InteractiveFly: GeneBrief

cabut: Biological Overview | References

BIOLOGICAL OVERVIEW

The Drosophila transcription factor Cabut/dTIEG (Cbt) is a growth regulator, whose expression is modulated by different stimuli. This study determined Cbt association with chromatin and identified Yorkie (Yki), the transcriptional co-activator of the Hippo (Hpo) pathway as its partner. Cbt and yki co-localize on common gene promoters, and the expression of target genes varies according to changes in Cbt levels. Down-regulation of Cbt suppresses the overgrowth phenotypes caused by mutations in expanded (ex) and yki over-expression, whereas its up-regulation promotes cell proliferation. These results imply that Cbt is a novel partner of yki that is required as a transcriptional co-activator in growth control (Ruiz-Romero, 2015).

Gene expression is regulated through the integrated action of, among others, many cis-regulatory elements, including core promoters and enhancers located at greater distances from transcription start sites (TSS). The combinatorial binding of transcription factors (TF) to these elements can lead to diverse types of transcriptional output, and an understanding of this mechanism is crucial, for example, in the context of development. In fact, the final size and shape of an organism require a complex genetic network of signaling molecules, the final outcome of which must be finely regulated in space and time to ensure a proper response (Ruiz-Romero, 2015).

The transcription factor Cabut/dTIEG (Cbt) is the fly ortholog of TGF-β-inducible early genes 1 and 2 (TIEG1 and TIEG2) in mammals, which belong to the evolutionary conserved TIEG family (Munoz-Descalzo, 2007). TIEGs are zinc finger proteins of the Krüppel-like factor (KLF) family that can function as either activators or repressors depending on the cellular context, the promoter to which they bind or the interacting partners. TIEG proteins participate in a wide variety of cellular processes, from development to cancer, and regulate genes that control proliferation, apoptosis, regeneration or differentiation (Ruiz-Romero, 2015).

Drosophila cbt was identified and characterized from an overexpression screen of EP lines conducted to determine genes involved in establishing epithelial planar cell polarity (Munoz-Descalzo, 2005). This TF is ubiquitously expressed in the wing disk, and its expression increases in response to metabolic, hormonal and stress signals. Cbt levels rise upon inhibition of TOR signaling, and it is among the most highly Mlx-regulated genes (Havula, 2013). Among its functions, it is known that Cbt is required during dorsal closure downstream of JNK signaling (Munoz-Descalzo, 2005), that it is a modulator of different signaling pathways involved in wing patterning and proliferation (Rodriguez 2011) and that it promotes ectopic cell cycling when overexpressed. Moreover, Cbt is a crucial downstream mediator gene of the JNK signaling required during wing disk regeneration (Blanco, 2010). In spite of this, little is known about its downstream target genes or its precise mechanism of action. This study reports a novel function for Cbt as a partner of yki (Yorkie). yki is the key effector of growth control and the downstream element of the highly conserved Hpo (Hippo) signaling pathway. The Hpo pathway limits organ size by phosphorylating and inhibiting Yki, a key regulator of proliferation and apoptosis. yki can also act as an oncogene, since it has potent growth-promoting activity. The results show a role for Cbt as a transcriptional activator with the capacity to modulate yki growth response (Ruiz-Romero, 2015).

To characterize Cbt target genes, chromatin immunoprecipitation and high-throughput sequencing (ChIP-Seq) were performed from third instar larval wing imaginal disks. Analysis of Cbt-bound regions in the entire genome revealed that approximately 70% of its peaks were located in proximal promoters or introns, consistent with its role as a transcriptional regulator. Thus, 2,060 putative target genes were identified in the wing disk. Gene Ontology (GO) analysis indicated that this subset of genes was enriched in transcriptional activity, cell migration, mitotic cell cycle and signaling pathways known to play a role in imaginal disk development. As expected, among Cbt targets previously described genes were found such as salm (spalt major) or cbt itself, but also several unidentified target genes such as wg (wingless) or vg (vestigial) (Ruiz-Romero, 2015).

Cbt association around the TSS may be an indication of its function as a primary regulatory element, but does not provide any information about its role as an activator or a repressor. To elucidate this question, published data on chromatin modifications as well as recently obtained RNA-Seq data from the wing disk were examined and Cbt targets were ranked according to their expression level. Although at different levels, target genes are mostly expressed in the wing disk. This positive correlation with gene expression was also detected in the extensive overlap between Cbt occupancy and trimethylated histone 3 lysine 4 (H3K4me3). In contrast, only 13% of Cbt target genes correlated with the repressive chromatin mark H3K27me3. Although 200 Cbt targets seemed to present both modifications, these may be coupled to the differential expression pattern of several genes in the wing disk (Ruiz-Romero, 2015).

To clarify whether Cbt binds to active or inactive genes, Cbt occupancy was examined of genes known to be differentially expressed in a subpopulation of cells within the wing disk tissue. The gene nub (nubbin) is expressed in the wing primordium. GFP expression in the wing pouch was examined using a nub-GAL4 driver and ChIP assays followed by quantitative PCR (qPCR) were performed in sorted cells. In the vicinity of the TSS of genes expressed in the wing pouch, such as rn (rotund) and nub, Cbt was only found in GFP-positive cells. Cbt was also present in the promoter of cycA (cyclin A), both in GFP-positive and GFP-negative cells, in accordance with its expression throughout the entire wing disk (wing pouch and notum). These observations indicate that Cbt might act as a positive activator of transcription in this tissue. To further confirm this, the expression of selected targets was examined after cbt overexpression. Induction of cbt in the dorsal domain of the wing using an ap-GAL4 (apterous) driver led to a clear increase in the expression levels of target genes, as detected by qPCR. cbt was also ectopically expressed in the ptc (patched) domain of the wing disk using the ptc-GAL4 driver, and the pattern of Wg (normally restricted to cells adjacent to the D/V boundary in the wing blade and to two rings in the hinge region) and Vg (expressed throughout the wing blade) was examined by immunostaining. After cbt induction, spread staining of Wg was observed in the boundary and ring regions. Likewise, analysis of Vg revealed increased protein levels in the region where cbt was upregulated. No ectopic expression of Wg or Vg was detected in regions far from where they are normally expressed, suggesting that cbt alone is not sufficient to ectopically activate transcription of these genes but can modulate or cooperate with factors that promote their basal expression. Taken together, these results suggest that Cbt functions as a transcriptional activator in the wing disk. Nevertheless, its contribution to repression in some contexts or through binding to different partners cannot be disregarded, as previous experiments have demonstrated the relevance of the Sin3A interaction domain for Cbt's repressive role (Ruiz-Romero, 2015).

TIEG factors contain three conserved C-terminal zinc finger motifs that seem to bind to GC-rich sequences in vertebrates. To characterize the set of motifs enriched within Cbt binding sites, different pattern discovery methods were used. Among others, GC sequences and the Sp1 motif, were detected as expected for a TIEG family member, but in addition, one of the most enriched motifs comprised GAGA-binding sequences. No enrichment of the proposed consensus TIEG motif 5'GGTGTG3' was found, which suggests that Cbt binds to degenerated or alternative motifs or may function through its interaction with other TFs. A recent study identified a novel Mad-like motif in promoters of Cbt-regulated genes. However, this new motif does not coincide with previously reported Cbt binding data (Ruiz-Romero, 2015).

Many studies have emphasized the complexity of yki and its mammalian homologs YAP and TAZ regulation, including multiple combinations with associate proteins in distinct target genes. Besides DNA-binding partners such Sd (Scalloped) and Hth (Homothorax) in Drosophila, yki can cooperate with other factors directly on target promoters, such as the cell cycle-related gene dE2F1. Remarkably, a recent report shows that Cbt and dE2F1 regulate an overlapping set of cell cycle genes (Song, 2014). In the Dpp pathway, Mad (Mothers against decapentaplegic) and yki interact to form a transcription complex to activate their common targets. This association is conserved through evolution, as YAP and TAZ interact with Smad proteins to potentiate transcriptional activity. Recent studies have also identified Mask (Multiple ankyrin repeats single KH domain) as a novel cofactor for Yki/YAP, required to induce target gene expression. The results highlight the role of Cbt as a new yki partner involved in the activation of some yki target gene expression. This function of Cbt may occur in part through association with GAF as well as chromatin remodeler complexes (Ruiz-Romero, 2015)

Since overexpression of cbt results in an increase in proliferation as well as wing size (Rodriguez, 2011), it was hypothesized that Cbt's role in size control could be mediated through its association with Yki. To address this question, cbt levels were depleted, and the effect on the growth of ex mutant clones and in clones overexpressing yki in wing and eye-antenna imaginal disks was examined. The yki target gene ex acts as an upstream positive modulator of the Hpo pathway, and in accordance with its role as a tumor suppressor, its loss-of-function mutation results in large clones. Expression of cbt RNAi in this mutant background caused a clear reduction in the clone size. In the same direction, the overgrowth known to occur by overexpression of a yki-activated form is prevented in a mutant cbt background as well as expressing cbt RNAi. Moreover, impaired growth caused by yki depletion could not be rescued increasing cbt levels and overexpression of yki and cbt triggered massive growth in imaginal tissues. Finally, depletion of cbt in adult organs (wings and eyes) also reduced Yki-mediated overgrowth, indicating a general function for Cbt in the regulation of Hippo pathway-mediated tissue growth (Ruiz-Romero, 2015).

In addition to its role during development, it has been shown that Cbt expression is highly regulated by stress and metabolic conditions (Bulow, 2010; Havula, 2013). Cbt has also been identified as a JNK-inducible gene during dorsal closure, and this study has shown that JNK and tissue damage trigger cbt transient overexpression to promote wing disk regeneration, indicating that its levels must be finely controlled during regenerative growth. Moreover, cbt heterozygous mutant disks fail to proliferate and do not regenerate, and it is known that during regeneration, the JNK pathway triggers yki translocation to the nucleus to promote the proliferative response. Altogether, these data support a model for Cbt acting as a modulator of yki activity in the transcriptional regulatory mechanisms that control tissue growth (Ruiz-Romero, 2015).

Hunting complex differential gene interaction patterns across molecular context

Heterogeneity in genetic networks across different signaling molecular contexts can suggest molecular regulatory mechanisms. This study describes a comparative chi-square analysis (CPΞ²) method, considerably more flexible and effective than other alternatives, to screen large gene expression data sets for conserved and differential interactions. CPΞ² decomposes interactions across conditions to assess homogeneity and heterogeneity. Theoretically, an asymptotic chi-square null distribution was proven for the interaction heterogeneity statistic. Empirically, on synthetic yeast cell cycle data, CPΞ² achieved much higher statistical power in detecting differential networks than alternative approaches. CPΞ² was applied to Drosophila melanogaster wing gene expression arrays collected under normal conditions, and conditions with overexpressed E2F and Cabut, two transcription factor complexes that promote ectopic cell cycling. The resulting differential networks suggest a mechanism by which E2F and Cabut regulate distinct gene interactions, while still sharing a small core network. Thus, CPΞ² is sensitive in detecting network rewiring, useful in comparing related biological systems (Song 2014).

Mondo/ChREBP-Mlx-regulated transcriptional network is essential for dietary sugar tolerance in Drosophila

Sugars are important nutrients for many animals, but are also proposed to contribute to overnutrition-derived metabolic diseases in humans. Understanding the genetic factors governing dietary sugar tolerance therefore has profound biological and medical significance. Paralogous Mondo transcription factors ChREBP and MondoA, with their common binding partner Mlx, are key sensors of intracellular glucose flux in mammals. This paper reports analysis of the in vivo function of Drosophila melanogaster Mlx and its binding partner Mondo (ChREBP) in respect to tolerance to dietary sugars. Larvae lacking mlx or having reduced mondo expression show strikingly reduced survival on a diet with moderate or high levels of sucrose, glucose, and fructose. mlx null mutants display widespread changes in lipid and phospholipid profiles, signs of amino acid catabolism, as well as strongly elevated circulating glucose levels. Systematic loss-of-function analysis of Mlx target genes reveals that circulating glucose levels and dietary sugar tolerance can be genetically uncoupled: Kruppel-like transcription factor Cabut and carbonyl detoxifying enzyme Aldehyde dehydrogenase type III are essential for dietary sugar tolerance, but display no influence on circulating glucose levels. On the other hand, Phosphofructokinase 2, a regulator of the glycolysis pathway, is needed for both dietary sugar tolerance and maintenance of circulating glucose homeostasis. Furthermore, evidence is shown that fatty acid synthesis, which is a highly conserved Mondo-Mlx-regulated process, does not promote dietary sugar tolerance. In contrast, survival of larvae with reduced fatty acid synthase expression is sugar-dependent. These data demonstrate that the transcriptional network regulated by Mondo-Mlx is a critical determinant of the healthful dietary spectrum allowing Drosophila to exploit sugar-rich nutrient sources (Havula, 2013).

Mono- and disaccharides, i.e. sugars, are an important source of nutritional energy, but animal species display marked differences in the degree of sugar utilization and tolerance. While the diet of carnivores is typically low in sugars, nectarivores, like hummingbirds, feed primarily on sugar-rich nectar. Sugars from fruits and honey have been part of the ancestral human diet. However, the large quantities of refined sugars consumed by modern humans far exceed those available in natural sources. In fact, it has been proposed that excessive added sugar in the diet, especially fructose, might contribute to the development of metabolic syndrome. Yet the genetic factors governing the delicate balance between healthful dietary sugar utilization and the sugar overload-induced metabolic disturbance are poorly understood (Havula, 2013).

Drosophila is a well-suited model for exploring the physiological consequences of sugar intake. Drosophila melanogaster is a generalist fruit breeder naturally performing well on a broad range of dietary sugars. However, excessive intake of sugars has been shown to cause diabetes-like metabolic changes in D. melanogaster, including insulin resistance, elevated circulating glucose and increased adiposity. Dietary sugars have also been shown to shorten Drosophila lifespan. The sugar-induced insulin resistance has been attributed to the JNK-regulated lipocalin Neural Lazarillo. Moreover, high sugar induced gene expression has been previously analysed. However, beyond these observations, the functional interactions between genotype and dietary sugar have remained poorly understood (Havula, 2013).

Elevated systemic glucose levels cause cellular stress and tissue damage. Animals therefore rapidly adapt their metabolism to fluctuating sugar intake, maintaining circulating glucose levels constant. A postprandial increase in circulating glucose triggers the release of insulin, which induces the rapid uptake of excess glucose by metabolic tissues including muscle, adipose tissue, and liver. Intracellular glucose is immediately converted into glucose-6-phosphate and further metabolized into glycogen and lipids or catabolised to release energy. Metabolic tissues are exposed to large variations in the flux of intracellular glucose and therefore need to regulate their metabolism accordingly (Havula, 2013).

The basic helix-loop-helix transcription factor paralogs ChREBP (Carbohydrate Response Element Binding Protein) and MondoA act together with their common binding partner Mlx (Max-like protein X) to mediate transcriptional responses to intracellular glucose in mammals. The ChREBP/MondoA-Mlx complex is activated by glucose-6-phosphate and other phosphorylated hexoses, and regulates gene expression by binding to target promoters containing a carbohydrate response element (ChoRE). ChREBP and MondoA regulate the majority of the global glucose-induced transcriptional responses and many of their target genes encode enzymes in glycolytic and lipogenic pathways. ChREBP and MondoA play differential tissue-specific roles in mammals: ChREBP functions in the liver, adipose tissue and pancreatic beta cells, while MondoA is predominantly expressed in the skeletal muscle (Havula, 2013).

Of the mammalian ChREBP/MondoA-Mlx complex, the role of ChREBP has been studied in a physiological setting using loss-of-function mice. While ChREBP is nonessential in terms of survival, the mutant mice display a number of metabolic phenotypes, including elevated plasma glucose and liver glycogen as well as reduced adiposity. ChREBP^-/- mice survive poorly on a diet with high levels of sugars, but the underlying reasons have remained unexplored. ChREBP is known to regulate a range of metabolic genes, including those involved in de novo lipogenesis. Which target genes contribute to the various physiological phenotypes and what is the causal interrelationship between the physiological phenotypes, are questions that require powerful genetics and are therefore challenging to address in vivo in mammals. Moreover, existence of another Mondo paralog, MondoA, might mask some phenotypes in the ChREBP^-/- mouse. To better understand the physiological roles of the Mondo/ChREBP/-Mlx complex and its target genes, this study has explored their role in Drosophila melanogaster. The Drosophila genome encodes one ortholog for each of ChREBP/MondoA and Mlx, which is called Mondo (alternative identifiers: CG18362, Mlx interactor, ChREBP) and Mlx (alternative identifiers: CG3350, Bigmax), respectively (Havula, 2013).

mlx null mutant flies, which displayed lethality in the late pupal stage were generated. D. melanogaster larvae can normally utilize high levels of dietary sugar but loss of Mlx or knockdown of Mondo caused striking intolerance towards sucrose, glucose and fructose. The mlx null mutant larvae also displayed extensive metabolic changes, with strongly elevated circulating glucose, signs of amino acid catabolism and altered lipid and phospholipid profiles. Systematic functional analysis of Mlx-regulated genes revealed three genes contributing to dietary sugar tolerance: cabut, encoding a Kruppel-like transcription factor, phosphofructokinase 2, a regulator of the glycolytic pathway, and Aldehyde dehydrogenase type III, which is linked to detoxification of reactive ald (Havula, 2013).

To test in an unbiased way if any of the genes downregulated in mlx1 mutants had an essential role in maintaining organismal sugar tolerance, 103 candidate genes were systematically targeted by RNAi. Intriguingly, ubiquitous knockdown of two Mlx-regulated genes identified on the microarray led to significant sugar intolerance. Transcription factor Cabut was among the most highly Mlx-regulated genes in the microarray. Ubiquitous knockdown of Cabut expression by RNAi during the larval stage caused a modest delay of pupation on low sugar diet. However, on high sugar diet (20% yeast-15% sucrose) Cabut knockdown led to prominent developmental delay and impaired survival. This suggests that Mondo-Mlx activates a hierarchical transcriptional network to regulate dietary sugar tolerance with Cabut as an essential downstream effector (Havula, 2013).

Another Mlx-regulated gene, which caused sugar intolerance upon ubiquitous knockdown, was Aldehyde dehydrogenase type III (Aldh-III, CG11140). Most Aldh-III knockdown animals reached the pharate stage on a low sugar diet, but died during early pupal stages on a high-sugar diet. Similar early pupal lethality was observed in mlx1 mutants on sugar concentrations that allowed pupation. Survival on a 20% sucrose-only diet was also significantly reduced upon Aldh-III knockdown. Tests were made to see whether restoring Aldh-III activity by transgenic expression would be sufficient to rescue impaired mlx1 mutant survival on high sugar diet. While transgenic expression of Aldh-III did not rescue mlx1 mutant pupation on 20% yeast-15% sucrose diet, larval survival of mlx1 mutants on 20% sucrose-only diet was significantly improved by transgenic Aldh-III. The same was true when Aldh-III expression was rescued only in the fat body. In conclusion, Aldh-III is essential and sufficient for providing dietary sugar tolerance (Havula, 2013).

Whether the sugar intolerance observed upon knockdown of Cabut and Aldh-III was associated with elevated circulating glucose levels was tested. Surprisingly, knockdown of either gene did not result in a significant increase in circulating glucose. This implies that impaired clearance of circulating glucose is not an essential prerequisite for intolerance to dietary sugars. Instead, these two Mondo-Mlx-regulated parameters can be uncoupled at the level of the downstream target genes. Based on these phenotypes, the Cabut-dependent branch of the transcriptional network mediates only a subset of Mondo-Mlx functions. The possibility that Cabut could be a direct regulator of Aldh-III was also studied; however the mRNA levels of Aldh-III were unchanged in Cabut RNAi larvae. Also the mRNA levels of Mondo and Mlx were unchanced in Cabut RNAi larvae, indicating that Cabut is not a feedback regulator of Mondo-Mlx. Notably, it is possible that Cabut is regulating a subset of common genes with Mondo-Mlx (Havula, 2013).

The transcription factor Cabut coordinates energy metabolism and the circadian clock in response to sugar sensing

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. This study characterizes cabut (cbtDrosophila. cbt was rapidly induced upon sugar feeding through direct regulation by Mondo-Mlx. CBT repressed several metabolic targets in response to sugar feeding, including both isoforms of phosphoenolpyruvate carboxykinase (pepck). Deregulation of pepck1 (CG17725) in mlx mutants underlay imbalance of glycerol and glucose metabolism as well as developmental lethality. Furthermore, cbt provided a regulatory link between nutrient sensing and the circadian clock. Specifically, a subset of genes regulated by the circadian clock were also targets of CBT. Moreover, perturbation of CBT levels led to deregulation of the circadian transcriptome and circadian behavioral patterns (Bartok, 2015).

This study establishes the Drosophila klf10 ortholog, cbt, as a repressive effector of the sugar sensing transcriptional network. Specifically, (1) cbt expression is activated by dietary sugars in mlx-dependent manner; (2) cbt is a direct target of Mlx; (3) many key metabolic genes are rapidly repressed by CBT upon sugar feeding; (4) CBT binds to the proximity of pepck genes; (5) pepck1 is dispensable for viability, but essential for glucose and glycerol homeostasis; (6) deregulation of pepck1 underlies lethality of mlx mutants, and (7) CBT modulates the circadian system by controlling the cycling of a subset of circadian output genes. Based on these findings, a model is proposed in which CBT serves as a repressive downstream effector of the Mondo-Mlx-mediated sugar sensing, which contributes to diet-induced physiological readjustment, including flux of central carbon metabolism and cycling of metabolic circadian clock targets (Bartok, 2015).

By uncovering the CBT-mediated repression of pepck isoforms downstream of Mondo-Mlx, This study provides a mechanistic explanation to the regulation of cataplerosis in response to intracellular sugar sensing. Drosophila Mondo-Mlx is known to drive activation of glycolysis, for example, by promoting the expression of phosphofructokinase 2 (Havula 2013). Placing the rate-limiting enzymes of gluconeogenesis downstream, the same sensor mechanism that activates glycolysis provides an elegant mechanism to adjust the direction of flux of glucose metabolism in response to sugar input. Such simple network topology provides a robust safeguard against loss of energy in futile cycles caused by simultaneous high activity of glycolysis and gluconeogenesis (Bartok, 2015).

Mondo-Mlx also activates the expression of lipogenic gene expression (e.g., FAS and ACC) in order to promote conversion of excess sugars into triglycerides (Sassu, 2012; Havula, 2013). In addition to fatty acid moieties, which are built by FAS and ACC, triglyceride biosynthesis requires glycerol-3-phosphate. Substrate-labeling studies in mammals have shown that a significant portion of glycerol in triglycerides is in fact derived from the PEPCK-dependent glyceroneogenesis pathway. This is supported by the current findings showing significantly lower circulating glycerol levels in well-fed pepck1-mutant larvae. The impact of glyceroneogenesis on triglyceride homeostasis is also likely reflected in the reduced triglyceride levels in pepck1 mutant flies. Similarly, mammalian studies have shown that elevated expression of PEPCK-C in adipose tissue increases fat mass, whereas reduced PEPCK-C expression leads to lower fat content. Moreover, in humans, adipose tissue expression of PEPCK-C positively correlates with adiposity and plasma triacylglycerol levels. Control of pepck through CBT places both branches of triglyceride biosynthesis under Mondo-Mlx. Inhibition of PEPCK-mediated cataplerosis upon high sugar intake allows maximal conversion of excess glucose-6-phosphate into the glycerol moieties of triglycerides through the glycolytic route of glycerol-3-phosphate synthesis. Simultaneous impairment of de novo lipogenesis and failure to suppress glyceroneogenesis likely leads to the breakage of glycerol homeostasis and massive accumulation of circulating glycerol, as observed in mlx mutants. Interestingly, a recent study showed that fasting serum levels of glycerol predicted development of hyperglycemia and type 2 diabetes. It will be interesting to learn whether this diagnostic marker is associated with deregulation of pepck isoforms and the activity of ChREBP/MondoA-Mlx and Klf10-dependent transcriptional network (Bartok, 2015).

According to the current view, Mondo/ChREBP and Mlx act mainly in nutrient sensing and metabolic regulation. In contrast, CBT and its human ortholog Klf10 are multifunctional transcription factors. In Drosophila, cbt was originally identified as a developmental regulator with an essential function in dorsal closure early in development. Moreover, cbt is a direct transcriptional target of the circadian transcription factor CLK, and this study establishes it is deeply involved in the control of the circadian transcriptional network. While CBT overexpression leads to strong behavioral abnormalities, they are not accompanied by noticeable changes in the oscillation of the core clock components in the fly heads. This suggests that it reflects a specific effect on circadian output. If the behavioral patterns were due to an effect in the general health of the animal, deregulation of core clock components would be expected. Despite the null effect of cbt overexpression in the expression of core clock genes, this study observed that cbt modulates the expression of an important subset of CLK and circadian-controlled genes, most of which are involved in metabolic functions. Strong effects of cbt downregulation were observed in circadian oscillation of metabolic genes, establishing CBT as a new regulator of the circadian transcriptome. Interestingly, downregulation of CBT in circadian cells decreases the amplitude of oscillation of a large number of circadian-controlled genes, providing a direct link between food intake, circadian gene expression, and behavior. Given the established link between feeding time, metabolism, and the circadian system in Drosophila, it will be interesting to further analyze the importance of CBT in this coordination (Bartok, 2015).

Although the functional analysis in this study largely focused on the metabolic role of pepck regulation by Mondo-Mlx-CBT network, microarray and RNA-seq analyses revealed other interesting CBT transcriptional targets including bmm. This gene is an ortholog of the human adipocyte triglyceride lipase gene, and it is an essential regulator of triglyceride stores in Drosophila. bmm expression is positively regulated by the Forkhead transcription factor FoxO, which is activated during starvation through inhibition of insulin-like signaling. The sugar-dependent repression of bmm expression by CBT is in perfect agreement with the lipogenic role of Mondo-Mlx (Bartok, 2015).

It has been proposed that CBT mammalian ortholog Klf10 acts as a negative feedback regulator for ChREBP-activated genes, including lipogenic genes FAS and ACC (Iizuka, 2011). This conclusion was based on suppression of ChREBP-activated transcription upon Klf10 overexpression in primary liver cells. This model was tested by analyzing the expression of Mondo-Mlx targets FAS and ACC, but no effect was observed by cbtRNAi. In contrast, genome-wide expression analysis of CBT loss-of-function flies revealed that the CBT-dependent branch of the sugar sensing transcriptional network mediates rapid repression of gene expression. It is interesting to note that while most metabolic targets of CBT are rapidly and persistently downregulated, cbt expression is rapidly attenuated during prolonged sugar feeding. This is likely due to the negative autoregulation demonstrated earlier and supported by ChIP data. The finding that most of the identified CBT-dependent mRNAs are stably repressed for many hours after cbt levels have significantly attenuated suggests that CBT-mediated repression might involve regulation at the chromatin level. This is in agreement with the possible involvement of Sin3A in CBT-mediated repression. Through such persistent regulatory marks, sugar feeding may have a long-lasting influence on metabolic homeostasis, which is a topic that certainly deserves to be more thoroughly analyzed in the future (Bartok, 2015).

In sum, this work provides a mechanistic explanation for the transcriptional repression upon Mondo-Mlx-mediated intracellular sugar sensing through the transcription factor CBT. The CBT-mediated repressive branch of the sugar sensing network is involved in securing the mutually exclusive activity of glycolysis and gluconeogenesis and coordination of fatty acid and glycerol biosynthesis with respect to dietary sugar intake. This study also establishes a mechanism for nutrient input into the circadian gene expression. As intracellular sugar-sensing and circadian regulation are highly conserved processes, the insight achieved in this study in the genetically tractable Drosophila model should provide a new conceptual framework for forthcoming studies in human subjects and mammalian model systems (Bartok, 2015).

Transcriptional activity and nuclear localization of Cabut, the Drosophila ortholog of vertebrate TGF-beta-inducible early-response gene (TIEG) proteins

Cabut (Cbt) is a C₂H₂-class zinc finger transcription factor involved in embryonic dorsal closure, epithelial regeneration and other developmental processes in Drosophila melanogaster. Cbt orthologs have been identified in other Drosophila species and insects as well as in vertebrates. Indeed, Cbt is the Drosophila ortholog of the group of vertebrate proteins encoded by the TGF-ss-inducible early-response genes (TIEGs), which belong to Sp1-like/Kruppel-like family of transcription factors. Several functional domains involved in transcriptional control and subcellular localization have been identified in the vertebrate TIEGs. However, little is known of whether these domains and functions are also conserved in the Cbt protein. To determine the transcriptional regulatory activity of the Drosophila Cbt protein, Gal4-based luciferase assays were performed in S2 cells and it was shown that Cbt is a transcriptional repressor and able to regulate its own expression. Truncated forms of Cbt were then generated to identify its functional domains. This analysis revealed a sequence similar to the mSin3A-interacting repressor domain found in vertebrate TIEGs, although located in a different part of the Cbt protein. Using beta-Galactosidase and eGFP fusion proteins, it was also shown that Cbt contains the bipartite nuclear localization signal (NLS) previously identified in TIEG proteins, although it is non-functional in insect cells. Instead, a monopartite NLS, located at the amino terminus of the protein and conserved across insects, is functional in Drosophila S2 and Spodoptera exigua Sec301 cells. Genetic interaction and immunohistochemical assays suggested that Cbt nuclear import is mediated by Importin-α2. These results constitute the first characterization of the molecular mechanisms of Cbt-mediated transcriptional control as well as of Cbt nuclear import, and demonstrate the existence of similarities and differences in both aspects of Cbt function between the insect and the vertebrate TIEG proteins (Belacortu, 2012).

Drosophila TIEG is a modulator of different signalling pathways involved in wing patterning and cell proliferation

Acquisition of a final shape and size during organ development requires a regulated program of growth and patterning controlled by a complex genetic network of signalling molecules that must be coordinated to provide positional information to each cell within the corresponding organ or tissue. The mechanism by which all these signals are coordinated to yield a final response is not well understood. This study has characterized the Drosophila ortholog of the human TGF-beta Inducible Early Gene 1 (dTIEG). TIEG are zinc-finger proteins that belong to the Kruppel-like factor (KLF) family and were initially identified in human osteoblasts and pancreatic tumor cells for the ability to enhance TGF-beta response. Using the developing wing of Drosophila as 'in vivo' model, the dTIEG function has been studied in the control of cell proliferation and patterning. These results show that dTIEG can modulate Dpp signalling. Furthermore, dTIEG also regulates the activity of JAK/STAT pathway suggesting a conserved role of TIEG proteins as positive regulators of TGF-β signalling and as mediators of the crosstalk between signalling pathways acting in a same cellular context (Rodriguez, 2011).

dTIEG, the Drosophila ortholog of TIEG1 protein, functions during the imaginal discs development. Similar to TIEG1 protein in humans, the dTIEG expression in the imaginal discs is ubiquitous although the transcriptional levels vary. dTIEG shares structural features with the vertebrate dTIEG proteins such as the three Zn-finger motifs and a serine- proline-rich region, where the R3 repression domain would be located. However, the R1 and R2 motifs are more divergent suggesting that these domains might not be completely conserved and therefore the repressor function of dTIEG could be compromised (Rodriguez, 2011).

Another important difference with respect to TIEG proteins is that dTIEG enhances BMP signalling, particularly the Dpp signalling pathway. The genetic analysis has provided evidence that dTIEG is a novel regulator of patterning and growth during wing development modulating positively both the Dpp and JAK/STAT pathways. When dTIEG and Sal are overexpressed, the wing phenotypes are similar. dTIEG controls Dpp/BMP2 signalling by modulating the expression of P-Mad and the target genes Sal and Omb. In Drosophila, there are two more BMP ligands; Scw that is required only in early embriogenesis and Gbb that contributes to BMP signalling with moderate effects in late patterning and cell proliferation during wing development. Similarly, the Activin pathway also functions during wing development although its role is less understood. Two different ligands dAct and Daw trigger signalling through the type I receptor Baboon and Smad2, both specific components of this pathway, to regulate cell proliferation and in a lesser extent patterning. Recent data indicate that Smad2 exerts an inhibitory effect on Mad signalling that suggest a role of Smad2 on vein formation and cell proliferation through Dpp/BMP2 signalling. Thus, according to the phenotypes described in this study the regulation of these pathways by dTIEG can be ruled out. Other KLF members identified in Drosophila such as Krüppel, Sp1 and Buttonhead are involved in developmental processes independent of Dpp/BMP2 signalling (Rodriguez, 2011).

Previous results had shown that Cabut is expressed in the embryo and regulates dpp expression acting downstream of the JNK pathway during dorsal closure. dTIEG modulates Dpp/BMP2 signalling during wing development. Several pieces of evidence support this conclusion. First, dTIEG overexpression enhances transcriptional activation of Dpp target genes such as sal and omb as it is the case with the overexpression of an active form of the TGF-β type II receptor Tkv. Target genes of other signalling pathways, such as Hedgehog or Wingless, do not seem to be directly affected. In contrast, the elimination of dTIEG function in somatic clones causes a down-regulation of sal and omb expression indicating a decrease of Dpp/BMP2 activity in the wing disc. Moreover, P-Mad expression is also reduced. Besides, the epistatic experiments revealed that dTIEG acts downstream of Tkv and requires Mad as a partner to exert its regulatory action on sal and omb genes. However, a slight decrease of dTIEG function caused by two independent lines of targeted expression of interference RNAs (UAS-dTIEGi) did not cause any discernible phenotype. These results indicate that dTIEG must be completely eliminated to exert its regulatory function on Dpp/BMP2 pathway and further reinforce the role of dTIEG as a modulator in contrast to other components of the pathway that have been shown to induce severe phenotypes when eliminated (Rodriguez, 2011).

Since the function of dTIEG on the Dpp/BMP2 pathway is reminiscent of the role of TIEG proteins in TGF-β signalling, the expression of Dpp/BMP2 repressors was also examined. The overexpression of TIEG1 and TIEG3 results in the repression of the inhibitory Smad7. In Drosophila, however, the elimination of dTIEG function did not cause detectable changes in the expression of either the I-Smad/Dad or Brk suggesting certain differences in the mechanism of action of dTIEG. These observations could be explained by the absent of two repressor domains (R1 and R2) in dTIEG. Moreover, recent studies in mouse myoblasts have showed that TIEG1 can be stimulated by both pathways: myostatin and TGF-β signalling. In this context the expression of Smad2 and Smad7 was unaffected in contrast to the changes observed when TGF-β signalling was activated. This suggests that myostatin signalling might compensate the TGF-β signalling on the regulation of Smad2 and Smad7. In Drosophila, the Myoglianin (Myg) is another TGF-β ligand related to Myostatin. 'In vitro' experiments indicate that Myg can trigger activin signalling through Wit, another TGF-β type II receptor, that binds both activin and BMP ligands through a mechanism that is poorly understood. These results indicate that many aspects about the mechanism of TIEG proteins still remain unknown and suggest that TIEG might be using alternative mechanisms in different cellular contexts (Rodriguez, 2011).

Misregulation of the Dpp pathway not only leads to alterations in patterning but also in cell proliferation. Whereas mutant cells (i.e., tkv clones) that cannot respond to the Dpp/BMP2 signal fail to proliferate and die, an increase of Dpp signalling promotes overproliferation. Previous studies have postulated different models to correlate the uniform cell growth in the wing disc with the slope of the Dpp gradient and brk activity. The existence of a still unknown inhibitor of cell proliferation has been suggested. However, other signalling pathways also contribute to wing proliferation and the integration of all these inputs must be considered although the mechanism by which the net balance arises remains unclear (Rodriguez, 2011).

The above results demonstrate that dTIEG controls cell proliferation. Ectopic dTIEG expression promotes overproliferation whereas elimination of dTIEG function in cell clones using a null allele produces a failure in cell proliferation. To assess that the loss of function phenotypes were caused by dTIEG and not for the adjacent med15 gene a genetic analysis of med15 was performed in the wing disc. The results are consistent with a role of MED15 as a co-activator required for the basal transcription of different genes that results essential for cell viability (Rodriguez, 2011).

On the other hand, dTIEG also regulates the expression of STAT92E, the main effector of the JAK/STAT pathway. The upregulation of STAT92E-lacZ expression in dTIEG mutant cells reflects a decrease in JAK/STAT activity indicating that dTIEG is also a positive regulator of this pathway. The result fits with the reduced size of dTIEG mutant clones respect to the sibling clones (wild-type cells) and the proliferative effect described for STAT92E in the wing disc. Thus, the JAK/STAT pathway might contribute to the defects in cell proliferation observed in dTIEG cells. Several pieces of evidence support a role for JAK/STAT in the regulation of other signalling pathways although in most of the cases the mechanism remains unknown. In other Drosophila developmental contexts, STAT92E can upregulate Dpp signalling and repress the Wingless and Hh pathways. Thus, dTIEG could play a role as a connector gene to integrate signalling from Dpp/TGF-β and JAK/STAT pathways. Indeed, the mild reduction of P-Mad levels observed in dTIEG mutant cells could reflect the net balance resulting from simultaneous changes in the JAK/STAT and Dpp/BMP2 activities. Supporting this observation, TIEG1, in addition to its role in the transcriptional control of Smad proteins, also regulates the activity of other genes by binding directly to their promoters (Rodriguez, 2011).

In conclusion, these results demonstrate an evolutionary conserved function of TIEG proteins regulating the activity of different TGF-β signals and mediating the crosstalk among different pathways in the control of differentiation and cell proliferation. Further experiments will be required for the acquisition of a better knowledge of the molecular mechanism involved in the process (Rodriguez, 2011).

Expression of Drosophila Cabut during early embryogenesis, dorsal closure and nervous system development

cabut (cbt) encodes a transcription factor involved in Drosophila dorsal closure (DC), and it is expressed in embryonic epithelial sheets and yolk cell during this process upon activation of the Jun N-terminal kinase (JNK) signaling pathway. Additional studies suggest that cbt may have a role in multiple developmental processes. To analyze Cbt localization through embryogenesis, a Cbt specific antibody was generated that has allowed detecting new Cbt expression patterns. Immunohistochemical analyses on syncytial embryos and S2 cells reveal that Cbt is localized on the surface of mitotic chromosomes at all mitotic phases. During DC, Cbt is expressed in the yolk cell, in epidermal cells and in the hindgut, but also in amnioserosal cells, which also contribute to the process, albeit cbt transcripts were not detected in that tissue. At later embryonic stages, Cbt is expressed in neurons and glial cells in the central nervous system, and is detected in axons of the central and peripheral nervous systems. Most of these expression patterns are recapitulated by GFP reporter gene constructs driven by different cbt genomic regions. Moreover, they have been further validated by immunostainings of embryos from other Drosophila species, thus suggesting that Cbt function during embryogenesis appears to be conserved in evolution (Belacortu, 2011).

The Drosophila FoxA ortholog Fork head regulates growth and gene expression downstream of Target of rapamycin

Forkhead transcription factors of the FoxO subfamily regulate gene expression programs downstream of the insulin signaling network. It is less clear which proteins mediate transcriptional control exerted by Target of rapamycin (TOR) signaling, but recent studies in nematodes suggest a role for FoxA transcription factors downstream of TOR. This study presents evidence that outlines a similar connection in Drosophila, in which the FoxA protein Fork head (FKH) regulates cellular and organismal size downstream of TOR. Ectopic expression and targeted knockdown of FKH in larval tissues elicits different size phenotypes depending on nutrient state and TOR signaling levels. FKH overexpression has a negative effect on growth under fed conditions, and this phenotype is not further exacerbated by inhibition of TOR via rapamycin feeding. Under conditions of starvation or low TOR signaling levels, knockdown of FKH attenuates the size reduction associated with these conditions. Subcellular localization of endogenous FKH protein is shifted from predominantly cytoplasmic on a high-protein diet to a pronounced nuclear accumulation in animals with reduced levels of TOR or fed with rapamycin. Two putative FKH target genes, CG6770, a nuclear DNA binding phosphoprotein, and cabut, are transcriptionally induced by rapamycin or FKH expression, and silenced by FKH knockdown. Induction of both target genes in heterozygous TOR mutant animals is suppressed by mutations in fkh. Furthermore, TOR signaling levels and FKH impact on transcription of the dFOXO target gene d4E-BP (Thor), implying a point of crosstalk with the insulin pathway. In summary, these observations show that an alteration of FKH levels has an effect on cellular and organismal size, and that FKH function is required for the growth inhibition and target gene induction caused by low TOR signaling levels (Bulow, 2010).

Identification and analysis of cabut orthologs in invertebrates and vertebrates

Cabut (cbt) is a Drosophila melanogaster gene involved in epidermal dorsal closure (DC). Its expression is dependent on the Jun N-terminal kinase (JNK) cascade, and it functions downstream of Jun regulating dpp expression in the leading edge cells. The Cbt protein contains three C₂H₂-type zinc fingers and a serine-rich domain, suggesting that it functions as a transcription factor. This study has identified single cbt orthologs in other Drosophila species, as well as in other insects and invertebrate organisms like ascidians and echinoderms, but not in nematodes. Gene structure and protein sequence are highly conserved among Drosophilidae, but are more diverged in the other species of invertebrates analyzed. According to this, it was demonstrated that cbt expression is detected in the embryonic lateral epidermis in several Drosophila species, as it occurs in D. melanogaster, thus suggesting that the cbt orthologs may have a conserved role in these species during DC. The genomes of several vertebrate species were examined, and the cbt orthologous genes were found in these organisms to encode proteins that belong to the TIEG family of Sp1-like/Kruppel-like transcription factors. Phylogenetic analysis of the invertebrate and vertebrate proteins identified indicates that they mainly follow the expected phylogeny of the species, and that the cbt gene was duplicated during vertebrate evolution. Because it was not possible to identify cbt orthologous either in yeast or in plants, the results suggest that this gene has been probably conserved throughout metazoans and that it may play a fundamental role in animal biology (Munoz-Descalzo, 2007).

Cabut, a C2H2 zinc finger transcription factor, is required during Drosophila dorsal closure downstream of JNK signaling

During dorsal closure, the lateral epithelia on each side of the embryo migrate dorsally over the amnioserosa and fuse at the dorsal midline. Detailed genetic studies have revealed that many molecules are involved in this epithelial sheet movement, either with a signaling function or as structural or motor components of the process. This paper report the characterization of cabut (cbt), a new Drosophila gene involved in dorsal closure. cbt is expressed in the yolk sac nuclei and in the lateral epidermis. The Cbt protein contains three C2H2-type zinc fingers and a serine-rich domain, suggesting that it functions as a transcription factor. cbt mutants die as embryos with dorsal closure defects. Such embryos show defects in the elongation of the dorsal-most epidermal cells as well as in the actomyosin cable assembly at the leading edge. A combination of molecular and genetic analyses demonstrates that cbt expression is dependent on the JNK cascade during dorsal closure, and it functions downstream of Jun regulating dpp expression in the leading edge cells (Munoz-Descalzo, 2005).

Search PubMed for articles about Drosophila Cabut

Bartok, O., Teesalu, M., Ashwall-Fluss, R., Pandey, V., Hanan, M., Rovenko, B.M., Poukkula, M., Havula, E., Moussaieff, A., Vodala, S., Nahmias, Y., Kadener, S. and Hietakangas, V. (2015). The transcription factor Cabut coordinates energy metabolism and the circadian clock in response to sugar sensing. EMBO J 34(11):1538-53. PubMed ID: 25916830

Belacortu, Y., Weiss, R., Kadener, S. and Paricio, N. (2011). Expression of Drosophila Cabut during early embryogenesis, dorsal closure and nervous system development. Gene Expr Patterns 11: 190-201. PubMed ID: 21109026

Belacortu, Y., Weiss, R., Kadener, S. and Paricio, N. (2012). Transcriptional activity and nuclear localization of Cabut, the Drosophila ortholog of vertebrate TGF-beta-inducible early-response gene (TIEG) proteins. PLoS One 7: e32004. PubMed ID: 22359651

Blanco, E., Ruiz-Romero, M., Beltran, S., Bosch, M., Punset, A., Serras, F. and Corominas, M. (2010). Gene expression following induction of regeneration in Drosophila wing imaginal discs. Expression profile of regenerating wing discs. BMC Dev Biol 10: 94. PubMed ID: 20813047

Bulow, M. H., Aebersold, R., Pankratz, M. J. and Junger, M. A. (2010). The Drosophila FoxA ortholog Fork head regulates growth and gene expression downstream of Target of rapamycin. PLoS One 5: e15171. PubMed ID: 21217822

Havula, E., Teesalu, M., Hyotylainen, T., Seppala, H., Hasygar, K., Auvinen, P., Oresic, M., Sandmann, T. and Hietakangas, V. (2013). Mondo/ChREBP-Mlx-regulated transcriptional network is essential for dietary sugar tolerance in Drosophila. PLoS Genet 9: e1003438. PubMed ID: 23593032

Iizuka, K., Takeda, J. and Horikawa, Y. (2011). Kruppel-like factor-10 is directly regulated by carbohydrate response element-binding protein in rat primary hepatocytes. Biochem Biophys Res Commun 412: 638-643. PubMed ID: 21856285

Munoz-Descalzo, S., Terol, J. and Paricio, N. (2005). Cabut, a C2H2 zinc finger transcription factor, is required during Drosophila dorsal closure downstream of JNK signaling. Dev Biol 287: 168-179. PubMed ID: 16198331

Munoz-Descalzo, S., Belacortu, Y. and Paricio, N. (2007). Identification and analysis of cabut orthologs in invertebrates and vertebrates. Dev Genes Evol 217: 289-298. PubMed ID: 17333257

Rodriguez, I. (2011). Drosophila TIEG is a modulator of different signalling pathways involved in wing patterning and cell proliferation. PLoS One 6: e18418. PubMed ID: 21494610

Ruiz-Romero, M., Blanco, E., Paricio, N., Serras, F. and Corominas, M. (2015). Cabut/dTIEG associates with the transcription factor Yorkie for growth control. EMBO Rep 16(3):362-9. PubMed ID: 25572844

Sassu, E. D., McDermott, J. E., Keys, B. J., Esmaeili, M., Keene, A. C., Birnbaum, M. J. and DiAngelo, J. R. (2012). Mio/dChREBP coordinately increases fat mass by regulating lipid synthesis and feeding behavior in Drosophila. Biochem Biophys Res Commun 426: 43-48. PubMed ID: 22910416

Song, M., Zhang, Y., Katzaroff, A. J., Edgar, B. A. and Buttitta, L. (2014). Hunting complex differential gene interaction patterns across molecular contexts. Nucleic Acids Res 42: e57. PubMed ID: 24482443

date revised: 20 June 2015

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