Biology subjects

Policastro, R. A.

Publications and source records attributed to Policastro, R. A..

8 recordsLinked to original sources

bioRxiv2024

The Drosophila Estrogen-Related Receptor promotes triglyceride storage within the larval fat body

The Estrogen-Related Receptor (ERR) family of nuclear receptors (NRs) serve key roles in coordinating triglyceride (TAG) accumulation with juvenile growth and development. In both insects and mammals, ERR activity promotes TAG storage during the post-embryonic growth phase, with loss-of-function mutations in mouse Esrra and Drosophila melanogaster dERR inducing a lean phenotype. However, the role of insect ERRs in controlling TAG accumulation within adipose tissue remains poorly understood, as previous transcriptomic and metabolomic studies relied on whole animal analyses. Here we address this shortcoming by using tissue-specific approaches to examine the role of dERR in regulating lipid metabolism within the Drosophila larval fat body. We find that dERR autonomously promotes TAG accumulation within fat body cells and regulates expression of genes involved in glycolysis, {beta}-oxidation, and mevalonate metabolism. As an extension of these results, we not only discovered that dERR mutant fat bodies exhibit decreased expression of known dHNF4 target genes but also found that dHNF4 activity is decreased in dERR mutants. Overall, our findings indicate that dERR plays a multifaceted role in the larval fat body to coordinate lipid storage with developmental growth and hint at a conserved mechanism by which ERR and HNF4 homologs coordinately regulate metabolic gene expression.

developmental biology↗

bioRxiv2024

Glycolytic Disruption Triggers Interorgan Signaling to Nonautonomously Restrict Drosophila melanogaster Larval Growth

Drosophila larval growth requires efficient conversion of dietary nutrients into biomass. Lactate Dehydrogenase (Ldh) and Glycerol-3-phosphate dehydrogenase (Gpdh1) support larval biosynthetic metabolism by maintaining NAD+/NADH redox balance and promoting glycolytic flux. Consistent with the cooperative functions of Ldh and Gpdh1, the loss of both enzymes, but neither single enzyme, induces a developmental arrest. However, Ldh and Gpdh1 exhibit complex and often mutually exclusive expression patterns, suggesting that the Gpdh1; Ldh double mutant lethal phenotype could be mediated nonautonomously. Here we find that the developmental arrest displayed by the double mutants extends beyond simple metabolic disruption and instead stems, in part, from changes in systemic growth factor signaling. Specifically, we demonstrate that this synthetic lethality is linked to the upregulation of Upd3, a cytokine involved in the Jak/Stat signaling pathway. Moreover, we demonstrate that either loss of the Upd3 or dietary administration of the steroid hormone 20-hydroxyecdysone (20E) rescue the synthetic lethal phenotype of Gpdh1; Ldh double mutants. Together, these findings demonstrate that metabolic disruptions within a single tissue can nonautonomously modulate interorgan signaling to ensure synchronous developmental growth. SUMMARY STATEMENTWe used the fruit fly Drosophila melanogaster to demonstrate that disruption of glycolysis within a single larval tissue alters systemic cytokine signaling and nonautonomously inhibits development of the entire animal.

developmental biology↗

bioRxiv2024

Renal L-2-hydroxyglutarate dehydrogenase activity promotes hypoxia tolerance and mitochondrial metabolism in Drosophila melanogaster

The mitochondrial enzyme L-2-hydroxyglutarate dehydrogenase (L2HGDH) regulates the abundance of L-2-hydroxyglutarate (L-2HG), a potent signaling metabolite capable of influencing chromatin architecture, mitochondrial metabolism, and cell fate decisions. Loss of L2hgdh activity in humans induces ectopic L-2HG accumulation, resulting in neurodevelopmental defects, altered immune cell function, and enhanced growth of clear cell renal cell carcinomas. To better understand the molecular mechanisms that underlie these disease pathologies, we used the fruit fly Drosophila melanogaster to investigate the endogenous functions of L2hgdh. Our studies revealed that while L2hgdh is not essential for growth or viability under standard culture conditions, L2hgdh mutants are hypersensitive to hypoxia and expire during the reoxygenation phase with severe disruptions of mitochondrial metabolism. Moreover, we find that the fly renal system (Malpighian tubules; MTs) is a key site of L2hgdh activity, as L2hgdh mutants that express a rescuing transgene within the MTs survive hypoxia treatment and exhibit normal levels of mitochondrial metabolites. We also demonstrate that even under normoxic conditions, L2hgdh mutant MTs experience significant metabolic stress and are sensitized to aberrant growth upon Egfr activation. Overall, our findings present a model in which renal L2hgdh activity limits systemic L-2HG accumulation, thus indirectly regulating the balance between glycolytic and mitochondrial metabolism, enabling successful recovery from hypoxia exposure, and ensuring renal tissue integrity.

genetics↗

bioRxiv2021

Flexible analysis of TSS mapping data and detection of TSS shifts with TSRexploreR

Heterogeneity in transcription initiation has important consequences for transcript stability and translation, and shifts in transcription start site (TSS) usage are prevalent in various disease and developmental contexts. Accordingly, numerous methods for global TSS profiling have been developed, including our recently published Survey of TRanscription Initiation at Promoter Elements with high-throughput sequencing (STRIPE-seq), a method to profile transcription start sites (TSSs) on a genome-wide scale with minimal cost and time. In parallel to our development of STRIPE-seq, we built TSRexploreR, an R package for end-to-end analysis of TSS mapping data. TSRexploreR provides functions for TSS and TSR detection, normalization, correlation, visualization, and differential TSS/TSR analysis. TSRexploreR is highly interoperable, accepting the data structures of TSS and TSR sets generated by several existing tools for processing and alignment of TSS mapping data, such as CAGEr for Cap Analysis of Gene Expression (CAGE) data. Lastly, TSRexploreR implements a novel approach for the detection of shifts in TSS distribution.

bioinformatics↗

bioRxiv2021

ChEC-seq produces robust and specific maps of transcriptional regulators

We previously introduced chromatin endogenous cleavage and high-throughput sequencing (ChEC-seq), which uses fusion of a chromatin-binding protein of interest to micrococcal nuclease (MNase) and high-throughput sequencing to generate genome-wide maps of factor binding. Here, we respond to concerns that have been raised about the specificity of the method relative to its negative control when a single long calcium incubation time is used. Through reanalysis of our previously published datasets, we show that short-duration ChEC-seq experiments provide robust, specific maps of transcriptional regulators across the budding yeast genome. Our analyses also confirm that consideration of MNase digestion kinetics is important for proper design and interpretation of ChEC-seq experiments.

genomics↗

bioRxiv2020

LSD1 promotes secretory cell specification to drive BRAF mutant colorectal cancer

Despite the connection to distinct mucus-containing colorectal cancer (CRC) histological subtypes, the role of secretory cells, including goblet and enteroendocrine (EEC) cells, in CRC progression has been underexplored. Analysis of TCGA and single cell RNA sequencing data demonstrates that multiple secretory progenitor populations are enriched in BRAF-mutant CRC patient tumors and cell lines. Enrichment of EEC progenitors in BRAF-mutant CRC is maintained by DNA methylation and silencing of NEUROD1, a key gene required for differentiation of EECs. Mechanistically, secretory cells and the factors they secrete, such as Trefoil factor 3, are shown to promote colony formation and activation of cell survival pathways in the entire cell population. We further identify LSD1 as a critical regulator of secretory cell specification in vitro and in a colon orthotopic xenograft model, where LSD1 loss reduces tumor growth and metastasis. This work establishes EEC progenitors, in addition to goblet cells, as targetable populations in BRAF-mutant CRC and identifies LSD1 as a therapeutic target in secretory lineage-containing CRC.

cancer biology↗

bioRxiv2020

Simple and efficient measurement of transcription initiation and transcript levels with STRIPE-seq

Accurate mapping of transcription start sites (TSSs) is key for understanding transcriptional regulation. However, current protocols for genome-wide TSS profiling are laborious and/or expensive. We present Survey of TRanscription Initiation at Promoter Elements with high-throughput sequencing (STRIPE-seq), a simple, rapid, and cost-effective protocol for sequencing capped RNA 5 ends from as little as 50 ng total RNA. Including depletion of uncapped RNA and SPRI bead cleanups, a STRIPE-seq library can be constructed in about five hours. We demonstrate application of STRIPE-seq to TSS profiling in yeast and human cells and show that it can also be effectively used for measuring transcript levels and differential gene expression analysis. In conjunction with our ready-to-use computational analysis workflows, STRIPE-seq is a straightforward, efficient means by which to probe the landscape of transcriptional initiation.

genomics↗

bioRxiv2018

A Cyclin A - Myb-MuvB - Aurora B network regulates the choice between mitotic cycles and polyploid endoreplication cycles.

Cells switch to polyploid endoreplication cycles during development, wound healing, and cancer. We used integrated approaches in Drosophila to determine how mitotic cycles are remodeled into endoreplication cycles, and how similar this remodeling is between developmental and induced endoreplicating cells (devECs and iECs). We found that while only devECs had a dampened E2F1 transcriptome, repression of a Cyclin A - Myb-MuvB - Aurora B mitotic network promoted endoreplication in both devECs and iECs. Cyclin A associated with and activated Myb-MuvB to induce transcription of mitotic genes, with expression of one, Aurora B, being key for mitotic commitment. Knockdown of Cyclin A, Myb, Aurora B, or downstream cytokinetic proteins induced distinct types of endoreplication, suggesting that repression of different mitotic network steps may explain the known diversity of polyploid cycles. These findings reveal how remodeling of a mitotic network promotes polyploid cycles that contribute to development, wound healing, and cancer.

developmental biology↗