SWI/SNF chromatin remodelers are represented by three biochemically distinct subcomplexes, the abundant cBAF and the less abundant PBAF and GBAF. Genetics have identified important roles for PBAF in development and disease; however, relating PBAF-mediated phenotypes to biochemical function in chromatin regulation and gene activation has been challenging. Here, we show that the PBRM1 subunit of PBAF is critical for the completion of TGFβ1-mediated epithelial-mesenchymal transition (EMT) of mammary cells in vitro as well as the metastasis of murine breast cancers in vivo. Using epigenomics to profile different stages of EMT, we find that PBRM1 is necessary for targeting PBAF to inducible promoters marked by H3K14ac. We further find that PBRM1 facilitates DNA accessibility at sites bound by TGFβ1-inducible transcription factors, such as Atf3, for the induction of genes involved in migration, cell survival, and inflammation, providing evidence that PBAF is a vulnerability in late-stage metastatic cancers.

S-Adenosylhomocysteinase (AHCY, also known as SAHH) is a highly conserved enzyme that catalyzes the reversible hydrolysis of SAH into adenosine and homocysteine. As the sole enzyme capable of catalyzing this reaction, AHCY modulates cellular methylation potential required for DNA, RNA, and protein methyltransferase activity. Recent discoveries, however, expand its role well beyond this canonical function, positioning AHCY as a metabolic gatekeeper that integrates one-carbon metabolism with epigenetic regulation, RNA processing, nucleotide balance, and redox signaling. This review brings together mechanistic, structural, and regulatory insights into AHCY while critically evaluating diverse biochemical and biophysical methods for assaying its activity. Comparative structural analyses uncover conserved tetrameric organization alongside species-specific adaptations in oligomeric state, NAD+ pocket accessibility, and C-terminal dynamics that shape enzyme catalytic efficiency and regulation. AHCY function is further fine-tuned through a wide spectrum of posttranslational modifications and small-molecule interactions, linking it to transcriptional control, stress adaptation, and viral infection. By linking SAH turnover to methylation capacity and adenosine/homocysteine flux, AHCY coordinates metabolism with chromatin regulation and stress responses. These cross-cutting roles highlight how a single metabolic enzyme bridges catalysis, regulation, and disease. In doing so, AHCY exemplifies the broader principle that metabolic enzymes can have a central role as regulators of metabolic flux and cellular regulation, offering both mechanistic depth and translational promise as a therapeutic target.

Alterations in biological rhythms are a common feature of aging, and disruption of circadian rhythms can exacerbate age-associated pathologies. The retina is critical for detecting light for both vision and for transmitting time-of-day information to the brain, synchronizing rhythms throughout the body. Disruption of circadian rhythms by manipulating the molecular clock leads to premature retinal degeneration in flies and mice, and gene expression rhythms are disrupted in models of age-associated ocular disease. Despite this, it is unknown how or why the gene expression rhythms of the retina change with age. Here, we show that ∼70% of the Drosophila transcriptome is rhythmically expressed throughout the diurnal cycle, with ∼40% of genes showing altered rhythms with age. These transcriptome-wide changes in aging photoreceptors are accompanied by shifts in the rhythmic patterns of RNA Polymerase II (Pol II) occupancy, histone H3 lysine 4 (H3K4) methylation, and chromatin accessibility, without major changes in occupancy of the circadian clock transcription factors Clock (Clk) and Cycle (Cyc). Instead, aging decreases genome-wide levels of several different histone methyl marks including H3K4 methylation, whose relative levels across the day correlate with the phase of rhythmic gene expression. Moreover, individual knockdown of the three H3K4 methyltransferases in young photoreceptors results in massive disruptions to rhythmic gene expression that resemble those observed during aging. We conclude that there are broad epigenetic shifts in the aging retina, including decreased histone methylation, that contribute to changes in biological rhythms even in the presence of a robust molecular circadian clock.

Glycine-N-methyltransferase (Gnmt) is highly upregulated in the aging Drosophila melanogaster eye. Gnmt regulates S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) availability by catalyzing the transfer of a methyl group from SAM to glycine, producing sarcosine and SAH. Gnmt is well studied in the liver; however, little is known about the consequences of increased Gnmt in neurons. We overexpressed Gnmt in the eye and profiled diurnal rhythmic gene expression and histone methylation in photoreceptor neurons. Here, we show that eye-specific Gnmt overexpression altered rhythmic gene expression without impacting photoreceptor viability. Gnmt overexpression decreased global repressive histone methylation but had no major effect on H3K4 methylation, suggesting that methylation reactions are selectively inhibited by Gnmt and that Gnmt disrupts rhythmic gene expression independently of H3K4 methylation. Gnmt overexpression did not alter rhythmicity of core clock genes and did not impact circadian behavior. These results suggest that Gnmt plays a role in the regulation of light-dependent rhythmic gene expression in photoreceptors that does not involve the molecular clock.

One-carbon metabolism influences gene expression by providing methyl units for DNA, RNA, and histone methylation. Robust methylation requires rapid hydrolysis of the methylation by-product S- adenosylhomocysteine (SAH) by S- adenosylhomocysteinase (Ahcy). Here, we show Ahcy is a redox-sensitive enzyme that is inhibited by oxidation of a conserved cysteine, C195, in vitro and in vivo . Transient oxidation of Ahcy is neuroprotective in a Drosophila light stress model where it results in rapid gene expression changes and protects against retinal degeneration. Thus, redox sensing by the one-carbon metabolic enzyme Ahcy enables rapid changes in gene expression in response to changes in redox homeostasis.

SWI/SNF chromatin remodelers utilize ATP to mobilize nucleosomes on DNA and are represented by three biochemically distinct subcomplexes, the more abundant cBAF and the less abundant PBAF and GBAF subcomplexes. Patient mutations and genetic studies have identified important roles for PBAF subunits in development and disease; however, relating PBAF-mediated phenotypes to biochemical function in chromatin regulation and gene expression has been challenging. Further complicating matters, cell-based systems often do not reflect the phenotypes and genotypes observed with PBAF mutations in vivo . Here we show that the PBRM1 subunit of PBAF is critical for the completion of TGFB1-mediated epithelial-mesenchymal transition of mammary cells in vitro as well as the metastasis of murine breast cancers in vivo. Using epigenomics to profile different stages of EMT, we find that PBRM1 is necessary for targeting PBAF to inducible promoters marked by H3K14ac alone. We further find that PBRM1 facilitates DNA accessibility at sites bound by TGFβ1-inducible transcription factors, such as Atf3, for the induction of genes involved in migration, cell survival, and inflammation. Our model allows us to separate constitutive vs inducible gene expression to help explain some of the context-dependent phenotypes observed with PBRM1 deletion. In addition, we provide evidence that while PBRM1 deletions can promote the initiation of certain cancers in early stages, PBAF may be a vulnerability in late-stage metastatic cancers.

One-carbon metabolism influences gene expression by providing methyl units for DNA, RNA, and histone methylation. Robust methylation requires rapid hydrolysis of the methylation by-product S-adenosylhomocysteine (SAH) by S-adenosylhomocysteinase (Ahcy). Ahcy is essential for maintaining methylation potential; however, the mechanisms governing its enzymatic activity, particularly in response to cellular stress, remain largely uncharacterized. Here, we show Ahcy is a redox-sensitive enzyme that is inhibited by oxidation of a conserved cysteine, C195, in vitro resulting in elevated SAH levels upon oxidative stress in vivo. We leveraged High-Throughput Desorption Electrospray Ionization Mass Spectrometry to directly quantify Ahcy enzymatic activity and observed that H2O2-induced oxidation significantly reduced its catalytic efficiency. Notably, while C195 is essential for enzymatic activity in Drosophila melanogaster and humans, this residue is not conserved in Caenorhabditis elegans Ahcy that is also insensitive to H2O2. Structural analysis revealed that C195 is positioned near NAD+ in the active site, close to a second cysteine residue that is also lacking in C. elegans Ahcy. Ahcy oxidation is neuroprotective in a Drosophila light stress model that increases oxidative stress. Moreover, Ahcy knockdown suppresses light stress-induced gene expression changes in photoreceptors, although this response is uncoupled from changes in H3K4me3 and H3K27me3 levels, which were previously reported to alter in response to Ahcy knockdown in cultured cells. Thus, the one-carbon metabolism enzyme Ahcy senses changes in cellular redox homeostasis through a conserved cysteine residue that alters its activity, enabling rapid changes in gene expression that enable a neuroprotective response.

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.

The retina is one of the highest oxygen-consuming tissues because visual transduction and light signaling pro-cesses require large amounts of ATP. Thus, because of the high energy demand, oxygen-rich environment, and tissue transparency, the eye is susceptible to excess production of reactive oxygen species (ROS) resulting in oxidative stress. Oxidative stress in the eye is associated with the development and progression of ocular diseases including cataracts, glaucoma, age-related macular degeneration, and diabetic retinopathy. ROS can modify and damage cellular proteins, but can also be involved in redox signaling. In particular, the thiol groups of cysteines can undergo reversible or irreversible oxidative post-translational modifications (PTMs). Identifying the redox -sensitive cysteines on a proteome-wide scale provides insight into those proteins that act as redox sensors or become irreversibly damaged upon exposure to oxidative stress. In this study, we profiled the redox proteome of the Drosophila eye under prolonged, high intensity blue light exposure and age using iodoacetamide isobaric label sixplex reagents (iodo-TMT) to identify changes in cysteine availability. Although redox metabolite analysis of the major antioxidant, glutathione, revealed similar ratios of its oxidized and reduced form in aged or light -stressed eyes, we observed different changes in the redox proteome under these conditions. Both conditions resulted in significant oxidation of proteins involved in phototransduction and photoreceptor maintenance but affected distinct targets and cysteine residues. Moreover, redox changes induced by blue light exposure were accompanied by a large reduction in light sensitivity that did not arise from a reduction in the photopigment level, suggesting that the redox-sensitive cysteines we identified in the phototransduction machinery might contribute to light adaptation. Our data provide a comprehensive description of the redox proteome of Drosophila eye tissue under light stress and aging and suggest how redox signaling might contribute to light adaptation in response to acute light stress.

Chromatin regulation plays an essential role in many nuclear processes and genome-wide chromatin profiling approaches contribute to understanding how chromatin regulates cell homeostasis. Chromatin dysregulation lies at the heart of many human diseases, most of which have a tissue-specific nature. Because of the physiological similarity of Drosophila and humans, tissue-specific studies can be performed using fruit flies. Here, we present an improved nuclear tagging approach that allows for efficient purification of cell-type specific nuclei from Drosophila increasing yield and stringency. Using this protocol, we purified photoreceptor neuron nuclei and demonstrate the feasibility and high quality of chromatin accessibility profiling (using Omni-ATAC) as well as profiling of histones and histone modifications using ChIP-seq, CUT&RUN, and CUT&Tag.

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.

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 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, our studies highlight the functional link between dysregulation of R-loop homeostasis, gene expression, and visual function during aging.

Age-related eye disease is a prevalent problem that is exacerbated by the rising, aging population. Drosophila melanogaster serves as a powerful model organism for studying age-related eye disease, as they share 60% of their genetic material with humans. Here, we seek to understand how changes in gene expression regulate photoreceptor neuron survival during aging. A preliminary RNA interference screen was carried out to identify genes necessary for cell survival throughout aging. Microscopy imaging and reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) were used to further investigate the role of histone methyltransferases Set1 and Set2 and the transcription factor Clock in photoreceptor neuron survival. Set1 and Set2 add methyl groups to lysine residues of histone H3, while Clock forms a heterodimer with the transcription factor cycle to activate transcription of genes involved in the circadian rhythm. Microscopy imaging was used to qualitatively and quantitatively assess loss of rhabdomeres, compartments of photoreceptor cells responsible for phototransduction, in flies lacking expression of either Set1, Set2, or a functional Clock-cycle complex. Flies expressing RNA interference against Set1 and Set2 show retinal degeneration at or before the age of 30 days. Flies that fail to form a functional Clock-cycle complex due to expression of a dominant negative form of Clock (ClkDN) show progressive retinal degeneration starting at the age of 5 days. Interestingly, this retinal degeneration is rescued when flies are raised in the dark. These phenotypes were validated by measuring target gene expression using RT-qPCR. RNA interference against mCherry and overexpression of LacZ were used as controls for Set1 and Set2 RNA interference and overexpression of ClkDN, respectively. These findings suggest that histone methylation and Clock-dependent gene expression play a role in age-related eye disease in both Drosophila and humans.

ABSTRACT The histone acetyltransferase Gcn5 is critical for gene expression and development. In Drosophila, Gcn5 is part of four complexes (SAGA, ATAC, CHAT and ADA) that are essential for fly viability and have key roles in regulating gene expression. Here, we show that although the SAGA, ADA and CHAT complexes play redundant roles in embryonic gene expression, the insect-specific CHAT complex uniquely regulates expression of a subset of developmental genes. We also identify a substantial decrease in histone acetylation in chiffon mutant embryos that exceeds that observed in Ada2b, suggesting broader roles for Chiffon in regulating histone acetylation outside of the Gcn5 complexes. The chiffon gene encodes two independent polypeptides that nucleate formation of either the CHAT or Dbf4-dependent kinase (DDK) complexes. DDK includes the cell cycle kinase Cdc7, which is necessary for maternally driven DNA replication in the embryo. We identify a temporal switch between the expression of these chiffon gene products during a short window during the early nuclear cycles in embryos that correlates with the onset of zygotic genome activation, suggesting a potential role for CHAT in this process. This article has an associated First Person interview with the first author of the paper.

Chiffon is the sole Drosophila ortholog of Dbf4, the regulatory subunit for the cell-cycle kinase Cdc7 that initiates DNA replication. In Drosophila, the chiffon gene encodes two polypeptides with independent activities. Chiffon-A contains the conserved Dbf4 motifs and interacts with Cdc7 to form the Dbf4-dependent Kinase (DDK) complex, which is essential for a specialized form of DNA replication. In contrast, Chiffon-B binds the histone acetyltransferase Gcn5 to form the Chiffon histone acetyltransferase (CHAT) complex, which is necessary for histone H3 acetylation and viability. Previous studies have shown that the Chiffon-B region is only present within insects. However, it was unclear how widely the interaction between Chiffon-B and Gcn5 was conserved among insect species. To examine this, we performed yeast two-hybrid assays using Chiffon-B and Gcn5 from a variety of insect species and found that Chiffon-B and Gcn5 interact in Diptera species such as Australian sheep blowfly and yellow fever mosquito. Protein domain analysis identified that Chiffon-B has features of acidic transcriptional activators such as Gal4 or VP16. We propose that the CHAT complex plays a critical role in a biological process that is unique to Dipterans and could therefore be a potential target for pest control strategies.

Advanced age is one of the leading risk factors for vision loss and eye disease. Photoreceptors are the primary sensory neurons of the eye. The extended photoreceptor cell lifespan, in addition to its high metabolic needs due to phototransduction, makes it critical for these neurons to continually respond to the stresses associated with aging by mounting an appropriate gene expression response. Here, we sought to untangle the more general neuronal age-dependent transcriptional signature of photoreceptors with that induced by light stress. To do this, we aged flies or exposed them to various durations of blue light, followed by photoreceptor nuclei-specific transcriptome profiling. Using this approach, we identified genes that are both common and uniquely regulated by aging and light induced stress. Whereas both age and blue light induce expression of DNA repair genes and a neuronal-specific signature of death, both conditions result in downregulation of phototransduction. Interestingly, blue light uniquely induced genes that directly counteract the overactivation of the phototransduction signaling cascade. Lastly, unique gene expression changes in aging photoreceptors included the downregulation of genes involved in membrane potential homeostasis and mitochondrial function, as well as the upregulation of immune response genes. We propose that light stress contributes to the aging transcriptome of photoreceptors, but that there are also other environmental or intrinsic factors involved in age-associated photoreceptor gene expression signatures.

Chromatin regulation plays an essential role in many nuclear processes, and genome-wide chromatin profiling approaches contribute to understanding how chromatin regulates cell homeostasis. Chromatin dysregulation lies in the heart of many human diseases, which most of them have a tissue-specific nature. Because of the physiological similarity of Drosophila and humans, tissue-specific studies can be performed using fruit flies. Here, we present an improved nuclear tagging approach that allows for efficient purification of cell-type specific nuclei from Drosophila increasing yield and stringency. Using this protocol, we purified photoreceptor neuron nuclei, and demonstrate the feasibility and high quality of chromatin accessibility profiling as well as profiling of histones and histone modifications, using Omni-ATAC and ChIP-seq, respectively. Last, we describe a modification to the nuclei purification protocol that allows for application of recently developed CUT&Tag and demonstrate that CUT&Tag outperforms traditional ChIP-seq, although protocol might require further optimization.

Spt7 belongs to the suppressor of Ty (SPT) module of the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex and is known as the yeast ortholog of human STAF65γ. Spt7 lacks intrinsic enzymatic activity but is responsible for the integrity and proper assembly of the SAGA complex. Here, we determined the role of the SAGA Spt7 subunit in cellular aging. We found that Spt7 was indispensable for a normal lifespan in both dividing and nondividing yeast cells. In the quiescent state of cells, Spt7 was required for the control of overall mRNA levels. In mitotically active cells, deletion of the SPT module had little effect on the recombination rate within heterochromatic ribosomal DNA (rDNA) loci, but loss of Spt7 profoundly elevated the plasmid-based DNA recombination frequency. Consistently, loss of Spt7 increased spontaneous Rad52 foci by approximately two-fold upon entry into S phase. These results provide evidence that Spt7 contributes to the regulation of the normal replicative lifespan (RLS) and chronological lifespan (CLS), possibly by controlling the DNA recombination rate and overall mRNA expression. We propose that the regulation of SAGA complex integrity by Spt7 might be involved in the conserved regulatory pathway for lifespan regulation in eukaryotes.

The histone acetyltransferase Gcn5 is conserved throughout eukaryotes where it functions as part of large multi-subunit transcriptional coactivator complexes that stimulate gene expression. Here, we describe how studies in the model insect Drosophila melanogaster have provided insight into the essential roles played by Gcn5 in the development of multicellular organisms. We outline the composition and activity of the four different Gcn5 complexes in Drosophila: the Spt-Ada-Gcn5 Acetyltransferase (SAGA), Ada2a-containing (ATAC), Ada2/Gcn5/Ada3 transcription activator (ADA), and Chiffon Histone Acetyltransferase (CHAT) complexes. Whereas the SAGA and ADA complexes are also present in the yeast Saccharomyces cerevisiae, ATAC has only been identified in other metazoa such as humans, and the CHAT complex appears to be unique to insects. Each of these Gcn5 complexes is nucleated by unique Ada2 homologs or splice isoforms that share conserved N-terminal domains, and differ only in their C-terminal domains. We describe the common and specialized developmental functions of each Gcn5 complex based on phenotypic analysis of mutant flies. In addition, we outline how gene expression studies in mutant flies have shed light on the different biological roles of each complex. Together, these studies highlight the key role that Drosophila has played in understanding the expanded biological function of Gcn5 in multicellular eukaryotes.

25 Age-related loss of cellular function and increased cell death are characteristic hallmarks of 26 aging. While defects in gene expression and RNA metabolism have been linked with age27 associated human neuropathies, it is not clear how the changes that occur in aging neurons 28 contribute to loss of gene expression homeostasis. R-loops are RNA-DNA hybrids that typically 29 form co-transcriptionally via annealing of the nascent RNA to the template DNA strand, 30 displacing the non-template DNA strand. Dysregulation of R-loop homeostasis has been 31 associated with both transcriptional impairment and genome instability. Importantly, a growing 32 body of evidence links R-loop accumulation with cellular dysfunction, increased cell death and 33 chronic disease onset. Here, we characterized the R-loop landscape in aging Drosophila 34 melanogaster photoreceptor neurons and showed that bulk R-loop levels increased with age. 35 Further, genome-wide mapping of R-loops revealed that transcribed genes accumulated R36 loops over gene bodies during aging, which correlated with decreased expression of long and 37 highly expressed genes. Importantly, while photoreceptor-specific down-regulation of Top3β, a 38 DNA/RNA topoisomerase associated with R-loop resolution, lead to decreased visual function, 39 overexpression of Top3β or nuclear-localized RNase H1, which resolves R-loops, enhanced 40 positive light response during aging. Together, our studies highlight the functional link between 41 dysregulation of R-loop homeostasis, gene expression and visual function during aging. 42