<b>Rhythmic Transcription and Aging: Insights from the </b><b><i>Drosophila </i></b><b>Photoreceptor Transcriptome and Epigenome </b>
Creators: name: McGovern, Sarah E.
Publisher: name: Purdue University Graduate School
Year: 2024
Subjects: classificationCode: 310504
subject: Epigenetics (incl. genome methylation and epigenomics)
classificationCode: 310902
subject: Animal cell and molecular biology

description: Across diverse organisms and tissues, aging cells undergo extensive rewiring on an epigenetic and transcriptomic level, leading to widespread changes in rhythmic gene expression. Rhythmic gene expression is dictated by the circadian rhythm, which is synchronized to the external environment through the detection of light by the eye. The photosensitive tissue of the eye, the retina, exhibits considerable age-dependent transcriptomic and epigenetic alterations. During aging, the prevalence of ocular disease increases along with a decline in visual function overall, predisposing older individuals to circadian rhythm desynchronization, which is associated with a host of pathologies including neurodegenerative disease and cancer. Despite links between the health of the eye during aging and circadian rhythm dysfunction, a cohesive understanding of the molecular underpinnings that tie these together has not been reached.To first understand the transcriptional mechanisms that are necessary to maintain photoreceptor viability and function during aging, we performed a targeted photoreceptor-specific RNAi screen in <i>Drosophila </i>to identify transcriptional regulators necessary for protection against premature, age-dependent retinal degeneration. Using RNAi lines targeting transcription factors, chromatin remodelers, and histone modifiers, we identified 18 targets necessary for protection against premature and progressive retinal degeneration. These targets were enriched for factors involved in the regulation of RNA polymerase II (Pol II) initiation, pausing, and elongation, suggesting regulation of the transcription cycle is critical for photoreceptor health during aging. Transcriptome profiling of photoreceptors from select RNAi lines revealed that knockdown of the pausing factor <i>Spt5 </i>or the chromatin remodeler <i>domino </i>resulted in similar transcriptome-wide changes to those observed in aged photoreceptors. Together, these data showed that transcriptional regulators are key in maintaining photoreceptor viability during aging, and age-dependent changes in gene expression not only correlate with but may also contribute to an increased risk of retinal degeneration. This research is presented in Chapter 2: “Targeted RNAi screen identifies transcriptional mechanisms that prevent premature degeneration of adult photoreceptors”.To determine how the chromatin landscape dictates changes in the photoreceptor transcriptome with aging, we profiled chromatin marks associated with active transcription in young and old <i>Drosophila </i>photoreceptors using ChIP-seq. Both H3K4me3 and H3K36me3 decrease globally across actively-expressed genes during aging independent of differential gene expression. Knockdown of the H3K36me3 methyltransferase Set2 in young photoreceptors led to substantial changes in splicing events similar to those observed in aging photoreceptors, impacting genes involved in phototransduction and neuronal function. Because proper splicing is essential for visual behavior, and <i>Drosophila </i>visual function decreases with age, H3K36me3 may play a role in maintaining visual function in the eye through the regulation of alternative splicing. This research is presented in Chapter 3: “Establishing the contribution of active histone methylation marks to the aging transcriptional landscape of Drosophila photoreceptors”.Since the molecular circadian clock is necessary for light-dependent photoreceptor survival in <i>Drosophila</i>, we sought to characterize the rhythmic gene expression changes during aging by performing nuclear RNA-seq on young and old <i>Drosophila </i>photoreceptors across the circadian day. RNA-seq revealed that over 50% of the photoreceptor transcriptome is rhythmic, and one-third of expressed genes showed altered rhythmicity with age. CUT&amp;RUN of the core molecular clock transcriptional activators CLOCK and CYCLE in young and old photoreceptors identified relatively few target genes, and that CLOCK and CYCLE occupancy on chromatin does not substantially change with age, suggesting other epigenetic factors underly the drastic shifts in the photoreceptor rhythmic transcriptome during aging. Profiling of H3K4me2/3, H3K36me3, H3K9me3, and H3K27me3 at a single time-point using CUT&amp;RUN showed distinct patterns of distribution across genes in young and old photoreceptors, in addition to a genome-wide loss in levels of all marks. We performed ATAC-seq and CUT&amp;RUN of Pol II, H3K4me1, H3K4me2, and H3K4me3 across the circadian day in young and old photoreceptors to observe their daily patterns. Chromatin accessibility and Pol II occupancy oscillate throughout the day at all expressed genes, and the phase of this oscillation shifts in old photoreceptors. This oscillating pattern occurred at all expressed genes regardless of the timing of rhythmic gene expression. H3K4me1, me2, and me3 showed different oscillating patterns at all expressed genes that were nearly abolished in old photoreceptors. Though the overall oscillating patterns of H3K4 methylation also did not correlate with the timing of rhythmic gene expression, levels of H3K4 methylation do correlate with when the gene is most highly expressed. CUT&amp;RUN of H3K4 methylation in young photoreceptors with a knockdown of either of the H3K4 methyltransferases Trr or Trx determined that they have overlapping yet non-redundant roles in maintaining H3K4 methylation genome-wide. Transcriptome profiling of young photoreceptors with Trr or Trx knockdown showed that they are necessary for the majority of rhythmic gene expression in young photoreceptors. Additionally, Trr or Trx knockdown led to loss and modulation of rhythmic gene expression similar to the changes observed in aging photoreceptors. Together, these data suggest that the genome-wide decreases in histone methylation in aging photoreceptors are the driving force behind shaping the rhythmic transcriptome. This research is presented in Chapter 4: “Histone methylation loss underlies the rewiring of the rhythmic transcriptome in aging photoreceptors”.