Title: The transcription factor Cabut coordinates energy metabolism and the circadian clock in response to sugar sensing
Abstract: Article27 April 2015free access The transcription factor Cabut coordinates energy metabolism and the circadian clock in response to sugar sensing Osnat Bartok Osnat Bartok Biological Chemistry Department, Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Mari Teesalu Mari Teesalu Department of Biosciences, University of Helsinki, Helsinki, Finland Institute of Biotechnology, University of Helsinki, Helsinki, Finland Search for more papers by this author Reut Ashwall-Fluss Reut Ashwall-Fluss Biological Chemistry Department, Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Varun Pandey Varun Pandey Biological Chemistry Department, Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Mor Hanan Mor Hanan Biological Chemistry Department, Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Bohdana M Rovenko Bohdana M Rovenko Department of Biosciences, University of Helsinki, Helsinki, Finland Institute of Biotechnology, University of Helsinki, Helsinki, Finland Search for more papers by this author Minna Poukkula Minna Poukkula Institute of Biotechnology, University of Helsinki, Helsinki, Finland Search for more papers by this author Essi Havula Essi Havula Department of Biosciences, University of Helsinki, Helsinki, Finland Institute of Biotechnology, University of Helsinki, Helsinki, Finland Search for more papers by this author Arieh Moussaieff Arieh Moussaieff Department of Cell Biology, Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel Center for Bioengineering, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Sadanand Vodala Sadanand Vodala Howard Hughes Medical Institute, Brandeis University, Waltham, MA, USA Search for more papers by this author Yaakov Nahmias Yaakov Nahmias Department of Cell Biology, Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel Center for Bioengineering, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Sebastian Kadener Corresponding Author Sebastian Kadener Biological Chemistry Department, Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Ville Hietakangas Corresponding Author Ville Hietakangas Department of Biosciences, University of Helsinki, Helsinki, Finland Institute of Biotechnology, University of Helsinki, Helsinki, Finland Search for more papers by this author Osnat Bartok Osnat Bartok Biological Chemistry Department, Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Mari Teesalu Mari Teesalu Department of Biosciences, University of Helsinki, Helsinki, Finland Institute of Biotechnology, University of Helsinki, Helsinki, Finland Search for more papers by this author Reut Ashwall-Fluss Reut Ashwall-Fluss Biological Chemistry Department, Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Varun Pandey Varun Pandey Biological Chemistry Department, Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Mor Hanan Mor Hanan Biological Chemistry Department, Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Bohdana M Rovenko Bohdana M Rovenko Department of Biosciences, University of Helsinki, Helsinki, Finland Institute of Biotechnology, University of Helsinki, Helsinki, Finland Search for more papers by this author Minna Poukkula Minna Poukkula Institute of Biotechnology, University of Helsinki, Helsinki, Finland Search for more papers by this author Essi Havula Essi Havula Department of Biosciences, University of Helsinki, Helsinki, Finland Institute of Biotechnology, University of Helsinki, Helsinki, Finland Search for more papers by this author Arieh Moussaieff Arieh Moussaieff Department of Cell Biology, Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel Center for Bioengineering, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Sadanand Vodala Sadanand Vodala Howard Hughes Medical Institute, Brandeis University, Waltham, MA, USA Search for more papers by this author Yaakov Nahmias Yaakov Nahmias Department of Cell Biology, Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel Center for Bioengineering, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Sebastian Kadener Corresponding Author Sebastian Kadener Biological Chemistry Department, Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Ville Hietakangas Corresponding Author Ville Hietakangas Department of Biosciences, University of Helsinki, Helsinki, Finland Institute of Biotechnology, University of Helsinki, Helsinki, Finland Search for more papers by this author Author Information Osnat Bartok1,‡, Mari Teesalu2,3,‡, Reut Ashwall-Fluss1, Varun Pandey1, Mor Hanan1, Bohdana M Rovenko2,3, Minna Poukkula3, Essi Havula2,3, Arieh Moussaieff4,5, Sadanand Vodala6, Yaakov Nahmias4,5, Sebastian Kadener 1 and Ville Hietakangas 2,3 1Biological Chemistry Department, Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel 2Department of Biosciences, University of Helsinki, Helsinki, Finland 3Institute of Biotechnology, University of Helsinki, Helsinki, Finland 4Department of Cell Biology, Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel 5Center for Bioengineering, The Hebrew University of Jerusalem, Jerusalem, Israel 6Howard Hughes Medical Institute, Brandeis University, Waltham, MA, USA ‡These authors contributed equally to this work *Corresponding author. Tel.: +358 2 94158001; E-mail: [email protected] *Corresponding author. Tel.: +972 2 6585099; E-mail: [email protected] The EMBO Journal (2015)34:1538-1553https://doi.org/10.15252/embj.201591385 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Nutrient sensing pathways adjust metabolism and physiological functions in response to food intake. For example, sugar feeding promotes lipogenesis by activating glycolytic and lipogenic genes through the Mondo/ChREBP-Mlx transcription factor complex. Concomitantly, other metabolic routes are inhibited, but the mechanisms of transcriptional repression upon sugar sensing have remained elusive. Here, we characterize cabut (cbt), a transcription factor responsible for the repressive branch of the sugar sensing transcriptional network in Drosophila. We demonstrate that cbt is rapidly induced upon sugar feeding through direct regulation by Mondo-Mlx. We found that CBT represses several metabolic targets in response to sugar feeding, including both isoforms of phosphoenolpyruvate carboxykinase (pepck). Deregulation of pepck1 (CG17725) in mlx mutants underlies imbalance of glycerol and glucose metabolism as well as developmental lethality. Furthermore, we demonstrate that cbt provides a regulatory link between nutrient sensing and the circadian clock. Specifically, we show that a subset of genes regulated by the circadian clock are also targets of CBT. Moreover, perturbation of CBT levels leads to deregulation of the circadian transcriptome and circadian behavioral patterns. Synopsis Sugar feeding in flies induces specific gene expression but also triggers a repressive branch via transcription factor Cabut. Induction of Cabut alters accumulation of the metabolic enzyme PEPCK and provides a regulatory link between nutrient sensing and the circadian clock. Transcriptional regulator Cabut is directly activated by Mondo-Mlx upon sugar feeding. Cabut represses metabolic genes upon sugar feeding. Cabut represses the expression of both isoforms of the phosphoenolpyruvate carboxykinase PEPCK. Deregulation of PEPCK1 contributes to the metabolic imbalance and lethality of mlx mutant animals. Cabut represses the cycling of metabolic target genes of the circadian clock. Introduction Animals respond to changes in food intake through nutrient sensing pathways that readjust metabolism to maintain homeostasis. Various signaling pathways and transcriptional responses are crucial in the adaptation of an organism to changing dietary conditions (Hietakangas & Cohen, 2009; Havula & Hietakangas, 2012). For example, the paralogous basic helix-loop-helix-leucine zipper (bHLHZ) transcription factors (TFs), carbohydrate-responsive element binding protein (ChREBP), and MondoA coordinate energy metabolism with dietary sugar intake (for review see Havula & Hietakangas, 2012; Filhoulaud et al, 2013). These TFs heterodimerize with their common binding partner Mlx to regulate gene expression in response to intracellular glucose-6-phosphate and other phosphorylated hexoses. Drosophila encodes single orthologs for ChREBP/MondoA and for Mlx, called Mondo (Mio, CG18362) and Mlx (Bigmax, CG3350), respectively. We have recently shown that loss of functional Mondo-Mlx in Drosophila leads to dramatic intolerance for dietary sugars and to impaired glucose and lipid metabolism (Havula et al, 2013). In humans, altered ChREBP activity in adipose tissue and liver is associated with severe obesity and polymorphisms of chrebp are linked to elevated circulating triglyceride levels (Kooner et al, 2008; Benhamed et al, 2012; Eissing et al, 2013). Excess dietary sugars are metabolized into lipids, and there is emerging evidence suggesting that high sugar intake, synergistically with genetic risk factors, contributes to obesity and metabolic syndrome (Qi et al, 2012; Stanhope et al, 2013). Sugar intake activates the ChREBP/Mondo-Mlx-dependent gene expression program to drive fatty acid biosynthesis. This is mediated by binding of ChREBP/Mondo-Mlx to the carbohydrate response elements (ChoREs) in the promoters of lipogenic genes including fatty acid synthase (Fas) and acetyl-CoA carboxylase (ACC) (Ishii et al, 2004). Flies lacking functional Mondo-Mlx display elevated circulating glucose, trehalose and reduced adiposity, but the metabolic effector genes underlying these phenotypes are incompletely understood (Sassu et al, 2012; Havula et al, 2013). In addition to fatty acids, biosynthesis of triglycerides requires glycerol-3-phosphate. Glycerol-3-phosphate is synthesized by three alternative pathways: glycolysis, phosphorylation of diet-derived glycerol by glycerol kinase, and from TCA cycle intermediates via the so-called glyceroneogenesis pathway (Ballard et al, 1967; Hanson & Reshef, 2003; Jin et al, 2013). A rate-limiting reaction in the glyceroneogenesis pathway is catalyzed by phosphoenol-pyruvate carboxykinase (PEPCK) (Hanson & Reshef, 2003). PEPCK controls the cataplerotic flux, which converts the oxaloacetate (OAA) from the TCA cycle into phosphoenolpyruvate (PEP). PEP can be further routed into glucose (trehalose in insects (Becker et al, 1996)) through gluconeogenesis or glycerol-3-phosphate through glyceroneogenesis. PEPCK is present as a cytoplasmic and a mitochondrial isoform, both of which have shown to contribute to gluconeogenesis (Stark et al, 2014). As pepck plays a key role in adjusting the direction of flux of central carbon metabolism, it is under tight nutritional control. It is well established in mammals that the hormonal insulin/glucagon axis controls pepck expression via transcription factors CREB and FoxO1, both of which are active during starvation (high glucagon, low insulin) (Oh et al, 2013). However, whether cell-intrinsic nutrient sensing mechanisms contribute to PEPCK regulation has remained poorly understood. Circadian clocks time most physiological and behavioral processes to 24-h rhythms (Hardin, 2011; Partch et al, 2014). Among these processes is metabolism, which is strongly controlled by the circadian system (Bass, 2012; Peek et al, 2012; Masri & Sassone-Corsi, 2014; Dibner & Schibler, 2015). In Drosophila, this control manifests mainly at the transcriptional level (Xu et al, 2008, 2011). Metabolism feeds back to the circadian system, as dietary cues (i.e., restriction feeding) and metabolites have profound impact on the circadian system (Mattson et al, 2014). Circadian clocks are spread throughout the body and are present in most cells. Circadian clocks work cell autonomously, although cell-to-cell communication is known to provide robustness to circadian behavior (Weiss et al, 2014). In Drosophila, circadian control of metabolism is achieved mainly in the fat body and it is involved in the timed expression of hundreds of metabolic genes, including the ones involved in sugar and fat metabolism (Xu et al, 2008, 2011). The importance of this regulation is highlighted by the fact that disruption of circadian rhythms either by genetic or environmental cause is strongly associated with metabolic imbalances, diabetes, and obesity (Bass, 2012; Peek et al, 2012). Transcription factors (TFs) form hierarchical regulatory networks, which allow the integration of different input signals and the generation of complex and robust biological responses. In the context of sugar sensing, our previous work demonstrated that ChREBP/MondoA-Mlx strongly regulates the TF cabut (cbt) in Drosophila, which is involved in sugar tolerance by a yet unknown mechanism (Havula et al, 2013). The closest mammalian homologs of CBT are Krüppel-like factors Klf10 and Klf11 (Subramaniam et al, 2010; Spittau & Krieglstein, 2012). klf10 expression is promoted by sugar in a ChREBP-dependent manner (Iizuka et al, 2011), whereas klf11 expression is elevated upon starvation (Zhang et al, 2013), suggesting that klf10 is the functional ortholog of cbt in the context of sugar sensing transcriptional network. Interestingly, the circadian clock can regulate klf10 levels, and hence, this transcription factor might act as a link between the circadian and metabolic systems (Guillaumond et al, 2010). Furthermore, a variant of the klf10 gene is proposed to contribute to the risk of type-2 diabetes (Gutierrez-Aguilar et al, 2007). Here, we found that CBT is a central mediator of a repressive branch of the intracellular sugar sensing transcriptional network in Drosophila. We demonstrate that cbt expression is activated upon sugar feeding through direct regulation by the sugar sensing TF complex Mondo-Mlx. Genome-wide analyses reveal that CBT contributes to rapid and persistent repression of metabolic genes upon sugar feeding. CBT directly represses both pepck isoforms, inhibiting cataplerosis during sugar feeding. Failure in this regulatory axis leads to loss of glucose and glycerol homeostasis and to lethality observed in animals lacking functional Mondo-Mlx. Last but not least, we found that CBT integrates feeding information into a subset of metabolic genes, which are regulated by the circadian clock. In sum, here we identify and characterize a transcriptional circuit that allows animals to readjust their central carbon metabolism as well as circadian clock with respect to sugar feeding. Results cbt is regulated by dietary sugar through Mondo-Mlx Based on our earlier findings on cbt being essential for sugar tolerance (Havula et al, 2013), we decided to explore how cbt is regulated upon sugar feeding. To this end, we measured cbt levels at different times after re-feeding with 5 or 10% sucrose. Indeed, we observed a twofold increase in cbt levels 6 h following sugar re-feeding in adults (Supplementary Fig S1A). In contrast, re-feeding with protein did not lead to increased cbt expression, but rather to a small decrease (Supplementary Fig S1B). Given our previous findings, we hypothesized that the sugar-induced expression of cbt might be mediated by Mondo-Mlx activity. As mlx null mutants (mlx1) are pupal lethal (Havula et al, 2013), we sought to explore cabut expression in the larvae. Indeed, cbt expression was upregulated in larvae that had been transferred to high sugar/high protein diet (10% yeast, 20% sucrose) compared to ones remaining in low sugar/high protein diet (10% yeast), and this upregulation was fully abrogated in mlx1 mutants (Fig 1A). These results suggest that the Mondo-Mlx dimer may directly regulate cbt expression. Supporting this possibility, we found two conserved consensus motifs for putative binding of this heterodimer, the carbohydrate-responsive elements (ChoRE), near the cbt transcription start site (TSS): one was located about one kilobase upstream and the other a few hundred bases downstream the annotated TSS (Fig 1B and C). We performed chromatin immunoprecipitation (ChIP) from Drosophila S2C cells using anti-Mlx antibodies or preimmune serum as a negative control. Indeed, Mlx strongly bound these two putative ChoREs in the cbt promoter region (Fig 1D). Moreover, we observed that Mlx binding to cbt promoter was significantly increased in high glucose media (Fig 1D, compare ± Glc). To functionally test the importance of the ChoREs, we constructed a luciferase reporter containing −1,961 to + 641 region of the cbt promoter. We also generated versions of this promoter in which the putative ChoREs were mutated. As the reporter failed to display glucose responsiveness in S2C cells (data not shown), we performed the experiments in human HepG2 hepatocellular carcinoma cells, which are a well-established model system for ChREBP-mediated gene regulation (Jeong et al, 2011). Indeed, the cbt promoter was activated by glucose stimulation, and the reporter activity was significantly inhibited when ChoRE2 was mutated (Fig 1E). In conclusion, our experiments show that cbt is induced by dietary sugars likely through direct binding of Mondo-Mlx to the ChoREs in the cbt promoter. Figure 1. cbt is a direct target of Mondo-Mlx-mediated sugar sensing cbt expression is sugar inducible and Mlx dependent. cbt mRNA levels in control (precise excision of P-element line) and mlx1 mutant 2nd instar larvae after 8 h of 10% yeast (Yeast) or 10% yeast and 20% sucrose feeding (Sugar). Actin was used as a reference gene. Error bars indicate SD, n = 3. *P < 0.05; **P < 0.01. Two putative ChoRE sites in the cbt gene region. Conservation of the putative ChoRE sites in the cbt gene region in Drosophila species. ChIP from S2C cells with anti-Mlx antibody or pre-immune serum in the presence or absence of 50 mM glucose (6 h). The percentage of input pulled down was determined by qPCR. Actin was used as a negative control. Error bars indicate SD, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001. ChoRE2 is required for full cbt promoter activity and sugar responsiveness. Luciferase reporter assay with cbt promoter in HepG2 cells grown in low (5.5 mM) or high (25 mM) glucose. Error bars indicate SD, n = 3. **P < 0.01. Download figure Download PowerPoint cbt regulates the expression of genes involved in metabolism CBT is known to repress its own expression (Belacortu et al, 2012), but beyond that, the transcriptional targets and function of CBT are still poorly understood. Therefore, we sought to identify CBT-regulated transcriptome in adult Drosophila. We utilized the GAL4-UAS system to generate flies in which cbt is down- or upregulated. We tested several GAL4-UAS drivers in combination with UAS-cbt or UAS-cbt RNAi. Most of these combinations resulted in high or total developmental lethality. We were able to obtain adult flies by using a GAL4 driver that expresses the gene of interest only in cells harboring an active circadian clock (tim-gal4 driver). The tim-gal4 driver expresses in approximately half of the cells of the fly head, including the brain, eyes, and the fat body, the fly equivalent of the mammalian liver and adipose tissue (Xu et al, 2008). To overexpress cbt, we utilized the tim-gal4 driver line in combination with a UAS-cbtFLAG transgene (Muñoz-Descalzo et al, 2005); these flies will be referred to as cbtOE flies. To downregulate cbt, we used the same driver in combination with a publicly available cbt RNAi transgene (NIG.4427R from NIG-Fly Stock Center), a UAS-dcr2 transgene, and a mutant cbt allele (cbtE1 (Muñoz-Descalzo et al, 2005)) in heterozygosis. We will call these cbtRNAi flies. After verifying the levels of CBT by Western blot (Supplementary Fig S2A), we assessed the transcriptomes in heads of control, cbtRNAi, and cbtOE flies using oligonucleotide microarrays. More than 1,000 genes were significantly affected by at least one of the manipulations in cbt levels (Fig 2A, Supplementary Dataset S1). Among them, genes related to neuronal function, immunity, and metabolism were specifically enriched (Fig 2B; Supplementary Fig S2B). In particular, we found that genes involved in carbohydrate metabolism were strongly affected by cbt up- or downregulation (Fig 2B and Supplementary Fig S3). Previously, a role of CBT in regulating circadian transcription was suggested by the fact that the circadian transcriptional activator CLK binds CBT promoter in a timely fashion (Abruzzi et al, 2011). Interestingly, we found a significant number of CLK-controlled genes among the genes downregulated by CBT (P < 0.0001), suggesting that in addition to metabolic gene expression cbt might regulate the circadian transcriptome (Abruzzi et al, 2011). Figure 2. CBT represses metabolic genes upon sugar feeding Heatmap representing the microarray data obtained from control, cbtRNAi, and cbtOE flies. From each strain, fly heads at two different time points during the day–night cycle (3 h after light on or light off) were collected. Based on statistical analysis, levels of 1,023 mRNAs were affected in one or more of the strains (FDR < 0.2 and fold change > 1.5). Histogram showing the fold change of the top 20 genes with expression altered by downregulation (top panel) or upregulation (bottom panel) of CBT. For each gene, the fold change was calculated as the ratio between the values obtained in cbtOE and cbtRNAi flies. Heatmap representing the 3′ RNA-seq data of control and cbtRNAi adult flies following 24 h of sugar refeeding (left). For clarity, selected transcripts are displayed. mRNAs were classified into three main groups depending on their response to sugar: genes that are downregulated similarly in control and cbtRNAi flies, genes that are not significantly affected in control but upregulated in cbtRNAi flies, and genes that are downregulated in control flies but are not differentially expressed following sugar intake in cbtRNAi flies. The regulation by sugar and CBT is represented on the right. Heatmap representing 3′ RNA-seq data of genes that change significantly following sugar intake. The transcriptome assay was performed from heads of adult flies that had been starved for 16 h and transferred to vials containing food with 5% sucrose. The arrow indicates the time when the flies were re-fed. The levels of 520 mRNAs were significantly changed at least in one of the time points. Two transcriptional modules that contain mRNAs that were strongly downregulated following sugar intake. The graphs were obtained by averaging normalized expression of all the genes in these modules. CBT targets are clustered into the module displaying rapid and persistent downregulation by sugar. Download figure Download PowerPoint CBT constitutes a repressive branch of the sugar sensing transcriptional network As cbt expression is induced by sugar intake, we decided to determine the role of CBT in the sugar-induced transcriptional response. To do so, we profiled the transcriptomes using 3′ RNA-seq of control and cbtRNAi flies after they had been starved and exposed to different levels of sucrose for 18 h (see scheme in Supplementary Fig S4A). After a very conservative analysis of the data, we found that levels of 72 transcripts were strongly affected by sugar intake at this time point. Most of these mRNAs (51) displayed decreased expression in response to this dietary manipulation (Supplementary Dataset S2). Of these 51 genes, 22 mRNAs were downregulated in a CBT-dependent manner (Fig 2C; Supplementary Dataset S2; Supplementary Fig S4B). In addition, we found eight more CBT-regulated mRNAs by looking at genes that are significantly more affected by sugar intake in cbtRNAi flies compared with control flies (Supplementary Dataset S3). Among the CBT-repressed targets, we found transcripts encoding proteins with function in lipolysis, gluconeogenesis, and glycerol metabolism including brummer (bmm) lipase, fructose-1,6-bisphosphatase (fbp), phosphoenolpyruvate carboxykinase (pepck), and glycerol kinase (Gyk) (Supplementary Fig S4B). Notably, the pepck, fbp, and Gyk encode proteins with related functions. PEPCK participates with FBP in gluconeogenesis, and Gyk and PEPCK control biosynthesis of glycerol-3-phosphate, albeit through two distinct pathways. These results demonstrate that CBT has a central role in repressing the expression of key metabolic genes in response to dietary sugars. In order to temporally characterize the transcriptional response to dietary sugars with respect to CBT function, we performed a transcriptomic analysis of adult fly heads harvested at different times following sugar intake after starvation. Our analysis identified 520 mRNAs that changed levels significantly at least at one of the time points (Fig 2D; Supplementary Dataset S4). Clustering analysis allowed us to identify ten transcriptional modules with different temporal patterns of expression following sugar intake (Fig 2D and E and Supplementary Fig S5; Supplementary Dataset S5; see 4 for details). Interestingly, the majority of CBT-dependent targets clustered into Group 1 containing genes that are repressed by sugar feeding in a rapid and persistent manner (Fig 2E). This further supports the conclusion that CBT is a sugar-activated transcriptional repressor. pepck isoforms are negatively regulated by the Mondo-Mlx/CBT network One of the strongest CBT-regulated genes across all our genomic analyses was pepck (CG17725) (Fig 2B and Supplementary Fig S4B). In mammals, PEPCK exists as cytoplasmic (PEPCK-C) and mitochondrial (PEPCK-M) isoforms, displaying partially redundant functions (Stark et al, 2014). Drosophila genome also encodes two genes homologous to mammalian PEPCK isoforms, namely CG17725 (called as Pepck in FlyBase) and the neighboring gene CG10924. CG17725 lacks an identifiable mitochondrial targeting sequence, while MitoProt II predicts that CG10924 is likely targeted to mitochondria (likelihood 0.82). We call the CG17725 and CG10924 as pepck1 and pepck2, respectively. In adult flies, cbt overexpression resulted in strong downregulation of pepck1 expression, whereas cbtRNAi flies displayed elevated pepck1 mRNA levels (Fig 3A). Moreover, sugar feeding rapidly and persistently downregulated the expression of pepck1, and this downregulation was prevented by RNAi-mediated depletion of CBT (Fig 3B and Supplementary Figs S5 and S6A). Feeding with protein caused only a modest downregulation of pepck1 expression (Supplementary Fig S6B). Similar to adults, sugar feeding led to downregulation of pepck1 expression in larvae (Supplementary Fig S6C). In fed larvae, two independent RNAi lines against CBT caused upregulation of pepck1 expression (Fig 3C; Supplementary Fig S6D), providing further evidence for CBT-mediated repression of pepck1. Consistent with the finding that CBT function is dependent on functional Mondo-Mlx, pepck1 expression was also strongly elevated in mlx mutant larvae (Fig 3C). Moreover, fat body-specific overexpression of CBT in the mlx mutant background was sufficient to downregulate pepck1 expression (Fig 3D), consistent with the model that CBT acts downstream of Mondo-Mlx to repress pepck1. CBT knockdown and loss of mlx led to comparable upregulation of pepck2 (Fig 3E). Moreover, fat body-specific overexpression of CBT in the mlx mutant also rescued pepck2 expression levels (Supplementary Fig S6E). In conclusion, our data demonstrate that CBT represses both Drosophila pepck isoforms. Figure 3. The Mondo-Mlx/CBT network negatively regulates the expression of pepck isoforms pepck1 mRNA levels in heads of cbtRNAi, control (yw; UAS-cbt), and cbtOE adult flies. pepck1 levels were normalized to rp49. Error bars indicate SD, n = 6. **P < 0.01. pepck1 mRNA levels in the heads of control (+/cbtE1-UAS-cbt RNAi) or cbtRNAi adult flies that were starved for 16 h and then exposed to food containing different concentrations of sucrose for 18 h. RNA-seq data. Error bars indicate SD, n = 3. *P < 0.05. pepck1 mRNA levels in 1st instar cbt knock-down and mlx1 mutant larvae. Actin was used as a reference gene. Control (Tub-GAL80ts; Tub-GAL4 > ), cbtRNAi (Tub-GAL80ts; Tub-GAL4 > cbtRNAi NIG 4427R-1), control for mlx1, and mlx1 mutants were grown on 20% yeast. Error bars indicate SD, n = 3. *P < 0.05, **P < 0.01. pepck1 mRNA levels in 1st instar control (precise excision of P-element line) larvae, mlx1 mutants, and in mlx1 mutants with fat body-specific over-expression of cbt (mlx1, r4-GAL4 > UAS-cbt). Actin was used as a reference gene. Error bars indicate SD, n = 3 (control and mlx1) and n = 7 (mlx1;