Systems-level design principles of metabolic rewiring in an animal

The regulation of metabolism is vital to any organism and can be achieved by transcriptionally activating or repressing metabolic genes. Although many examples of transcriptional metabolic rewiring have been reported, a systems-level study of how metabolism is rewired in response to metabolic perturbations is lacking in any animal. Here we apply Worm Perturb-Seq (WPS)-a high-throughput method combining whole-animal RNA-interference and RNA-sequencing to around 900 metabolic genes in the nematode Caenorhabditis elegans. We derive a metabolic gene regulatory network (mGRN) in which 385 perturbations are connected to 9,414 genes by more than 110,000 interactions. The mGRN has a highly modular structure in which 22 perturbation clusters connect to 44 gene expression programs. The mGRN reveals different modes of transcriptional rewiring from simple reaction and pathway compensation to rerouting and more complex network coordination. Using metabolic network modelling, we identify a design principle of transcriptional rewiring that we name the compensation-repression (CR) model. The CR model explains most transcriptional responses in metabolic genes and reveals a high level of compensation and repression in five core metabolic functions related to energy and biomass. We provide preliminary evidence that the CR model may also explain transcriptional metabolic rewiring in human cells. In this study, we applied Worm Perturb-seq (WPS), a massively parallel RNAi and RNA-seq technology, to perturb around 900 metabolic genes in nematode C. elegans, followed by a RNA-seq readout. The technology generates multiplexed RNA-seq libraries, each of which contains the triplicate samples of around 16 single gene perturbations and six empty vector controls (total ~50 samples per library). The RNA-seq library construction follows an adapted CEL-seq2 protocol, giving a multiplexed bulk RNA-seq library for 3'-end sequencing of C. elegans genes. The read 1 is used to encode barcode information and read 2 maps to the 3' end region of C. elegans transcripts. Barcode information can be found from the Supplementary Table "barcode_and_metadata_full_dataset.csv". These libraries can be directly processed by the WPS data processing pipeline (https://github.com/XuhangLi/WPS) providing the barcode information attached. A custom processing of the data is feasible following the typical demultiplexing methods for single-cell RNA-seq data that are able to process CEL-seq2 library. Please refer to our manuscript for decription of the design of read 1.