Transcriptome profiling of aging Drosophila photoreceptors reveals gene expression trends that correlate with visual senescence.

BACKGROUND: Aging 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. RESULTS: To 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. CONCLUSIONS: Overall, 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.

Lola-I is a promoter pioneer factor that establishes de novo Pol II pausing during development.

While the accessibility of enhancers is dynamically regulated during development, promoters tend to be constitutively accessible and poised for activation by paused Pol II. By studying Lola-I, a Drosophila zinc finger transcription factor, we show here that the promoter state can also be subject to developmental regulation independently of gene activation. Lola-I is ubiquitously expressed at the end of embryogenesis and causes its target promoters to become accessible and acquire paused Pol II throughout the embryo. This promoter transition is required but not sufficient for tissue-specific target gene activation. Lola-I mediates this function by depleting promoter nucleosomes, similar to the action of pioneer factors at enhancers. These results uncover a level of regulation for promoters that is normally found at enhancers and reveal a mechanism for the de novo establishment of paused Pol II at promoters.

Chromatin accessibility in the Drosophila embryo is determined by transcription factor pioneering and enhancer activation.

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 Drosophila as a model, we analyzed high-resolution TF binding and chromatin accessibility data with interpretable deep learning and performed genetic validation experiments. We identify a hierarchical relationship between the pioneer TF Zelda and the TFs involved in axis patterning. Zelda consistently pioneers chromatin accessibility proportional to motif affinity, whereas patterning TFs augment chromatin accessibility in sequence contexts where they mediate enhancer activation. We conclude that chromatin accessibility occurs in two tiers: one through pioneering, which makes enhancers accessible but not necessarily active, and the second when the correct combination of TFs leads to enhancer activation.

Transcriptome Profiling Identifies Multiplexin as a Target of SAGA Deubiquitinase Activity in Glia Required for Precise Axon Guidance During Drosophila Visual Development.

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.