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.

Fluorescence and Excited-State Conformational Dynamics of the Orange Carotenoid Protein.

The orange carotenoid protein (OCP) mediates nonphotochemical quenching (NPQ) mechanisms in cyanobacteria. A bound ketocarotenoid serves as a sensor of midvisible light intensity and as a quencher of phycocyanobilin excitons in the phycobilisome. The photochemical mechanism that triggers conversion of the protein from a resting, orange state (OCPO) to an active, red state (OCPR) after optical preparation of the S2 state of the carotenoid remains an open question. We report here that the fluorescence spectrum and quantum yield of the bound carotenoids in OCPO report important details of the motions that follow optical preparation of the S2 state. The fluorescence spectra from OCPO preparations containing 3'-hydroxyechinenone (3hECN) or canthaxanthin (CAN) are markedly mirror asymmetric with respect to the absorption line shape and more than an order of magnitude more intense than for carotenoids in solution. Further, 3hECN exhibits a narrower fluorescence line shape and a larger quantum yield than CAN because its excited-state motions are hindered by a hydrogen bonding interaction between the 3'-hydroxyl group on its ß2 ring and Leu37 in the N-terminal domain. These results show that large-amplitude motions of the carotenoid's ß2-cyclohexene ring and of the conjugated polyene backbone initiate photochemistry in OCPO.