Genomic Regulation of the Aging Drosophila Eye
Aging is characterized by changes in transcriptional outputs that correlate with physiological changes observed as we age, including decreased function, and increased cell death. Importantly, many of these changes are conserved across tissues and organisms . Because one of the molecular hallmarks of aging is epigenetic dysregulation, we are interested in understanding how age-associated changes in chromatin contribute to the aging transcriptome. To accomplish this, we use the Drosophila visual system as a model for aging, with a particular focus on photoreceptor neurons.
To perform cell-type specific genomic studies in Drosophila, we previously developed a nuclei immuno-enrichment method that was compatible with RNA-seq. However, due to low nuclei yields, this protocol was not amenable to chromatin-based studies, such as ChIP-seq and ATAC-seq. In Chapter 1, we developed an improved approach to isolate Drosophila melanogaster nuclei tagged with a GFPKASH protein that increased yields without compromising efficiency. We further demonstrate that this protocol is compatible with several chromatin profiling techniques, such as Assay of Transposable-Accessible Chromatin (ATAC)-seq, Chromatin Immunoprecipitation (ChIP-seq), and CUT&Tag.
Chromatin accessibility is enriched for transcription factors. Thus, in Chapter 2, we profiled accessible chromatin in aging photoreceptors and integrated this data with RNA-seq to identify transcription factors that showed differential activity in aging Drosophila photoreceptors. Surprisingly, we found that 57 transcription factors had differential binding activity during aging, including 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.
Previous work in the Weake lab found that long, highly expressed genes were more susceptible to be downregulated with age. DNA:RNA hybrids are co-transcriptional structures that form when the nascent RNA hybridizes with the template strand, resulting in a displaced non-template ssDNA. Importantly, accumulation of R-loops is associated with transcriptional inhibition and genomic instability, both hallmarks of aging. In Chapter 3, I characterized R-loop in maintaining proper transcriptional outputs and regulating visual function during aging. Bulk assays to measure R-loop levels revealed a significant increase in nuclear R-loops 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 Top3β, a DNA/RNA topoisomerase associated with R-loop resolution, lead to decreased visual function, over-expression of Top3β or nuclear-localized RNase H1, which resolves R-loops, enhanced positive light response during aging.
Together, these studies underscore the importance of understanding how age-related changes in genomic processes, such as circadian transcription and maintenance of R-loops, contribute to physiological changes observed during aging.
R-LOOPS AS A NOVEL DRIVER OF PHOTORECEPTOR AGING
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Epigenetic Regulation of Gene Expression in the Aging Eye
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