Targeted RNAi screen identifies transcriptional mechanisms that prevent premature degeneration of adult photoreceptors.

Aging is associated with a decline in visual function and increased prevalence of ocular disease, correlating with changes in the transcriptome and epigenome of cells in the eye. Here, we sought to identify the transcriptional mechanisms that are necessary to maintain photoreceptor viability and function during aging. To do this, we performed a targeted photoreceptor-specific RNAi screen in Drosophila to identify transcriptional regulators whose knockdown results in premature, age-dependent retinal degeneration. From an initial set of 155 RNAi lines each targeting a unique gene and spanning a diverse set of transcription factors, chromatin remodelers, and histone modifiers, we identified 18 high-confidence target genes whose decreased expression in adult photoreceptors leads to premature and progressive retinal degeneration. These 18 target genes were enriched for factors involved in the regulation of transcription initiation, pausing, and elongation, suggesting that these processes are essential for maintaining the health of aging photoreceptors. To identify the genes regulated by these factors, we profiled the photoreceptor transcriptome in a subset of lines. Strikingly, two of the 18 target genes, Spt5 and domino, show similar changes in gene expression to those observed in photoreceptors with advanced age. Together, our data suggest that dysregulation of factors involved in transcription initiation and elongation plays a key role in shaping the transcriptome of aging photoreceptors. Further, our findings indicate that the age-dependent changes in gene expression not only correlate but might also contribute to an increased risk of retinal degeneration.

Establishing the contribution of active histone methylation marks to the aging transcriptional landscape of Drosophila photoreceptors.

Studies in multiple organisms have shown that aging is accompanied by several molecular phenotypes that include dysregulation of chromatin. Since chromatin regulates DNA-based processes such as transcription, alterations in chromatin modifications could impact the transcriptome and function of aging cells. In flies, as in mammals, the aging eye undergoes changes in gene expression that correlate with declining visual function and increased risk of retinal degeneration. However, the causes of these transcriptome changes are poorly understood. Here, we profiled chromatin marks associated with active transcription in the aging Drosophila eye to understand how chromatin modulates transcriptional outputs. We found that both H3K4me3 and H3K36me3 globally decrease across all actively expressed genes with age. However, we found no correlation with changes in differential gene expression. Downregulation of the H3K36me3 methyltransferase Set2 in young photoreceptors revealed significant changes in splicing events that overlapped significantly with those observed in aging photoreceptors. These overlapping splicing events impacted multiple genes involved in phototransduction and neuronal function. Since proper splicing is essential for visual behavior, and because aging Drosophila undergo a decrease in visual function, our data suggest that H3K36me3 could play a role in maintaining visual function in the aging eye through regulating alternative splicing.

Tissue-Specific, Genome-wide Mapping of R-loops in Drosophila Using MapR.

R-loops, or RNA:DNA hybrids, are structures that arise co-transcriptionally when a nascent RNA hybridizes back with the template ssDNA, leading to a displaced ssDNA. Because accumulation of R-loops can lead to genomic instability and loss of cellular homeostasis, it is important to determine the genome-wide distribution of R-loops in different physiological conditions. Current R-loop mapping strategies are based on R-loop enrichment-mediated by the S9.6 antibody, such as DRIP-seq, or by the exonuclease RNase H1, such as MapR-or the latest R-loop CUT&Tag, based on an artificial R-loop sensor derived from an RNase H1 sub-domain. Because some of these techniques often require high input material or expensive reagents, we sought to apply MapR, which does not require expensive reagents and has been shown to be compatible with low input samples. Importantly, we demonstrate that incorporation of improved CUT&RUN steps into the MapR protocol yields R-loop-enriched DNA when using low input Drosophila nuclei. Graphical abstract: Workflow for mapping tissue-specific, genome-wide R-loops in Drosophila . Purify GST-tagged and catalytically inactive RNase H1 tethered MapR enzymes, GST-ΔRH-MNase, and GST-MNase, from transformed E. coli. Perform tissue-specific nuclei immuno-enrichment from UAS-EGFP.KASH-Msp300 Drosophila using magnetic bead-bound green fluorescent protein (GFP) antibody. Incubate isolated nuclei with MapR enzymes and activate MNase DNA cleavage with low salt/high calcium buffers. Purify released, R-loopenriched DNA fragments and generate sequencing-ready libraries. Align MapR data to reference genome and compare R-loop enrichment peaks in genome browser.

The Clock:Cycle complex is a major transcriptional regulator of Drosophila photoreceptors that protects the eye from retinal degeneration and oxidative stress.

The aging eye experiences physiological changes that include decreased visual function and increased risk of retinal degeneration. Although there are transcriptomic signatures in the aging retina that correlate with these physiological changes, the gene regulatory mechanisms that contribute to cellular homeostasis during aging remain to be determined. Here, we integrated ATAC-seq and RNA-seq data to identify 57 transcription factors that showed differential activity in aging Drosophila photoreceptors. These 57 age-regulated transcription factors include two circadian regulators, Clock and Cycle, that showed sustained increased activity during aging. When we disrupted the Clock:Cycle complex by expressing a dominant negative version of Clock (ClkDN) in adult photoreceptors, we observed changes in expression of 15-20% of genes including key components of the phototransduction machinery and many eye-specific transcription factors. Using ATAC-seq, we showed that expression of ClkDN in photoreceptors leads to changes in activity of 37 transcription factors and causes a progressive decrease in global levels of chromatin accessibility in photoreceptors. Supporting a key role for Clock-dependent transcription in the eye, expression of ClkDN in photoreceptors also induced light-dependent retinal degeneration and increased oxidative stress, independent of light exposure. Together, our data suggests that the circadian regulators Clock and Cycle act as neuroprotective factors in the aging eye by directing gene regulatory networks that maintain expression of the phototransduction machinery and counteract oxidative stress.

Proper control of R-loop homeostasis is required for maintenance of gene expression and neuronal function during aging.

Age-related loss of cellular function and increased cell death are characteristic hallmarks of aging. While defects in gene expression and RNA metabolism have been linked with age-associated human neuropathies, it is not clear how the changes that occur in aging neurons contribute to loss of gene expression homeostasis. R-loops are RNA-DNA hybrids that typically form co-transcriptionally via annealing of the nascent RNA to the template DNA strand, displacing the non-template DNA strand. Dysregulation of R-loop homeostasis has been associated with both transcriptional impairment and genome instability. Importantly, a growing body of evidence links R-loop accumulation with cellular dysfunction, increased cell death, and chronic disease onset. Here, we characterized the R-loop landscape in aging Drosophila melanogaster photoreceptor neurons and showed that bulk R-loop levels increased with age. Further, genome-wide mapping of R-loops revealed that transcribed genes accumulated R-loops over gene bodies during aging, which correlated with decreased expression of long and highly expressed genes. Importantly, while photoreceptor-specific down-regulation of Top3ß, a DNA/RNA topoisomerase associated with R-loop resolution, lead to decreased visual function, over-expression of Top3ß or nuclear-localized RNase H1, which resolves R-loops, enhanced positive light response during aging. Together, our studies highlight the functional link between dysregulation of R-loop homeostasis, gene expression, and visual function during aging.

In vivo tissue-specific chromatin profiling in Drosophila melanogaster using GFP-tagged nuclei.

The chromatin landscape defines cellular identity in multicellular organisms with unique patterns of DNA accessibility and histone marks decorating the genome of each cell type. Thus, profiling the chromatin state of different cell types in an intact organism under disease or physiological conditions can provide insight into how chromatin regulates cell homeostasis in vivo. To overcome the many challenges associated with characterizing chromatin state in specific cell types, we developed an improved approach to isolate Drosophila melanogaster nuclei tagged with a GFPKASH protein. The perinuclear space-localized KASH domain anchors GFP to the outer nuclear membrane, and expression of UAS-GFPKASH can be controlled by tissue-specific Gal4 drivers. Using this protocol, we profiled chromatin accessibility using an improved version of Assay for Transposable Accessible Chromatin followed by sequencing (ATAC-seq), called Omni-ATAC. In addition, we examined the distribution of histone marks using Chromatin immunoprecipitation followed by sequencing (ChIP-seq) and Cleavage Under Targets and Tagmentation (CUT&Tag) in adult photoreceptor neurons. We show that the chromatin landscape of photoreceptors reflects the transcriptional state of these cells, demonstrating the quality and reproducibility of our approach for profiling the transcriptome and epigenome of specific cell types in Drosophila.