An efficient cloning method to expand vector and restriction site compatibility of Golden Gate Assembly.

Golden Gate Assembly is an efficient and rapid cloning method but requires dedicated vectors. Here, we modified Golden Gate to expand its compatibility to a broader range of destination vectors while maintaining its strengths. Our Expanded Golden Gate (ExGG) assembly adds to the insert(s) type IIS restriction sites that generate protruding ends compatible with traditional type IIP sites on the recipient vector. The ligated product cannot be cleaved again, owing to a single-base change near the junction. This allows the reaction to proceed in a single tube without an intermediate purification step. ExGG can be used to introduce multiple fragments into a vector simultaneously, including shorter fragments (<100 bp) and fragments with shared sequences, which can be difficult to assemble with other fast cloning strategies. Thus, ExGG extends the convenience of Golden Gate to a much larger space of pre-existing vectors designed for conventional cloning.

Ensheathing glia promote increased lifespan and healthy brain aging.

Glia have an emergent role in brain aging and disease. In the Drosophila melanogaster brain, ensheathing glia function as phagocytic cells and respond to acute neuronal damage, analogous to mammalian microglia. We previously reported changes in glia composition over the life of ants and fruit flies, including a decline in the relative proportion of ensheathing glia with time. How these changes influence brain health and life expectancy is unknown. Here, we show that ensheathing glia but not astrocytes decrease in number during Drosophila melanogaster brain aging. The remaining ensheathing glia display dysregulated expression of genes involved in lipid metabolism and apoptosis, which may lead to lipid droplet accumulation, cellular dysfunction, and death. Inhibition of apoptosis rescued the decline of ensheathing glia with age, improved the neuromotor performance of aged flies, and extended lifespan. Furthermore, an expanded ensheathing glia population prevented amyloid-beta accumulation in a fly model of Alzheimer's disease and delayed the premature death of the diseased animals. These findings suggest that ensheathing glia play a vital role in regulating brain health and animal longevity.

Genome annotation with long RNA reads reveals new patterns of gene expression and improves single-cell analyses in an ant brain.

BACKGROUND: Functional genomic analyses rely on high-quality genome assemblies and annotations. Highly contiguous genome assemblies have become available for a variety of species, but accurate and complete annotation of gene models, inclusive of alternative splice isoforms and transcription start and termination sites, remains difficult with traditional approaches. RESULTS: Here, we utilized full-length isoform sequencing (Iso-Seq), a long-read RNA sequencing technology, to obtain a comprehensive annotation of the transcriptome of the ant Harpegnathos saltator. The improved genome annotations include additional splice isoforms and extended 3' untranslated regions for more than 4000 genes. Reanalysis of RNA-seq experiments using these annotations revealed several genes with caste-specific differential expression and tissue- or caste-specific splicing patterns that were missed in previous analyses. The extended 3' untranslated regions afforded great improvements in the analysis of existing single-cell RNA-seq data, resulting in the recovery of the transcriptomes of 18% more cells. The deeper single-cell transcriptomes obtained with these new annotations allowed us to identify additional markers for several cell types in the ant brain, as well as genes differentially expressed across castes in specific cell types. CONCLUSIONS: Our results demonstrate that Iso-Seq is an efficient and effective approach to improve genome annotations and maximize the amount of information that can be obtained from existing and future genomic datasets in Harpegnathos and other organisms.

Unprogrammed epigenetic variation mediated by stochastic formation of ectopic heterochromatin.

Changes in gene expression via chromatin-mediated mechanisms are important for reprogramming and differentiation, but uncontrolled changes can potentially lead to harmful or adaptive phenotypic alteration. Thus, diversification of the genome-wide chromatin state must be strictly limited, but the underlying mechanism of this regulation is largely unknown. In this review, we focused on distribution of heterochromatin, a tight chromatin structure that negatively regulates gene expression. Heterochromatin is characterized by methylation of histone H3 at lysine 9, and its formation and spreading are controlled by H3K9-specific methyltransferases and reversal factors such as histone demethylases. We summarize recent findings and discuss how variability in the heterochromatin distribution is controlled in the unicellular eukaryote fission yeast. In this context, we recently found that the anti-silencing factor Epe1 plays a key role in the formation of the individual-specific heterochromatin distribution. In conclusion, recent studies revealed that there are many potential heterochromatin formation sites in the fission yeast genome, and several proteins contribute to suppression of spreading and genome-wide dispersal of heterochromatin; knowledge from fission yeast studies may provide insights into the mechanisms regulating epigenetic diversification in multicellular eukaryotes.

Regulation of ectopic heterochromatin-mediated epigenetic diversification by the JmjC family protein Epe1.

H3K9 methylation (H3K9me) is a conserved marker of heterochromatin, a transcriptionally silent chromatin structure. Knowledge of the mechanisms for regulating heterochromatin distribution is limited. The fission yeast JmjC domain-containing protein Epe1 localizes to heterochromatin mainly through its interaction with Swi6, a homologue of heterochromatin protein 1 (HP1), and directs JmjC-mediated H3K9me demethylation in vivo. Here, we found that loss of epe1 (epe1Δ) induced a red-white variegated phenotype in a red-pigment accumulation background that generated uniform red colonies. Analysis of isolated red and white colonies revealed that silencing of genes involved in pigment accumulation by stochastic ectopic heterochromatin formation led to white colony formation. In addition, genome-wide analysis of red- and white-isolated clones revealed that epe1Δ resulted in a heterogeneous heterochromatin distribution among clones. We found that Epe1 had an N-terminal domain distinct from its JmjC domain, which activated transcription in both fission and budding yeasts. The N-terminal transcriptional activation (NTA) domain was involved in suppression of ectopic heterochromatin-mediated red-white variegation. We introduced a single copy of Epe1 into epe1Δ clones harboring ectopic heterochromatin, and found that Epe1 could reduce H3K9me from ectopic heterochromatin but some of the heterochromatin persisted. This persistence was due to a latent H3K9me source embedded in ectopic heterochromatin. Epe1H297A, a canonical JmjC mutant, suppressed red-white variegation, but entirely failed to remove already-established ectopic heterochromatin, suggesting that Epe1 prevented stochastic de novo deposition of ectopic H3K9me in an NTA-dependent but JmjC-independent manner, while its JmjC domain mediated removal of H3K9me from established ectopic heterochromatin. Our results suggest that Epe1 not only limits the distribution of heterochromatin but also controls the balance between suppression and retention of heterochromatin-mediated epigenetic diversification.