BackgroundAging is associated with functional decline of neurons and increased incidence of both neurodegenerative and ocular disease. Photoreceptor neurons in Drosophila melanogaster provide a powerful model for studying the molecular changes involved in functional senescence of neurons since decreased visual behavior precedes retinal degeneration. Here, we sought to identify gene expression changes and the genomic features of differentially regulated genes in photoreceptors that contribute to visual senescence.ResultsTo identify gene expression changes that could lead to visual senescence, we characterized the aging transcriptome of Drosophila sensory neurons highly enriched for photoreceptors. We profiled the nuclear transcriptome of genetically-labeled photoreceptors over a 40 day time course and identified increased expression of genes involved in stress and DNA damage response, and decreased expression of genes required for neuronal function. We further show that combinations of promoter motifs robustly identify age-regulated genes, suggesting that transcription factors are important in driving expression changes in aging photoreceptors. However, long, highly expressed and heavily spliced genes are also more likely to be downregulated with age, indicating that other mechanisms could contribute to expression changes at these genes. Lastly, we identify that circular RNAs (circRNAs) strongly increase during aging in photoreceptors.ConclusionsOverall, we identified changes in gene expression in aging Drosophila photoreceptors that could account for visual senescence. Further, we show that genomic features predict these age-related changes, suggesting potential mechanisms that could be targeted to slow the rate of age-associated visual decline.Electronic supplementary materialThe online version of this article (10.1186/s12864-017-4304-3) contains supplementary material, which is available to authorized users.

Chromatin accessibility is integral to the process by which transcription factors (TFs) read out cis-regulatory DNA sequences, but it is difficult to differentiate between TFs that drive accessibility and those that do not. Deep learning models that learn complex sequence rules provide an unprecedented opportunity to dissect this problem. Using zygotic genome activation in the Drosophila embryo as a model, we generated high-resolution TF binding and chromatin accessibility data, analyzed the data with interpretable deep learning, and performed genetic experiments for validation. We uncover a clear hierarchical relationship between the pioneer TF Zelda and the TFs involved in axis patterning. Zelda consistently pioneers chromatin accessibility proportional to motif affinity, while patterning TFs augment chromatin accessibility in sequence contexts in which they mediate enhancer activation. We conclude that chromatin accessibility occurs in two phases: one through pioneering, which makes enhancers accessible but not necessarily active, and a second when the correct combination of transcription factors leads to enhancer activation.

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The Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex is a transcriptional coactivator with histone acetylase and deubiquitinase activities that plays an important role in visual development and function. In Drosophila melanogaster, four SAGA subunits are required for the deubiquitination of monoubiquitinated histone H2B (ubH2B): Nonstop, Sgf11, E(y)2, and Ataxin 7. Mutations that disrupt SAGA deubiquitinase activity cause defects in neuronal connectivity in the developing Drosophila visual system. In addition, mutations in SAGA result in the human progressive visual disorder spinocerebellar ataxia type 7 (SCA7). Glial cells play a crucial role in both the neuronal connectivity defect in nonstop and sgf11 flies, and in the retinal degeneration observed in SCA7 patients. Thus, we sought to identify the gene targets of SAGA deubiquitinase activity in glia in the Drosophila larval central nervous system. To do this, we enriched glia from wild-type, nonstop, and sgf11 larval optic lobes using affinity-purification of KASH-GFP tagged nuclei, and then examined each transcriptome using RNA-seq. Our analysis showed that SAGA deubiquitinase activity is required for proper expression of 16% of actively transcribed genes in glia, especially genes involved in proteasome function, protein folding and axon guidance. We further show that the SAGA deubiquitinase-activated gene Multiplexin (Mp) is required in glia for proper photoreceptor axon targeting. Mutations in the human ortholog of Mp, COL18A1, have been identified in a family with a SCA7-like progressive visual disorder, suggesting that defects in the expression of this gene in SCA7 patients could play a role in the retinal degeneration that is unique to this ataxia.

As the human population ages, there is an increased risk for ocular diseases such as age‐related macular degeneration (AMD), glaucoma, and diabetic retinopathy. Specifically, AMD is characterized by deterioration of the macula of the eye and is the leading cause of the loss of central vision in those individuals over 60. Twin studies have previously shown that non‐genetic, environmental factors contribute more than 50% of the overall risk for developing age‐related ocular diseases such as AMD. For this reason, epigenetic mechanisms have been thought to play a major role in the environmental‐based risks associated with AMD. The epigenetic factors that contribute to vision loss in the aging eye have yet to be identified, but are thought to play a pivotal role in maintaining proper ocular ability, as misregulation of these factors could lead to transcriptional changes in gene expression. We hypothesize that as photoreceptor cells age, there are changes in gene expression that contribute to the loss of vision seen later in life. Because age‐related changes in gene expression are cell autonomous, Drosophila melanogaster photoreceptor neurons were chosen as a model system due to the likelihood that these post‐mitotic cells are more sensitive to changes in gene expression. Photoreceptor cells are also likely to be at an increased vulnerability to the effects of aging because they must constantly express high levels of proteins involved in phototransduction in order to maintain their function throughout the adult lifespan. Using these Drosophila photoreceptors, we are conducting a study on the effects of aging on gene expression. Experiments to globally characterize the transcriptome of isolated nuclei from the R1‐R6 photoreceptor cells of male Drosophila were performed using RNA‐sequencing technology. We then used cluster analysis on this sequencing data to identify genes that exhibited statistically significant changes in gene expression at specific fly‐age time points. Using this cluster analysis data, we will prioritize genes from each cluster to generate subsets of genes to test through qRT‐PCR experiments on both dissected eye and isolated nuclear samples. This qRT‐PCR will allow us to validate and quantify the age‐related changes in gene expression observed by RNA‐sequencing in photoreceptor nuclei. From this, we can begin to ask questions about the role of epigenetic factors in regulating these changes in gene expression and how these factors are affected by aging.Support or Funding InformationNational Institutes of Health