Genomic Profiling of Estrogen-Related Receptor Identifies Adult Adipocyte-Specific Targets in Drosophila

Problem Statement
Adipose tissue is a critical metabolic organ responsible for energy storage and lipid mobilization. The estrogen-related receptor (ERR) family of nuclear receptors is a known regulator of glycolytic and lipid metabolism across multiple species, including Drosophila. Previous studies established that whole-body loss of ERR in Drosophila leads to decreased transcription of glycolytic and pentose phosphate pathway (PPP) enzymes and reduced lipid accumulation. However, a critical gap remained: it was unclear whether these observed changes in mRNA expression were due to the direct regulation of these genes by ERR within specific cell types. Furthermore, the cell-type-specific targets of ERR in adult adipose tissue had not been fully elucidated, particularly given that the Drosophila fat body is a compound tissue containing both adipocytes and hepatocyte-like oenocytes, making bulk tissue analysis insufficient for identifying adipocyte-specific binding events.

Methodology
To address the challenge of identifying transcription factor targets in specific cell types without requiring large tissue dissections or specific antibodies, the authors employed a modified targeted DamID approach called NanoDam.

• Genetic System: The study utilized an endogenously C-terminally GFP-tagged ERR protein (ERR::eGFSTF) crossed with a UAS-NanoDam transgene. The NanoDam construct consists of a Dam methyltransferase fused to a GFP nanobody.
• Cell-Type Specificity: Expression of NanoDam was restricted to adult adipocytes using the 3.1Lsp2-Gal4 driver, controlled by a temperature-sensitive tubGal80ts system. Flies were raised at 18°C (permissive for Gal80ts, preventing methylation) and shifted to 29°C for 14 hours in adulthood to induce NanoDam expression specifically in adipocytes.
• Data Generation: Adenine methylation occurs only where the NanoDam and the endogenous ERR overlap. Genomic DNA was extracted, methylated DNA was enriched, and libraries were sequenced on an Illumina NextSeq2000 platform.
• Analysis: Reads were mapped to the Drosophila genome (r6.42). Differentially abundant DpnI fragments (indicating ERR binding) were identified using DESeq2, with significance defined as an adjusted P-value < 0.05 and a log2 fold change ≥ 2. Gene ontology and pathway enrichment were performed using PANGEA.

Key Contributions and Results
The study successfully mapped ERR occupancy specifically in adult male and female adipocytes, identifying over 900 binding sites in both sexes.

• Global Binding Profile: ERR-NanoDam identified over 950 genomic regions (representing >1,000 unique genes) in females and 1,580 regions (1,480 unique genes) in males. Approximately 925 genes were common to both sexes.
• Pathway Enrichment: Common binding sites were significantly enriched for metabolic pathways, specifically carbohydrate metabolism, glycogen metabolism, glycolysis, trehalose biosynthesis, and the pentose phosphate pathway (PPP).
• Glycolysis: ERR was found to bind loci encoding seven of 13 glycolytic enzymes in both sexes. While some sex-specific differences were noted (males showed higher binding for Pgi, Gapdh1, and Gapdh2), the core set of glycolytic targets was conserved. These binding events largely overlapped with genes downregulated in previous whole-body ERR knockout studies.
• Pentose Phosphate Pathway (PPP): ERR bound to four of eight PPP enzymes (Pgls, Rpe, Tkt, Taldo) in both sexes. Notably, Zw (encoding G6PD), which is downregulated in ERR mutants, was not bound by ERR in adipocytes, suggesting it may be regulated indirectly or in other tissues.
• Gluconeogenesis: ERR bound to three of four gluconeogenesis enzyme genes and the trehalose transporter gene (Tret1). However, it did not bind the Fbp locus.
• Beta-Oxidation: ERR bound to six of 11 genes involved in fatty acid beta-oxidation. Four of these (CG5321, CG3902, scully, yip2) were also downregulated in ERR mutant larval adipocytes.
• Sexual Dimorphism: While a core set of metabolic targets was shared, the study observed sex-specific enrichment. ERR binding in males was enriched for fatty acid and carbohydrate metabolism genes, whereas female binding showed enrichment for glycogen and carbohydrate metabolic genes.

Significance and Claims
The paper claims that ERR functions centrally in adipose tissue to regulate multiple metabolic pathways, including glycolysis, the PPP, and fatty acid oxidation. The primary significance of this work lies in demonstrating that cell-type-specific genomic profiling (NanoDam) can identify direct transcriptional targets that are obscured in bulk RNA-seq analyses of whole-body or mixed-tissue samples. The results predict that ERR activity is required specifically in the adipocytes of both sexes to maintain energy homeostasis. The authors conclude that ERR targets are likely sexually dimorphic and can vary by developmental stage, reinforcing the conserved role of ERR in regulating energy metabolism across species while highlighting the necessity of tissue-specific analysis to fully understand transcriptional regulation.