Supplemental Tables for "Hypoxia-inducible factor 1alpha is required to establish the larval glycolytic program in Drosophila melanogaster"

Objectives: The rapid growth that occurs during Drosophila larval development requires a dramatic rewiring of central carbon metabolism to support biosynthesis. Larvae achieve this metabolic state, in part, by coordinately up-regulating the expression of genes involved in carbohydrate metabolism. The resulting metabolic program exhibits hallmark characteristics of aerobic glycolysis and establishes a physiological state that supports growth. To date, the only factor known to activate the larval glycolytic program is the Drosophila Estrogen-Related Receptor (dERR). However, dERR is dynamically regulated during the onset of this metabolic switch, indicating that other factors must be involved. Here we examine the possibility the Drosophila ortholog of Hypoxia inducible factor 1a (Hif1α) is also required to activate the larval glycolytic program.Methods: CRISPR/Cas9 was used to generate new loss-of-function alleles in the Drosophila gene similar (sima), which encodes the sole fly ortholog of Hif1a. The resulting mutant strains were analyzed using a combination of metabolomics and RNAseq for defects in carbohydrate metabolism.Results: Our studies reveal that sima mutants fail to activate aerobic glycolysis and die during larval development with metabolic phenotypes that mimic those displayed by dERR mutants. Moreover, we demonstrate that dERR and Sima/Hif1α protein accumulation is mutually dependent, as loss of either transcription factor results in decreased abundance the other protein.Conclusions: These findings demonstrate that Sima/HIF1a is required during embryogenesis to coordinately up-regulate carbohydrate metabolism in preparation for larval growth. Notably, our study also reveals that the Sima-dependent gene expression profile shares considerable overlap with that observed in dERR mutant, suggesting that Sima/HIF1a and dERR cooperatively regulate embryonic and larval glycolytic gene expression.研究目的：果蝇幼虫发育过程中的快速生长，需要中枢碳代谢发生剧烈重编程以支撑生物合成。幼虫可通过协同上调碳水化合物代谢相关基因的表达，部分实现这一代谢状态的转变。由此产生的代谢程序具备有氧糖酵解的典型特征，并建立了支持生长的生理状态。目前已知唯一可激活幼虫糖酵解程序的因子是果蝇雌激素相关受体（Drosophila Estrogen-Related Receptor，dERR）。然而，dERR在该代谢转换启动阶段存在动态调控，提示必然存在其他调控因子。本研究旨在探讨果蝇缺氧诱导因子1α（Hypoxia inducible factor 1a，Hif1α）的同源基因是否同样参与激活幼虫糖酵解程序。 研究方法：本研究利用CRISPR/Cas9技术，在果蝇sima基因（编码Hif1α唯一的果蝇同源蛋白）中构建新型功能缺失等位基因。随后结合代谢组学与RNA测序（RNAseq）技术，对获得的突变株系的碳水化合物代谢缺陷开展分析。 研究结果：本研究发现，sima突变体无法激活有氧糖酵解，且在幼虫发育阶段死亡，其代谢表型与dERR突变体高度相似。此外，本研究证实dERR与Sima/Hif1α蛋白的积累存在相互依赖关系：任意一种转录因子的缺失，都会导致另一种蛋白的丰度降低。 研究结论：上述结果表明，胚胎发育阶段的Sima/HIF1α是协同上调碳水化合物代谢、为幼虫生长做好准备所必需的。值得注意的是，本研究还发现Sima依赖的基因表达谱与dERR突变体中的基因表达谱存在大量重叠，提示Sima/HIF1α与dERR可协同调控胚胎及幼虫阶段的糖酵解相关基因表达。