morning than at night and was enhanced by forced locomotion via the dp38 pathway. Thus, dATF-2 is activated by the locomotor while it increases sleep, suggesting a role for dATF-2 as a regulator to connect sleep with locomotion.