The function of Retinoblastoma tumor suppressor (pRB) is greatly influenced by the cellular context, therefore the consequences of pRB inactivation are cell-type-specific. Here we employ single cell RNA-sequencing (scRNA-seq) to profile the impact of an Rbf mutation during Drosophila eye development. First, we build a catalogue of 11,500 wild type eye disc cells containing major known cell types. We find a transcriptional switch occurring in differentiating photoreceptors at the time of axonogenesis. Next, we map a cell landscape of Rbf mutant and identify a mutant-specific cell population that shows intracellular acidification due to increase in glycolytic activity. Genetic experiments demonstrate that such metabolic changes, restricted to this unique Rbf mutant population, sensitize cells to apoptosis and define the pattern of cell death in Rbf mutant eye disc. Thus, these results illustrate how scRNA-seq can be applied to dissect mutant phenotypes.

The transcription factor Forkhead box M1 (FoxM1) is overexpressed in breast cancers and correlates with poor prognosis. Mechanistically, FoxM1 associates with CBP to activate transcription and with Rb to repress transcription. Although the activating function of FoxM1 in breast cancer has been well documented, the significance of its repressive activity is poorly understood. Using CRISPR–Cas9 engineering, we generated a mouse model that expresses FoxM1-harboring point mutations that block binding to Rb while retaining its ability to bind CBP. Unlike FoxM1-null mice, mice harboring Rb-binding mutant FoxM1 did not exhibit significant developmental defects. The mutant mouse line developed PyMT-driven mammary tumors that were deficient in lung metastasis, which was tumor cell-intrinsic. Single-cell RNA-seq of the tumors revealed a deficiency in prometastatic tumor cells and an expansion of differentiated alveolar type tumor cells, and further investigation identified that loss of the FoxM1/Rb interaction caused enhancement of the mammary alveolar differentiation program. The FoxM1 mutant tumors also showed increased Pten expression, and FoxM1/Rb was found to activate Akt signaling by repressing Pten. In human breast cancers, expression of FoxM1 negatively correlated with Pten mRNA. Furthermore, the lack of tumor-infiltrating cells in FoxM1 mutant tumors appeared related to decreases in pro-metastatic tumor cells that express factors required for infiltration. These observations demonstrate that the FoxM1/Rb-regulated transcriptome is critical for the plasticity of breast cancer cells that drive metastasis, identifying a prometastatic role of Rb when bound to FoxM1. Significance: This work provides new insights into how the interaction between FoxM1 and Rb facilitates the evolution of metastatic breast cancer cells by altering the transcriptome.

The E2F transcription factors play a critical role in controlling cell fate. In Drosophila, the inactivation of E2F in either muscle or fat body results in lethality, suggesting an essential function for E2F in these tissues. However, the cellular and organismal consequences of inactivating E2F in these tissues are not fully understood. Here, we show that the E2F loss exerts both tissue-intrinsic and systemic effects. The proteomic profiling of E2F-deficient muscle and fat body revealed that E2F regulates carbohydrate metabolism, a conclusion further supported by metabolomic profiling. Intriguingly, animals with E2F-deficient fat body had a lower level of circulating trehalose and reduced storage of fat. Strikingly, a sugar supplement was sufficient to restore both trehalose and fat levels, and subsequently rescued animal lethality. Collectively, our data highlight the unexpected complexity of E2F mutant phenotype, which is a result of combining both tissue-specific and systemic changes that contribute to animal development.

Lipid metabolism and the serine, one-carbon, glycine (SOG) and methionine pathways are independently and significantly correlated with estrogen receptor–negative breast cancer (ERneg BC). Here, we propose a link between lipid metabolism and ERneg BC through phosphoglycerate dehydrogenase (PHGDH), the rate-limiting enzyme in the de novo serine pathway. We demonstrate that the metabolism of the paradigmatic medium-chain fatty acid octanoic acid leads to a metabolic shift toward the SOG and methionine pathways. PHGDH plays a role in both the forward direction, contributing to the production of S-adenosylmethionine, and the reverse direction, generating the oncometabolite 2-hydroxyglutarate, leading to epigenomic reprogramming and phenotypic plasticity. The methionine cycle is closely linked to the transsulfuration pathway. Consequently, we observe that the shift increases the antioxidant glutathione, which mitigates reactive oxygen species (ROS), enabling survival of a subset of cells that have undergone DNA damage. These metabolic changes contribute to several hallmarks of cancer.

The canonical role of the transcription factor E2F is to control the expression of cell cycle genes by binding to the E2F sites in their promoters. However, the list of putative E2F target genes is extensive and includes many metabolic genes, yet the significance of E2F in controlling the expression of these genes remains largely unknown. Here, we used the CRISPR/Cas9 technology to introduce point mutations in the E2F sites upstream of five endogenous metabolic genes in Drosophila melanogaster . We found that the impact of these mutations on both the recruitment of E2F and the expression of the target genes varied, with the glycolytic gene, Phosphoglycerate kinase ( Pgk) , being mostly affected. The loss of E2F regulation on the Pgk gene led to a decrease in glycolytic flux, tricarboxylic acid cycle intermediates levels, adenosine triphosphate (ATP) content, and an abnormal mitochondrial morphology. Remarkably, chromatin accessibility was significantly reduced at multiple genomic regions in Pgk ^ΔE2F mutants. These regions contained hundreds of genes, including metabolic genes that were downregulated in Pgk ^ΔE2F mutants. Moreover, Pgk ^ΔE2F animals had shortened life span and exhibited defects in high-energy consuming organs, such as ovaries and muscles. Collectively, our results illustrate how the pleiotropic effects on metabolism, gene expression, and development in the Pgk ^ΔE2F animals underscore the importance of E2F regulation on a single E2F target, Pgk .

Robust genetic systems to control the expression of transgenes in a spatial and temporal manner are a valuable asset for researchers. The GeneSwitch system induced by the drug RU486 has gained widespread use in the Drosophila community. However, some concerns were raised as negative effects were seen depending on the stock, transgene, stage, and tissue under study. Here, we characterized the adverse effects triggered by activating the GeneSwitch system in adult muscles using the MHC-GS-GAL4 driver. When a control, mock UAS-RNAi transgene was induced by feeding adult flies with RU486, we found that the overall muscle structure, including myofibrils and mitochondrial shape, was significantly disrupted and led to a significant reduction in the lifespan. Remarkably, lifespan was even shorter when 2 copies of the driver were used even without the mock UAS-RNAi transgene. Thus, researchers should be cautious when interpreting the results given the adverse effects we found when inducing RU486-dependent MHC-GS-GAL4 in adult muscles. To account for the impact of these effects we recommend adjusting the dose of RU486, setting up additional control groups, such as a mock UAS-RNAi transgene, as comparing the phenotypes between RU486-treated and untreated animals could be insufficient.