Repressed by TOR: Biological Overview | References
Gene name - Repressed by TOR
Cytological map position - 96A7-96A9
Function - transcription factor
Keywords - together with its binding partner encoded by REPTOR-BP, mediates much of the transcriptional response observed upon Tor complex 1 inhibition - plays critical roles in maintaining energy homeostasis and promoting animal survival upon nutrient restriction
Symbol - REPTOR
FlyBase ID: FBgn0039209
Genetic map position - chr3R:24,557,368-24,582,864
Classification - Basic leucine zipper (bZIP) domain
Cellular location - nuclear and cytoplasmic
TORC1, in Drosophila consisting of Target of rapamycin, Raptor and Lst8) regulates growth and metabolism, in part, by influencing transcriptional programs. This study has identified REPTOR and REPTOR-BP, both leucine zipper DNA-binding proteins, as transcription factors downstream of TORC1 that are required for approximately 90% of the transcriptional induction that occurs upon TORC1 inhibition in Drosophila. Thus, REPTOR and REPTOR-BP are major effectors of the transcriptional stress response induced upon TORC1 inhibition, analogous to the role of FOXO downstream of Akt. When TORC1 is active, it phosphorylates REPTOR on Ser527 and Ser530, leading to REPTOR cytoplasmic retention. Upon TORC1 inhibition, REPTOR becomes dephosphorylated in a PP2A-dependent manner, shuttles into the nucleus, joins its partner REPTOR-BP to bind target genes, and activates their transcription. In vivo functional analysis using knockout flies reveals that REPTOR and REPTOR-BP play critical roles in maintaining energy homeostasis and promoting animal survival upon nutrient restriction (Tiebe, 2015).
Target of rapamycin complex 1 (TORC1) integrates information on energy and nutrient status in eukaryotic cells. Under high-nutrient and -energy conditions, TORC1 drives translation, ribosome biogenesis, mitochondrial activity, lipid synthesis, nucleotide synthesis, and glycolysis. TORC1, thereby, couples activity of cellular anabolic and catabolic pathways to nutrient and energy supply. TORC1 is frequently mis-regulated in diseases such as cancer, diabetes, obesity, and neurodegeneration (Tiebe, 2015).
TORC1 regulates growth and metabolism by phosphorylating target proteins, such as S6K and 4E-BP, involved in translational regulation. Phosphorylation of targets changes very rapidly upon altered TORC1 activity, allowing cells to adapt quickly to changing environmental conditions. In addition, TORC1 also has long-lasting impact on cellular behavior through the control of transcriptional programs. This occurs by directly or indirectly modulating the activity of transcription factors such as SREBP, HIF1a, PGC-1a, TIF1a, PPARa, Atf4 (CREB2), TFEB, and TFE3 (Tiebe, 2015).
The TORC1 signaling pathway is highly conserved through evolution, thereby enabling the use of model organisms such as Drosophila for discovery of novel pathway components. Recent studies in Drosophila analyzed the impact of TORC1 signaling on cellular transcription. In Drosophila S2 cells, inhibition of TORC1 with rapamycin leads to numerous transcriptional changes. Genes involved in anabolic processes such as ribosome biogenesis are strongly repressed upon TORC1 inhibition. Previous work showed that this occurs via downregulation of myc activity (Teleman, 2008). A second class of genes is activated upon TORC1 inhibition. Although the function of these genes is less understood, they probably represent the genes needed for cells to adapt to conditions yielding reduced TORC1 activity, such as low nutrient availability. This study aimed to find the transcription factor responsible for mediating this upregulation upon TORC1 inhibition. The discovery is reported of these factors, which, surprisingly, are required for mediating most of the transcriptional induction that takes place upon TORC1 inhibition and play important roles in maintaining energy homeostasis in vivo (Tiebe, 2015).
This study has identify two uncharacterized genes, CG13624 and CG18619, which are termed REPTOR and REPTOR-BP, respectively, as transcription factors mediating circa 90% of the transcriptional repression downstream of TORC1 in Drosophila, which indicates that they are major effectors of TORC1. REPTOR is inhibited by TORC1-mediated phosphorylation and cytoplasmic retention by 14-3-3 proteins. Upon nutrient deprivation and low TORC1 activity, REPTOR becomes active, accumulating in the nucleus and binding target genes, a process that requires its partner, REPTOR-BP (Tiebe, 2015).
REPTOR is repressed when TORC1 activity is high, as is the case during larval stages when animals are feeding and growing. Hence, genetic removal of REPTOR during larval stages of well-fed animals has little phenotypic consequences, including no growth defects. In contrast, REPTOR is somewhat activated in (1) pupae and adults, which eat significantly less than larvae; (2) in larvae growing in low-nutrient conditions; and (3) in S2 cells growing in standard culture conditions. Hence, under these conditions, REPTOR loss of function leads to transcriptional and physiological phenotypes. The strongest REPTOR phenotypes become apparent when animals are starved, as these are the conditions where TORC1 is most inactive and, hence, REPTOR is most active (Tiebe, 2015).
The REPTOR regulatory system is analogous to another nutrient sensitive pathway-that of Akt and FOXO. When nutrients are low, Akt becomes inactive due to reduced systemic insulin signaling. This leads to FOXO dephosphorylation, release from 14-3-3 proteins, and nuclear accumulation, thereby activating target genes that mount a stress response. FOXO and REPTOR can be thought of as the respective counterparts for insulin and TORC1 signaling, which sense nutrients at the systemic and cell-autonomous levels, respectively. FOXO and REPTOR also have common target genes and bind near each other in shared enhancers. In sum, FOXO and REPTOR appear to have overlapping functions; indeed, genetic removal of both REPTOR and FOXO is synthetic lethal, indicating that they compensate for loss of each other (Tiebe, 2015).
As an effector of TORC1 function, REPTOR mediates some of the physiological effects of reduced TORC1 activity. Body-wide inhibition of TORC1 signaling leads to increased TAG levels and animals of reduced size. These phenotypes are, in part, due to activation of REPTOR, since removal of REPTOR strongly rescues them. Thus, TORC1 promotes growth during development not only by activating S6K but also by keeping REPTOR/REPTOR-BP repressed. Inhibition of TORC1 also extends lifespan. This could potentially be mediated, in part, via activation of REPTOR/REPTOR-BP, since both REPTOR and REPTOR-BP KO animals have significantly reduced lifespans (Tiebe, 2015).
Triglyceride levels were quantified in TOR2L1/2L19 hypomorphic larvae and found to be significantly elevated compared to those in controls, in line with a number of reports from adult flies. These results do not fit with one report that dTOR7/P mutant larvae are lean. The reason for this discrepancy or whether it has to do with the particular nature of the dTOR and/or dTOR[P] alleles is unknown. Further work will be required to examine this carefully (Tiebe, 2015).
Both REPTOR and REPTOR-BP proteins have DBDs. Hence, the DNA-binding specificity of the REPTOR/REPTOR-BP dimer likely reflects the combined action of the two proteins. Since REPTOR-BP can also homodimerize, the REPTOR-BP homodimer might bind DNA with a pattern distinct from that of the REPTOR/REPTOR-BP dimer (Tiebe, 2015).
Many of the genes repressed by rapamycin in larvae (~80%) were no longer repressed by rapamycin in REPTOR KO larvae, raising the possibility that these genes are also REPTOR targets. It is not thought, however, that this is the case for several reasons. (1) In S2 cells, almost no genes require REPTOR to be repressed by rapamycin. If REPTOR were also involved in transcriptional repression, this would likely be seen also in S2 cells. (2) The REPTOR-induced genes are in common between S2 cells and larvae, whereas the REPTOR-repressed ones are not, suggesting their regulation might result from indirect effects. (3) Transactivation assays with REPTOR and REPTOR-BP only show strong transcriptional activation of the reporters but no repression. That said, many transcription complexes have both activating and repressive activities, so further investigation might find that REPTOR and REPTOR-BP also have repressive functions (Tiebe, 2015).
Surprisingly, REPTOR and REPTOR-BP have attracted little attention to date. Microarray studies found that expression of CG13624 and CG18619 are regulated by nutrient conditions in the fly; however, no information regarding their function was available. Using BLAST to compare the human proteome with REPTOR and REPTOR-BP identifies Crebrf and Crebl2, respectively, which could potentially be human orthologs. It will be interesting to study them in light of the Drosophila data (Tiebe, 2015).
In summary, this study identifies REPTOR and REPTOR-BP as dedicated transcription factors that control the transcriptional repression downstream of TORC1 in Drosophila. Since these transcription factors mediate part of the functional output of TORC1, it will be interesting to assess their contribution toward the role that TORC1 plays in cancer, diabetes, and aging (Tiebe, 2015).
Luman/CREB3 recruitment factor (LRF or CREBRF) was identified as a regulator of Luman (or CREB3) that is involved in the unfolded protein response during endoplasmic reticulum stress. Luman is implicated in a multitude of functions ranging from viral infection and immunity to cancer. The biological function of LRF, however, is unknown. This paper reports that uteri of pregnant mice and embryos displayed enhanced LRF expression at all stages, and the expressed LRF was found to be localized specifically at implantation sites. On the other hand, uteri of mice induced for delayed implantation or pseudopregnant mice showed low levels of LRF expression, suggesting that LRF mediates uterine receptivity during implantation. Further, expression of LRF was found to be modulated by steroid hormones such as progesterone and estradiol. This study thereby identifies a potential role for LRF in the process of implantation in uteri and development of preimplantation embryos in mice (Yan, 2013).
Previous work has identified Drosophila REPTOR and REPTOR-BP as transcription factors downstream of mTORC1 that play an important role in regulating organismal metabolism (Tiebe, 2015). The mammalian ortholog of REPTOR-BP is Crebl2. This study finds that Crebl2 mediates part of the transcriptional induction caused by mTORC1 inhibition. In C2C12 myoblasts, Crebl2 knockdown leads to elevated glucose uptake, elevated glycolysis as observed by lactate secretion, and elevated triglyceride biosynthesis. In Hepa1-6 hepatoma cells, Crebl2 knockdown also leads to elevated triglyceride levels. In sum, this works identifies Crebl2 as a regulator of cellular metabolism that can link nutrient sensing via mTORC1 to the metabolic response of cells (Tiebe, 2019).
Metabolic pathways need to be tightly regulated to match a cell's metabolic demands to the nutrients and energy supply provided by the environment. Since metabolic pathways are highly complex networks of enzymes with many components, their regulation is often controlled by transcription factors that have the potential to activate or repress multiple genes at the same time. One of those transcription factors is cAMP response element binding protein (CREB). CREB, among other functions, adapts metabolic demand to energy supply during starvation and has been implicated in diabetes. For instance, CREB is activated upon starvation to promote gluconeogenesis and lipid oxidation in the liver. One distinguishing characteristic of CREB is its basic leucine zipper (bZIP) domain that allows CREB to bind to CREs (cAMP responsive elements). Based on this domain, several CREB-like transcription factors have been identified that are involved in a variety of processes. Previous work has identified two such CREB-like factors REPTOR (CG13624) and REPTOR-BP (CG18619) as transcription factors downstream of mTORC1 that play an important role in regulating metabolism in Drosophila. This study reports that when mTORC1 is active, it phosphorylates REPTOR, leading to its cytoplasmic retention. When mTORC1 activity drops, REPTOR enters the nucleus where it binds REPTOR-BP to induce transcription of a battery of genes that regulate fat metabolism, glycogen metabolism, and the response of adult flies to starvation. In the absence of REPTOR or REPTOR-BP, Drosophila are very lean and extremely sensitive to starvation (Tiebe, 2019).
The mammalian orthologs of REPTOR and REPTOR-BP are Crebrf and Crebl2, respectively. In mice, Crebrf was first described as LRF (Luman recruiting factor) based on the observation that LRF can bind Luman and suppress its function in the unfolded protein response (UPR). Crebrf KO female mice show a lack of instinct to tend their pups and reduced prolactin levels. In this context, Crebrf was found to bind to and repress the glucocorticoid receptor, thereby regulating lactating behavior. Recently, an allele of Crebrf (p.Arg457Gln) was linked to increased BMI and protection from diabetes in Pacific populations of Polynesia. Indeed, this SNP in Crebrf has an effect size that is much larger than that of any other known common BMI risk variant. Hence, like its fly homolog, Crebrf also regulates metabolic traits in humans (Tiebe, 2019).
In contrast to Crebrf, the role of Crebl2 in regulating cellular or organismal metabolism has been less studied. Crebl2 knockout mice have not been reported thus far, however 3T3-L1 cells lacking Crebl2 fail to differentiate into adipocytes. Conversely, overexpression of Crebl2 was sufficient to drive adipogenesis in these cells, and Crebl2 was also shown to bind CREB in this context24. Similar to CREB, Crebl2 can also be phosphorylated by AMPK. Hence Crebl2 plays a role in adipocyte differentiation (Tiebe, 2019).
The role of Crebl2 in regulating cellular metabolism was studied. Like its Drosophila ortholog, Crebl2 binds Crebrf. It was found that loss of Crebl2 has strong consequences on cellular metabolism in both myocytes and hepatocytes, where it regulates glucose uptake and lipid accumulation. This work identifies Crebl2 as a regulator of cellular metabolism and suggests that Crebl2 knockout mice would be worth studying in the future (Tiebe, 2019).
This work identifies Crebl2 is a metabolic regulator. Knockdown of Crebl2 causes a strong lipid metabolism phenotype, leading to a doubling in cellular triglyceride (TAG) stores in C2C12 myoblasts and Hepa1-6 cells. Although Crebl2 regulates lipid metabolism in both C2C12 and Hepa1-6 cells, it does so somewhat differently in these two cell types. In C2C12 cells, Crebl2 knockdown leads to increased glucose uptake. This increased glucose uptake is sufficient to fuel more energy storage in the form of TAGs, as well as elevated glycolysis and lactate secretion. In Hepa1-6 cells, Crebl2 knockdown does not cause increased glucose uptake, nor does it lead to elevated lactate secretion. Hence it seems to only reroute glucose towards energy storage. One possible explanation for this difference could be that Crebl2 knockdown might enhance GLUT4 function leading to increased glucose uptake. Since GLUT4 is expressed in muscle cells but not in hepatocytes, which take up most glucose via GLUT2, this might explain why glucose uptake is unchanged in Hepa1-6 cells upon Crebl2 knockdown. Nonetheless, the increased TAG is a common phenotype in both cell types. Since Crebl2 knockout mice have not been reported, it will be interesting to study the physiological consequences of Crebl2 loss-of-function in an organismal setting. Indeed, a SNP near human Crebl2 was found to be significantly associated with the metabolic parameter Homeostasis Model Assessment of beta-cell function (HOMA-b) (Tiebe, 2019).
Although Crebl2 regulates metabolism, it seems to do so in a tissue-specific manner. A previous report showed that in 3T3-L1 cells Crebl2 is required for adipogenesis. Knockdown of Crebl2 leads to a defect in adipocyte differentiation, and as a consequence impaired TAG accumulation. Hence the final consequence of Crebl2 knockdown in adipocytes is the opposite to what was observed in muscle or liver cells, which is an increase in TAG levels. Furthermore, the genes found dysregulated upon Crebl2 knockdown in C2C12 cells shows little overlap with the genes dysregulated in Hepa1-6 cells. These results highlight that metabolic regulation can be tissue specific, and in particular that Crebl2 likely has tissue specific effects that will add up in a complex manner at the organismal level (Tiebe, 2019).
This study shows that Crebl2 and Crebrf can form a heteromeric complex, analogous to the one formed by their Drosophila orthologs, suggesting they may be functioning together. Indeed, both Crebl2 and Crebrf promote adipogenesis. Hence it is likely that Crebrf also plays a metabolic regulatory role in muscle and liver cells. Furthermore, Crebl2 also binds CREB hence Crebl2 may be acting as a cofactor for several metabolically relevant transcription factors of the CREB family (Tiebe, 2019).
In Drosophila it was found that the orthologous proteins REPTOR and REPTOR-BP are regulated by mTORC1 activity. When mTORC1 activity is high, this leads to phosphorylation and cytosolic retention of REPTOR. When mTORC1 activity drops, REPTOR enters the nucleus where it binds REPTOR-BP and activates expression, thereby altering organismal metabolism. There are both similarities and differences between the fly and mammalian proteins. Both Crebl2 and REPTOR-BP are required for the induction of most of the genes that are transcriptionally induced upon mTORC1 inhibition. Both the fly and human genes regulate metabolism. Both REPTOR and Crebrf are induced upon mTORC1 inhibition. However, in the case of REPTOR this happens via phosphorylation whereas for Crebrf this occurs at least in part via transcriptional induction. It cannot be excluded that Crebrf might also be regulated by mTORC1 at the post-translational level. Unfortunately, it was not possible to assess Crebrf subcellular localization because generating Crebrf antibody was unsuccessful despite several attempts, and the localization of epitope-tagged Crebrf depends on the tag that is used. Although relocalization of epitope-tagged Crebrfupon rapamycin treatment was not seen, this could be due to overexpression, or to interference by the epitope tags. Hence further work will be required to address this issue by detecting the endogenous protein (Tiebe, 2019).
Circa 61 genes were found that are transcriptionally induced by rapamycin in MEFs, and the induction of many of these is blunted upon Crebl2 knockdown. These target genes can be grouped into two classes. Genes in the major class have low expression in the absence of Crebl2, and reduced induction in response to rapamycin, such as Bloc1s1. Genes in the second class have elevated basal expression upon Crebl2 knockdown and consequently blunted induction, such as Cxcl12. One possible explanation for this second class of targets is that perhaps Crebl2 suppresses their expression, and this repression is alleviated by mTORC1 inhibition. However, in this case Crebl2 would need to be activated by mTORC1 (for the 2nd class) and repressed by mTORC1 (for the 1st class) which makes this scenario unlikely. The second possible explanation is that Crebl2 always induces gene expression and is activated by rapamycin, but for some genes, basal expression levels increase upon loss of Crebl2 due to compensatory increases in other signaling pathways. Indeed, this has been seen this in the past, for instance for FOXO where loss of FOXO leads to increased basal levels of the bona-fide target Lk6 rather than reduced levels of the induced state (Tiebe, 2019).
How does Crebl2 knockdown cause increased cellular TAG levels? Crebl2 targets were tested in both C2C12 and Hepa1-6 cells to see if any of them reproduces the high-TAG phenotype caused by Crebl2 knockdown. Although many showed a tendency towards increasing cellular TAG levels, none of them gave as strong and as consistent a phenotype as Crebl2 itself. Hence it is believed most likely the phenotype caused by Crebl2 knockdown represents the combined sum of several different target genes. Surprisingly, the most enriched Gene Ontology category for both up-regulated and down-regulated transcripts upon Crebl2 knockdown in C2C12 cells are mRNAs having to do with protein secretion and glycosylation. Encoded by these mRNAs are both proteins involved in the secretory process, as well as proteins that are secreted themselves. The fact that some are up-regulated and some are down-regulated suggests that Crebl2 knockdown leads to a change in the cells' secretome. The biological significance of this, and the relationship to metabolism, is unclear, and a topic for future study (Tiebe, 2019).
In sum, this study shows that Crebl2 mediates part of the transcriptional response caused by mTORC1 inhibition and identifies Crebl2 as a metabolic regulator (Tiebe, 2019).
Search PubMed for articles about Drosophila REPTOR
Teleman, A. A., Hietakangas, V., Sayadian, A. C. and Cohen, S. M. (2008). Nutritional control of protein biosynthetic capacity by insulin via Myc in Drosophila. Cell Metab 7(1): 21-32. PubMed ID: 18177722
Tiebe, M., Lutz, M., De La Garza, A., Buechling, T., Boutros, M. and Teleman, A. A. (2015). Reptor and Reptor-BP regulate organismal metabolism and transcription downstream of TORC1. Dev Cell 33: 272-284. PubMed ID: 25920570
Tiebe, M., Lutz, M., Senyilmaz Tiebe, D. and Teleman, A. A. (2019). Crebl2 regulates cell metabolism in muscle and liver cells. Sci Rep 9(1): 19869. PubMed ID: 31882710
date revised: 10 May 2020
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