InteractiveFly: GeneBrief

CREB-regulated transcription coactivator: Biological Overview | References

BIOLOGICAL OVERVIEW

In fasted mammals, glucose homeostasis is maintained through induction of the cAMP response element-binding protein coactivator transducer of regulated CREB activity 2 (preferred name: CREB-regulated transcription coactivator; aka TORC2), which stimulates the gluconeogenic program in concert with the forkhead factor FOXO1 (see Drosophila Foxo). Starvation also triggers TORC activation in Drosophila, where it maintains energy balance through induction of CREB target genes in the brain. TORC mutant flies have reduced glycogen and lipid stores and are sensitive to starvation and oxidative stress. Neuronal TORC expression rescued stress sensitivity as well as CREB target gene expression in TORC mutants. During refeeding, increases in insulin signaling inhibited TORC activity through the salt-inducible kinase 2 (SIK2)-mediated phosphorylation and subsequent degradation of TORC. Depletion of neuronal SIK2 increased TORC activity and enhanced stress resistance. As disruption of insulin signaling also augments TORC activity in adult flies, these results illustrate the importance of an insulin-regulated pathway that functions in the brain to maintain energy balance (Wang, 2008).

Fasting triggers concerted changes in behavior, physical activity, and metabolism that are remarkably well conserved through evolution. In mammals, such responses are often coordinated by transcriptional coactivators that are themselves targets for regulation by environmental cues, but the extent to which these coactivators function in model organisms such as Drosophila is less clear (Wang, 2008).

In the basal state, mammalian TORCs are phosphorylated by salt-inducible kinases (SIKs) and sequestered in the cytoplasm via phosphorylation-dependent association with 14-3-3 proteins (Koo, 2005; Screaton, 2004). During fasting, elevations in circulating pancreatic glucagon promote TORC dephosphorylation via the protein kinase A (PKA)-mediated phosphorylation and inhibition of SIK2 (Wang, 2008).

Increases in intracellular calcium have also been found to stimulate cAMP response element-binding protein (CREB) target gene expression through the activation of calcineurin/PP2B, a calcium/calmodulin-dependent serine/threonine (Ser/Thr) phosphatase that binds directly to and dephosphorylates mammalian TORCs (Koo, 2005; Screaton, 2004). Indeed, cAMP and calcium signals stimulate TORC dephosphorylation cooperatively through their effects on SIKs and PP2B, respectively. Following their liberation from 14-3-3 proteins, dephosphorylated TORCs shuttle to the nucleus, where they mediate cellular gene expression by associating with CREB over relevant promoters (Wang, 2008).

TORC2 is thought to function in parallel with FOXO1 to maintain energy balance during fasting. Knockdown and knockout studies support a critical role for both proteins in regulating catabolic programs in the liver (Dentin, 2007, Koo, 2005; Matsumoto, 2007). In Drosophila, starvation promotes the mobilization of glycogen and lipid stores in response to increases in circulating adipokinetic hormone (AKH), the fly homolog of mammalian glucagon (Kim, 2004; Lee, 2004). In parallel, decreases in insulin/IGF signaling (IIS) also stimulate the dephosphorylation and nuclear translocation of Drosophila FOXO, which in turn stimulates a wide array of nutrient-regulated genes (Wang, 2008).

The accumulation of lipid and glycogen stores in adult flies is highly correlated with resistance to starvation in Drosophila (Djawdan, 1998). Indeed, disruption of the IIS pathway promotes lipid accumulation and correspondingly increases resistance to starvation and oxidative stress. Although FOXO does not appear to be required for starvation resistance in adult flies, overexpression of FOXO has been found to mimic the starvation phenotype in larvae (Wang, 2008).

This study addresses the importance of Drosophila TORC, the single Drosophila homolog of mammalian TORCs, in metabolic regulation. It was found that increases in TORC activity during starvation enhance survival through the activation of CREB target genes in the brain. During feeding, increases in insulin signaling inhibit TORC activity through phosphorylation by a Drosophila homolog of mammalian SIK2. These studies indicate that TORC is part of an insulin-regulated pathway that functions in concert with FOXO to promote energy balance and stress resistance (Wang, 2008).

Drosophila TORC shares considerable sequence homology with mammalian TORCs in its CREB binding domain (CBD), transactivation domain (TAD), calcineurin (Cn) recognition motif, and regulatory site (Ser157), which is phosphorylated by members of the AMPK family of stress- and energy-sensing Ser/Thr kinases in mammals. Drosophila TORC protein is expressed at low levels during larval and pupal stages, with the highest amounts detected in adults. TORC mRNA levels are also increased in adults relative to larvae, although to a lesser extent (Wang, 2008).

In the basal state, Drosophila TORC is highly phosphorylated at Ser157 and localized to the cytoplasm in Drosophila S2 and KC-167 cells. Demonstrating the importance of Ser157 phosphorylation in sequestering TORC, Ser157Ala mutant TORC shows only low-level binding to 14-3-3 proteins relative to wild-type TORC in HEK293T cells. Exposure to the adenylyl cyclase activator forskolin (FSK) or to staurosporine (STS), an inhibitor of SIKs and other protein kinases, promotes TORC dephosphorylation, liberation from 14-3-3 proteins, and nuclear translocation (Wang, 2008).

Consistent with these changes, overexpression of wild-type Drosophila TORC potentiated CRE-luciferase (CRE-luc) reporter activity following exposure of HEK293T cells to FSK, whereas phosphorylation-defective Ser157Ala TORC stimulated CRE-luc activity under basal as well as FSK-induced conditions. CRE-luc activity is blocked by coexpression of the dominant-negative CREB inhibitor ACREB. Taken together, these results indicate that Drosophila TORC modulates CREB target gene expression following its dephosphorylation at Ser157 and nuclear entry in response to cAMP (Wang, 2008).

Based on the ability of mammalian TORCs to promote fasting metabolism (Koo, 2005), this study examined whether Drosophila TORC performs a similar function in adult flies. Amounts of dephosphorylated, active TORC increased progressively during water-only starvation. Feeding adult flies paraquat, a respiratory chain inhibitor that stimulates the production of reactive oxygen species, also promoted the accumulation of dephosphorylated TORC, suggesting a broader role for this coactivator in stress resistance. Similar to mammalian TORCs (Dentin, 2007), the upregulation of TORC in Drosophila appears to reflect an increase in TORC protein stability, as amounts of TORC mRNA did not change significantly in response to fasting or paraquat treatment (Wang, 2008).

Insulin signaling regulates lipid and glucose metabolism in both C. elegans and Drosophila in part by inhibiting FOXO-dependent transcription. Lipid stores are increased in flies with mutations in the IIS pathway; these animals are resistant to starvation as well as oxidative stress. This study found that TORC enhances survival during starvation in part by stimulating target gene expression in neurons. Although TORC appears to act in parallel with FOXO, the increase in FOXO activity observed in TORC mutant flies indicates that TORC may also impact on this pathway (Wang, 2008).

TORC appears to be required for the expression of genes that promote lipid and glucose metabolism, amino acid transport, and proteolysis. Consistent with this idea, paralogs for a number of TORC-regulated genes (TrxT, CAT, and UCP4c) appear to be required for starvation and oxidative stress resistance (Chen, 2004; Fridell, 2005; Mockett, 2003; Svensson, 2007). Superimposed on these effects, neuronal TORC may also promote systemic resistance to starvation and oxidative stress by modulating the expression of neuropeptide hormones and other circulating factors that regulate peripheral glucose and lipid metabolism (Wang, 2008).

In mammals, refeeding has been found to increase SIK2 kinase activity during refeeding through the AKT-mediated phosphorylation of SIK2 (Dentin, 2007). Phosphorylated TORC2 is subsequently ubiquitinated and degraded through the E3 ligase COP1. Supporting a similar mechanism in Drosophila, RNAi-mediated knockdown of AKT in Drosophila is sufficient to increase TORC activity. Conversely, depletion of neuronal SIK2 enhances TORC activity and increases resistance to both starvation and paraquat feeding. Although a Drosophila homolog for COP1 has not been identified, it is imagined that the ubiquitin-dependent degradation of Drosophila TORC is also critical in modulating its activity in brain as well as other tissues (Wang, 2008).

Based on its ability to potentiate CREB target gene expression in neurons, TORC may function in other biological settings. Indeed, Drosophila CREB appears to have an important role in learning and memory, circadian rhythmicity, rest homeostasis, and addictive behavior. Future studies should reveal the extent to which TORC participates in these contexts as well (Wang, 2008).

CRTC potentiates light-independent timeless transcription to sustain circadian rhythms in Drosophila

Light is one of the strongest environmental time cues for entraining endogenous circadian rhythms. Emerging evidence indicates that CREB-regulated transcription co-activator 1 (CRTC1) is a key player in this pathway, stimulating light-induced Period1 (Per1) transcription in mammalian clocks. This study demonstrates a light-independent role of Drosophila CRTC in sustaining circadian behaviors. Genomic deletion of the crtc locus causes long but poor locomotor rhythms in constant darkness. Overexpression or RNA interference-mediated depletion of CRTC in circadian pacemaker neurons similarly impairs the free-running behavioral rhythms, implying that Drosophila clocks are sensitive to the dosage of CRTC. The crtc null mutation delays the overall phase of circadian gene expression yet it remarkably dampens light-independent oscillations of TIMELESS (TIM) proteins in the clock neurons. In fact, CRTC overexpression enhances CLOCK/CYCLE (CLK/CYC)-activated transcription from tim but not per promoter in clock-less S2 cells whereas CRTC depletion suppresses it. Consistently, TIM overexpression partially but significantly rescues the behavioral rhythms in crtc mutants. Taken together, these data suggest that CRTC is a novel co-activator for the CLK/CYC-activated tim transcription to coordinate molecular rhythms with circadian behaviors over a 24-hour time-scale. The study proposes that CRTC-dependent clock mechanisms have co-evolved with selective clock genes among different species (Kim, 2016).

CREB-dependent transcription has long been implicated in different aspects of circadian gene expression. In mammalian clocks, light exposure triggers intracellular signaling pathways that activate CREB-dependent Per1 transcription, thereby adjusting the circadian phase of master circadian pacemaker neurons in the suprachiasmatic nucleus (SCN). The phase-resetting process involves the specific CREB coactivator CRTC1 and its negative regulator SIK1, constituting a negative feedback in the photic entrainment via a CREB pathway (Sakamoto, 2013; Jagannath, 2013). This report demonstrates a novel role of Drosophila CRTC that serves to coordinate circadian gene expression with 24-hour locomotor rhythms even in the absence of light. CRTC may regulate several clock-relevant genes, including those clock output genes that might be involved in the rhythmic arborizations and PDF cycling of the circadian pacemaker neurons. However, tim transcription was identified as one of the primary targets of Drosophila CRTC to sustain circadian rhythms in the free-running conditions, thus defining its light-independent clock function (Kim, 2016).

CREB could employ another transcriptional coactivator CBP (CREB-binding protein) to activate CRE-dependent transcription. In fact, CBP is a rather general coactivator recruited to gene promoters by other DNA-binding transcription factors. Previous studies have shown that Drosophila CBP associates with CLK, titrating its transcriptional activity. Mammalian CBP and the closely related coactivator p300 also form a complex with CLOCK-BMAL1, a homolog of the Drosophila CLK-CYC heterodimer, to stimulate their transcriptional activity. One possible explanation for CRTC-activated tim transcription is that Drosophila CRTC may analogously target the CLK-CYC heterodimer to stimulate CLK-CYC-tivate CRE-dependent transcription. Under these circumstances, a circadian role of light-sensitive TIM might have degenerated, while-induced clock genes. Moreover, CRTC associates with the bZIP domain in CREB protein, whereas CBP/p300 binds CREB through the phosphorylated KID domain, indicating that they might not necessarily target the same transcription factors apart from CREB. Finally, a protein complex of CLK and CRTC could not be detected in Drosophila S2 cells. Thus, it is likely that CRTC and CBP/p300 play unique roles in circadian transcription through their interactions with different DNA-binding transcription factors (Kim, 2016).

If CRTC augments CLK-CYC-dependent tim transcription indirectly, then why do crtc effects require CLK? A recent study suggested that mammalian CLOCK-BMAL1 may regulate the rhythmic access of other DNA-binding transcription factors to their target promoters in the context of chromatin, acting as a pioneer-like transcription factor. Given the structural and functional homology between Drosophila CLK-CYC and mammalian CLOCK-BMAL1, the presence of CLK-CYC in the tim promoter might allow the recruitment of additional transcription factors (e.g., CREB) and their co-activators including CRTC for maximal tim transcription. The transcriptional context of tim promoter might thus define its sensitivity to crtc effects among other clock promoters. In addition, the differential assembly of transcription factors on the tim promoter could explain tissue-specific effects of crtc on TIM oscillations (i.e., circadian pacemaker neurons versus peripheral clock tissues). Interestingly, chromatin immunoprecipitation with V5-tagged CLK protein revealed that CLK-CYC heterodimers associate with both tim and Sik2 gene promoters in fly heads. In LD cycles, however, their rhythmic binding to the Sik2 promoter is phase-delayed by ~4.5 hours compared with that to the tim promoter. These modes of transcriptional regulation may gate crtc effects on tim transcription in a clock-dependent manner, particularly in the increasing phase of tim transcription (Kim, 2016).

Transcription from CREB-responsive reporter genes shows daily oscillations, both in Drosophila and mammals, implicating this transcriptional strategy in the evolution of molecular clocks. In fact, cAMP signaling and CRE-dependent transcription constitute the integral components of core molecular clocks, serving to regulate daily rhythmic transcription of circadian clock genes. For instance, reciprocal regulation of dCREB2 and per at the transcription level has been reported to sustain free-running circadian rhythms in Drosophila. During fasting in mammals, a transcriptional program for hepatic gluconeogenesis is induced by CREB phosphorylation and CRTC2 dephosphorylation. Fasting-activated CREB-CRTC2 then stimulates Bmal1 expression61, whereas CLOCK-BMAL1-induced CRY rhythmically gates CREB activity in this process by modulating G protein-coupled receptor activity and inhibiting cAMP-induced CREB phosphorylation62. This molecular feedback circuit thus mutually links mammalian clocks and energy metabolism in terms of CREB-dependent transcription (Kim, 2016).

On the basis of these observations, a model is proposed for the evolution of CRTC-dependent clocks to explain the distinctive circadian roles of CRTC homologs (see A model for the evolution of CRTC-dependent clocks). CRTC is a transcriptional effector that integrates various cellular signals (Altarejos, 2011). It was reasoned that ancestral clocks may have employed CREB-CRTC-mediated transcription to sense extracellular time cues cell-autonomously and integrate this timing information directly into the earliest transcription-translation feedback loop (TTFL). This strategy would have generated simple but efficient molecular clocks to tune free-running molecular rhythms in direct response to environmental zeitgebers, such as light and the availability of nutrients. A circadian role of CRTC then has differentially evolved along with a selective set of clock targets. In poikilothermic Drosophila, light is accessible directly to circadian pacemaker neurons in the adult fly brain. Therefore, TIM degradation by the blue-light photoreceptor CRY plays a major role in the light entrainment of Drosophila clocks, although the photic induction of CLK/CYC-dependent tim transcription has been reported specifically at lower temperatures. Accordingly, Drosophila CRTC retained a constitutive co-activator function from the ancestral TTFL to support CLK/CYC-activated tim transcription and sustain free-running circadian behaviors. In homeothermic mammals, light input to the SCN is indirectly mediated by neurotransmitter release from presynaptic termini of the retinohypothalamic tract (RHT). Intracellular signaling relays in the SCN converge on the dephosphorylation and nuclear translocation of CRTC1 to activate CRE-dependent transcription. Under these circumstances, a circadian role of light-sensitive TIM might have degenerated, while per took over a role in the light-entrainment pathway by retaining CREB-CRTC1-dependent transcriptional regulation from the primitive TTFL. Consequently, mammalian clocks have lost a homolog of the Drosophila-like cry gene family, but instead evolved CRY homologs of the vertebrate-like cry gene family with transcriptional repressor activities in CLOCK-BMAL1-dependent transcription (Kim, 2016).

Regulation of metabolism and stress responses by neuronal CREB-CRTC-SIK pathways has been well documented in Drosophila. Given the demonstration of a circadian role of CRTC in the pacemaker neurons, it is possible that CRTC might sense metabolic cues in the context of circadian neural circuits to entrain molecular clocks cell-autonomously. Alternatively, but not exclusively, CRTC could participate in the regulation of clock-relevant metabolism as clock outputs from pacemaker neurons. These hypotheses remain to be validated in future studies (Kim, 2016).

Shifting transcriptional machinery is required for long-term memory maintenance and modification in Drosophila mushroom bodies

Accumulating evidence suggests that transcriptional regulation is required for maintenance of long-term memories (LTMs). This study characterized global transcriptional and epigenetic changes that occur during LTM storage in the Drosophila mushroom bodies (MBs), structures important for memory. Although LTM formation requires the CREB transcription factor and its coactivator, CBP, subsequent early maintenance requires CREB and a different coactivator, CRTC. Late maintenance becomes CREB independent and instead requires the transcription factor Beadex, also know as LIM-only. Bx expression initially depends on CREB/CRTC activity, but later becomes CREB/CRTC independent. The timing of the CREB/CRTC early maintenance phase correlates with the time window for LTM extinction and this study identified different subsets of CREB/CRTC target genes that are required for memory maintenance and extinction. Furthermore, it was found that prolonging CREB/CRTC-dependent transcription extends the time window for LTM extinction. These results demonstrate the dynamic nature of stored memory and its regulation by shifting transcription systems in the MBs (Hirano, 2016).

This study has identified Bx and Smr as LTM maintenance genes and has characterize a shift in transcription between CREB/CRTC-dependent maintenance (1-4 days) to Bx-dependent maintenance (4-7 days). In addition, a biological consequence of this shift was identified in defining a time window during which LTM can be modified, β-Spec was identified as being required for memory extinction (Hirano, 2016).

LTM maintenance mechanisms change dynamically during storage. In particular, CRTC, which is not required during memory formation, becomes necessary during 4-day LTM maintenance and then becomes dispensable again. Consistent with this, CRTC translocates from the cytoplasm to the nucleus of MB neurons during 4-day LTM maintenance and returns to the cytoplasm within 7 days. On the other hand, Bx expression is increased at both phases, suggesting that transcriptional regulation of memory maintenance genes may change between these two phases. Supporting this idea, it was found that Bx expression requires CRTC during 4-day LTM maintenance but becomes independent of CRTC 7 days after training. It is proposed that CREB/CRTC activity induces Bx expression, which subsequently activates a feedback loop where Bx maintains its own expression and that of other memory maintenance genes (Hirano, 2016).

Although it is proposed that the shifts in transcriptional regulation that were observed occur temporally in the same cells, the possibility cannot be discounted that LTM lasting 7 days is maintained in different cells from LTM lasting 4 days. MB Kenyon cells can be separated into different cell types, which exert differential effects on learning, short-term memory and LTM. Thus, it is possible that LTM itself consists of different types of memory that can be separated anatomically. In this case, CRTC in one cell type may exert non-direct effects on another cell type to activate downstream genes including Bx and Smr. However, as that CRTC binds to the Bx gene locus to promote Bx expression and both CRTC and Bx are required in the same α/β subtype of Kenyon cells, it is likely that the shift from CRTC-dependent to Bx-dependent transcription occurs within the α/β neurons (Hirano, 2016).

Currently, it is proposed that the alterations in histone acetylation and transcription that were uncovered are required for memory maintenance. However, it is noted that decreases in memory after formation could be caused by defects in retrieval and maintenance. Thus, it remains formally possible that the epigenetic and transcriptional changes reported in this study are required for recall, but not maintenance. However, this is unlikely, as inhibition of CRTC from 4 to 7 days after memory formation does not affect 7 day memory, whereas inhibition from 1 to 4 days does. This suggests that at least one function of CRTC is to maintain memory for later recall (Hirano, 2016).

Consistent with a previous study in mice, which suggests distinct transcriptional regulations in LTM formation and maintenance (Halder, 2016), the data indicate that memory formation and maintenance are distinct processes. Although the HAT, CBP, is required for formation but dispensable for maintenance, other HATs, GCN5 and Tip60, are required for maintenance but dispensable for formation. Through ChIP-seq analyses, those downstream genes, Smr and Bx, were identified as LTM maintenance genes and these are not required for LTM formation. Collectively, these results suggest differential requirements of histone modifications between LTM formation and maintenance. Although other histone modifiers besides GCN5 and Tip60 were identified in the screen, knockdown of these histone modifiers did not affect LTM maintenance. There are ~50 histone modifiers encoded in the fly genome, raising the possibility that the lack of phenotype in some knockdown lines is due to compensation by other modifiers (Hirano, 2016).

The results indicate some correlation of increase in CRTC binding with histone acetylation and gene expression. Interestingly, DNA methylation shows higher correlation to gene expression in comparison with histone acetylation in mice. Notably, flies lack several key DNA methylases and lack detectable DNA methylation patterns. Hence, histone acetylation rather than DNA methylation may have a higher correlation with transcription in flies. Reduction in histone acetylation was detected, overlapping with increase in CRTC binding. Those reductions could be due to CRTC interacting with a repressor isoform of CREB, CREB2b or other transcriptional repressor that binds near CREB/CRTC sites. These interactions would decrease histone acetylation and gene expression, and may be related to LTM maintenance. Although this study focused on the upregulation of gene expression through CREB/CRTC, downregulation of gene expression by transcriptional repressors may also be important in understanding the transcriptional regulation in LTM maintenance. The results demonstrate the importance of HATs for LTM maintenance; however, the data do not conclude that histone acetylation is a determinant for gene expression, but rather it might be a passive mark of gene expression. HATs also target non-histone proteins and also interact with various proteins, both of which could support gene expression in LTM maintenance (Hirano, 2016).

Similar to traumatic fear memory in rodents, this study found that aversive LTM in flies can be extinguished by exposing them to an extinction protocol specifically during 4-day LTM maintenance. These observations suggest the time-limited activation of molecules that allows LTM extinction only during the early storage. Supporting this concept, it was found that CRTC is activated during the extinguishable phase of LTM maintenance and prolonging CRTC activity extends the time window for extinction. Thus, CRTC is the time-limited activated factor determining the time window for LTM extinction in flies. In cultured rodent hippocampal neurons, CRTC nuclear translocation is not sustained, suggesting that other transcription factors may function in mammals to restrict LTM extinction (Hirano, 2016).

This work demonstrates that LTM formation and maintenance are distinct, and involve a shifting array of transcription factors, coactivators and HATs. A key factor in this shift is CRTC, which shows a sustained but time-limited translocation to the nucleus after spaced training. Thus, MB neurons recruit different transcriptional programmes that enable LTM to be formed, maintained and extinguished (Hirano, 2016).

Neuronal energy-sensing pathway promotes energy balance by modulating disease tolerance

The starvation-inducible coactivator cAMP response element binding protein (CREB)-cAMP-regulated transcription coactivator (Crtc) has been shown to promote starvation resistance in Drosophila by up-regulating CREB target gene expression in neurons, although the underlying mechanism is unclear. This study found that Crtc and its binding partner CREB enhance energy homeostasis by stimulating the expression of short neuropeptide F (sNPF), an ortholog of mammalian neuropeptide Y, which was shown to be a direct target of CREB and Crtc. Neuronal sNPF was found to promote energy homeostasis via gut enterocyte sNPF receptors, which appear to maintain gut epithelial integrity. Loss of Crtc-sNPF signaling disrupts epithelial tight junctions, allowing resident gut flora to promote chronic increases in antimicrobial peptide (AMP) gene expression that compromised energy balance. Growth on germ-free food reduces AMP gene expression and rescues starvation sensitivity in Crtc mutant flies. Overexpression of Crtc or sNPF in neurons of wild-type flies dampens the gut immune response and enhances starvation resistance. These results reveal a previously unidentified tolerance defense strategy involving a brain-gut pathway that maintains homeostasis through its effects on epithelial integrity (Shen, 2016).

Disruptions in energy balance are a component of the collateral damage associated with mounting an immune response. In addition to regulating the magnitude of an immune response, energy allocation must be properly regulated to minimize physiological damage during infection. This study found that Drosophila sNPF, a mammalian NPY homolog, is regulated by CrebB/Crtc within the CNS, where it promotes energy balance by maintaining epithelial integrity and thereby attenuating overexuberant immune activation in the gut. The effects of sNPF were unexpected, given its role in food-seeking behavior. Indeed, food intake appears comparable between Crtc mutants and control flies (Shen, 2016).

The effects of sNPF are mediated by enterocyte sNPF-Rs, suggesting that the sNPF brain-gut signal is released by a subset of the sNPF+ neurons that directly innervate the gut. Although neuronal activity is known to contribute to energy homeostasis, the results suggest that the modulation of the gut immune system by CrebB/Crtc is a critical component in this process (Shen, 2016).

Epithelial tissues are typically colonized by both commensal and invasive microbes. sNPF appears to be actively expressed and released from the CNS in times of stress, providing nonautonomous control of gut immunity from the brain. Based on its widespread expression in the midgut, sNPF-R may provide ubiquitous attenuation of the innate immune response. Consistent with observations in Drosophila, activation of the NPY receptor ortholog (NPR-1) in Caenorhabditis elegans also down-regulates inflammatory gene expression. The current studies extend these findings by showing how a neuronal fasting-inducible pathway modulates energy balance via its effects on the gut immune system (Shen, 2016).

Following their activation, sNPF-Rs appear to promote energy balance by enhancing epithelial integrity. Although the mechanism underlying these effects is unclear, it is noted that disruption of the tight junction protein Bbg in flies also causes constitutive up-regulation of innate immunity genes. Future studies should reveal whether sNPF-R modulates the activity of Bbg or related proteins in enterocytes (Shen, 2016).

In mammals, inflammatory bowel diseases, such as ulcerative colitis, are often associated with profound weight loss, due, in part, to the chronic activation of the immune system. By reducing inflammatory gene expression and enhancing energy homeostasis, gut neuropeptides, such as NPY, may provide therapeutic benefit in this setting (Shen, 2016).

Fasting launches CRTC to facilitate long-term memory formation in Drosophila

Canonical aversive long-term memory (LTM) formation in Drosophila requires multiple spaced trainings, whereas appetitive LTM can be formed after a single training. Appetitive LTM requires fasting prior to training, which increases motivation for food intake. However, this study found that fasting facilitates LTM formation in general; aversive LTM formation also occurred after single-cycle training when mild fasting was applied before training. Both fasting-dependent LTM (fLTM) and spaced training-dependent LTM (spLTM) requires protein synthesis and cyclic adenosine monophosphate response element-binding protein (CREB) activity. However, spLTM requires CREB activity in two neural populations--mushroom body and dorsal-anterior-lateral (DAL) neurons--whereas fLTM required CREB activity only in mushroom body neurons. fLTM uses the CREB coactivator CREB-regulated transcription coactivator (CRTC), whereas spLTM uses the coactivator CBP. Thus, flies use distinct LTM machinery depending on their hunger state (Hirano, 2013).

In Drosophila, canonical aversive long-term memory (LTM), which is dependent on de novo gene expression and protein synthesis, is generated after multiple rounds of spaced training. In contrast, appetitive LTM can be formed by single-cycle training. Because both aversive and appetitive LTM require protein synthesis and activation of CREB, it is likely that both types of LTM are formed by similar mechanisms. Appetitive and aversive LTM are known to differ (i.e., octopamine is specifically involved in appetitive but not aversive memory formation). However, it remains unclear why single-cycle training is sufficient for appetitive but not aversive LTM formation. Appetitive LTM cannot form unless fasting precedes training. Although fasting increases motivation for food intake (a requirement for appetitive memory) it was suspected that fasting may activate a second, motivation-independent, memory mechanism that facilitates LTM formation after single-cycle training (Hirano, 2013).

Flies were deprived of food for various periods of time and then subjected to aversive single-cycle training. Fasting prior to training significantly enhanced 1-day memory, with a peak at 16 hours of fasting and a return to nonfasting levels at 20 to 24 hours of fasting. In contrast, 16 hours of fasting did not increase short-term memory (STM, measured 1 hour after training). In this protocol, flies were returned to food vials after training, raising a possibility that the perception of food as a reward after training may enhance the previous aversive memory. This possibility was tested by inserting refeeding periods between food deprivation and training. Although fasting followed by a 4-hour refeeding period failed to induce appetitive LTM, it significantly enhanced aversive 1-day memory; this finding suggests that enhancement of aversive memory occurs through a mechanism unrelated to increased motivation or perception of food as a reward. A 6-hour refeeding period was sufficient to prevent aversive memory enhancement. Continuous food deprivation after training suppressed aversive memory enhancement, which indicates that both fasting before training and feeding after training are required to enhance aversive memory (Hirano, 2013).

Administration of the protein synthesis inhibitor cycloheximide (CHX) abolished 1-day memory enhancement but had no effect on 1-hour memory, supporting the idea that memory enhancement consists of an increase of LTM. Memory remaining after CHX treatment is likely to be protein synthesis-independent, anesthesia-resistant memory (ARM). Fasting for 16 hours neither enhanced protein synthesis-independent memory nor canonical aversive LTM generated by spaced training (spLTM). Furthermore, fasting-dependent memory decayed within 4 days, and food deprivation did not enhance 4-day spLTM, indicating that fasting-dependent memory is physiologically different from spLTM (Hirano, 2013).

Fasting-dependent memory was blocked by acute, dose-dependent, expression of CREB2-b, a repressor isoform of CREB, in the mushroom bodies (MBs). Expression of the repressor from two copies of UAS-CREB2-b under control of the MB247-Switch (MBsw) GAL4 driver, which induces UAS transgene expression upon RU486 feeding, significantly suppressed fasting-dependent memory upon RU486 feeding, whereas expression from one copy of UAS-CREB2-b did not. Defects in LTM formation are highly correlated with CREB2-b amounts. Significantly higher MBsw-dependent expression of CREB proteins was found in flies carrying two copies of UAS-CREB2-b relative to flies carrying one copy. MBsw-dependent CREB2-b expression did not affect STM in either fed or food-deprived conditions. Because the aversive memory enhanced by fasting is mediated by protein synthesis and CREB, this memory is referred to as fasting-dependent LTM (fLTM). Similar to the results in aversive fLTM, MBsw-dependent CREB2-b expression also decreased appetitive LTM but not appetitive STM (Hirano, 2013).

A recent study concluded that CREB activity in MB neurons is not required for spLTM. In that study, CREB2-b was expressed using the OK107 MB driver and GAL80ts was used to restrict CREB2-b expression to 30°C. However, this study found that the GAL80ts construct still inhibited expression of CREB considerably at 30°C. When higher amounts of CREB2-b were acutely expressed in MBs using MBsw, a significant decrease was observed in 1-day spLTM, indicating that CREB activity in the MBs is likely to be required for spLTM (Hirano, 2013).

Consistent with the previous results expression of CREB2-b in two dorsal-anterior-lateral (DAL) neurons impaired aversive spLTM. In contrast, expression of CREB2-b in DAL neurons did not affect aversive fLTM. Moreover, appetitive LTM was also not affected by expression of CREB2-b in DAL neurons. MBsw did not express GAL4 in DAL neurons (Hirano, 2013).

CREB requires coactivators, including CBP (CREB-binding protein), to activate transcription needed for LTM formation. Acute expression of an inverted repeat of CBP (CBP-IR) in MBs significantly impaired spLTM without affecting either STM or 1-day memory after multiple massed trainings, which do not lead to LTM formation. However, neither aversive fLTM nor appetitive LTM was impaired by CBP-IR expression, indicating that an alternative coactivator may be required for fasting-dependent memory (Hirano, 2013).

Recent studies demonstrate the involvement of a cAMP-regulated transcriptional coactivator (CRTC) in hippocampal plasticity. In metabolic tissues, phosphorylated CRTC is sequestered in the cytoplasm while dephosphorylated CRTC translocates to the nucleus to promote CREB-dependent gene expression. Fasting causes CRTC dephosphorylation and activation. In line with this, significant accumulation of hemagglutinin (HA)-tagged CRTC (CRTC-HA) was found within MB nuclei after 16 hours of food deprivation. Subcellular fractionation indicated that food deprivation causes CRTC-HA nuclear translocation without affecting total CRTC-HA amounts (Hirano, 2013).

To examine the role of CRTC in fLTM and spLTM, a CRTC inverted repeat (CRTC-IR) was acutely expressed using MBsw, and suppression of aversive fLTM was observed but no effect was seen on STM. CHX treatment did not further decrease 1-day aversive memory, and CRTC-IR expression from a second MB driver, OK107, also impaired fLTM formation. CRTC-IR expression from MBsw also impaired appetitive LTM without affecting appetitive STM. In contrast, CRTC-IR expression from MBsw did not impair spLTM. CRTC-IR expression in DAL neurons had no effect on either aversive fLTM or appetitive LTM. Consistent with these results showing lack of fLTM after 24-hour fasting, 1-day aversive memory after 24-hour fasting did not decrease upon CRTC-IR expression in MBs (Hirano, 2013).

To examine the effects of spaced training on fLTM and the effects of fasting on spLTM, fed or fasted flies expressing either CBP-IR or CRTC-IR were space-trained. When CBP-IR was expressed to impair spLTM, 1-day memory after spaced training was impaired in fed conditions but not in fasting conditions, which suggested that spaced training protocols do not block fLTM. When CRTC-IR was expressed to impair fLTM formation, 1-day memory after spaced training was not affected by fasting, which suggested that mild fasting does not impair spLTM formation (Hirano, 2013).

Is activation of CRTC sufficient to generate fLTM in the absence of fasting? HA-tagged constitutively active CRTC (CRTC-SA-HA) was expressed from MBsw, and its nuclear accumulation was observed in the absence of fasting. Acute expression of CRTC-SA-HA from MBsw increased 1-day aversive memory after single-cycle training in fed flies, and this increase was not further enhanced by fasting. In contrast, expression of control CRTC-HA did not alter the fasting requirement for memory enhancement. CRTC-SA-HA expression did not affect feeding itself, which suggested that the memory enhancement is not due to impaired feeding. Taken together, CRTC activity in MBs is necessary and sufficient to form fLTM. Similar to the effects of fasting, CRTC-SA-HA expression did not affect STM or 4-day spLTM (Hirano, 2013).

In mammalian metabolic tissues, CRTC is phosphorylated by insulin signaling, which is suppressed by fasting (see Wang, 2008). CRTC phosphorylation is also regulated by insulin signaling in flies (Wang, 2008). To determine whether reduced insulin signaling activates CRTC and promotes fLTM formation, heterozygous mutants for chico, which encodes an adaptor protein required for insulin signaling, were tested. Although chico¹ null mutants are semilethal and defective for olfactory learning, heterozygous chico¹/+ mutants are viable and display normal learning (Hirano, 2013).

CRTC accumulated in MB nuclei in chico¹/+ mutants in the absence of food deprivation. Under conditions where flies were fed, chico¹/+ flies had significantly greater 1-day memory after single-cycle training relative to control flies, whereas 1-hour memory was unaffected. Enhanced 1-day memory in chico¹/+ flies was not further enhanced by fasting. Because the chico¹/+ mutation does not affect feeding itself, the memory enhancement would not seem to be attributable to impaired feeding. The increased 1-day memory in chico¹/+ mutants was suppressed by CHX treatment and CRTC-IR expression using MBsw, which suggests that reduced insulin signaling mimics fLTM through activation of CRTC in MBs (Hirano, 2013).

Single-cycle training after mild fasting generates both appetitive and aversive LTM, and CRTC in the MBs plays a key role in both types of LTM. A CRTC-dependent LTM pathway is unlikely to be involved in increasing motivation required to form appetitive memory, because CRTC knockdown did not affect appetitive STM and because CRTC-SA expression was not sufficient to form appetitive LTM without prior fasting. Although mild 16-hour fasting induced aversive fLTM, longer 24-hour fasting impaired aversive fLTM but not appetitive LTM. Thus, although aversive and appetitive fLTM share mechanistic similarities, they may be regulated by different inputs controlling motivation and fasting time courses. Because nuclear translocation of CRTC was sustained even after 24 hours of food deprivation, prolonged fasting may suppress a CRTC-independent step in aversive fLTM formation. spLTM was not affected by 24-hour fasting prior to training, which suggests that the unknown inhibitory effect of 24-hour fasting does not occur after spaced training. Continuous food deprivation after training suppressed aversive fLTM. In another study, Placais (2013) reports that continuous food-deprivation after spaced training suppresses spLTM as well (Hirano, 2013).

Suppression of aversive LTM by prolonged fasting may ensure that starving flies pursue available food, with less concern for safety. Although the biological importance of aversive fLTM in natural environments is currently unclear, the current results indicate that different physiological states may induce different types of LTM in flies (Hirano, 2013).

Drosophila salt-inducible kinase (SIK) regulates starvation resistance through cAMP-response element-binding protein (CREB)-regulated transcription coactivator

Salt-inducible kinase (SIK), one of the AMP-activated kinase (AMPK)-related kinases, has been suggested to play important functions in glucose homeostasis by inhibiting the cAMP-response element-binding protein (CREB)-regulated transcription coactivator (CRTC). To examine the role of SIK in vivo, a Drosophila SIK mutant was generated and was found to have higher amounts of lipid and glycogen stores and to be resistant to starvation. Interestingly, SIK transcripts are highly enriched in the brain, and neuron-specific expression of exogenous SIK was found to fully rescued lipid and glycogen storage phenotypes as well as starvation resistance of the mutant. Using genetic and biochemical analyses, it was demonstrated that CRTC Ser-157 phosphorylation by SIK is critical for inhibiting CRTC activity in vivo. Furthermore, double mutants of SIK and CRTC became sensitive to starvation, and the Ser-157 phosphomimetic mutation of CRTC reduced lipid and glycogen levels in the SIK mutant, suggesting that CRTC mediates the effects of SIK signaling. Collectively, these results strongly support the importance of the SIK-CRTC signaling axis that functions in the brain to maintain energy homeostasis in Drosophila (Choi, 2011).

Activation of cAMP response element-mediated gene expression by regulated nuclear transport of TORC proteins

The CREB family of proteins are critical mediators of gene expression in response to extracellular signals and are essential regulators of adaptive behavior and long-term memory formation. The TORC proteins were recently described as potent CREB coactivators, but their role in regulation of CREB activity remained unknown. TORC proteins were found to be exported from the nucleus in a CRM1-dependent fashion. A high-throughput microscopy-based screen was developed to identify genes and pathways capable of inducing nuclear TORC accumulation. Expression of the catalytic subunit of PKA and the calcium channel TRPV6 relocalized TORC1 to the nucleus. Nuclear accumulation of the three human TORC proteins was induced by increasing intracellular cAMP or calcium levels. TORC1 and TORC2 translocation in response to calcium, but not cAMP, is mediated by calcineurin, and TORC1 is directly dephosphorylated by calcineurin. TORC function was shown to be essential for CRE-mediated gene expression induced by cAMP, calcium, or GPCR activation, and nuclear transport of TORC1 is sufficient to activate CRE-dependent transcription. Drosophila TORC is translocated in response to calcineurin activation in vivo. Thus, TORC nuclear translocation is an essential, conserved step in activation of cAMP-responsive genes (Bittinger, 2004).

The subcellular localization of the single ancestral Drosophila TORC (dTORC) was examined to determine whether regulated nuclear translocation is a general property of TORC proteins. An inducible eGFP-dTORC transgene was constructed and stably integrated into the Drosophila genome. Examination of intact salivary tissue from transgenic larvae demonstrated that dTORC is predominantly present in the cytoplasm in untreated cells and rapidly translocates to the nucleus upon ionomycin exposure, with the majority of the dTORC shuttling to the nucleus 10 min after ionomycin exposure. Cotreatment with calcineurin inhibitor CsA completely blocked dTORC translocation in response to ionomycin, indicating that participation of calcineurin in the calcium-mediated cAMP response element binding protein (CREB) response is conserved between vertebrates and insects (Bittinger, 2004).

Bipartite functions of the CREB co-activators selectively direct alternative splicing or transcriptional activation

The CREB regulated transcription co-activators (CRTCs) regulate many biological processes by integrating and converting environmental inputs into transcriptional responses. Although the mechanisms by which CRTCs sense cellular signals are characterized, little is known regarding how CRTCs contribute to the regulation of cAMP inducible genes. This study shows that these dynamic regulators, unlike other co-activators, independently direct either pre-mRNA splice-site selection or transcriptional activation depending on the cell type or promoter context. Moreover, in other scenarios, the CRTC co-activators coordinately regulate transcription and splicing. Mutational analyses showed that CRTCs possess distinct functional domains responsible for regulating either pre-mRNA splicing or transcriptional activation. Interestingly, the CRTC1-MAML2 oncoprotein lacks the splicing domain and is incapable of altering splice-site selection despite robustly activating transcription. The differential usage of these distinct domains allows CRTCs to selectively mediate multiple facets of gene regulation, indicating that co-activators are not solely restricted to coordinating alternative splicing with increase in transcriptional activity (Amelio, 2009).

The idea that the CRTC co-activators perform more than one function is in accord with those of other co-regulators that possess multiple activities. For example, the CREB co-activator CBP mediates gene regulation through its intrinsic and associated histone acetyl transferase activity and by promoting the formation of the pre-initiation complex (PIC) through interactions with components of the core transcriptional machinery. Moreover, the transcriptional co-activators PGC, COAA and CAPER function as transcriptional activators, as well as regulators of transcriptionally coupled pre-mRNA processing. However, in contrast to other co-activators involved in splicing, the CRTCs lack any conserved, definable RNA-binding domain (e.g., an RNA recognition motif (RRM), an arginine-serine (RS) domain, or a K homology (KH) domain). Rather, it is hypothesized that CRTCs provide a scaffold for the assembly of a larger complex of proteins that may directly bind the transcript. This idea is supported by CRTC IP MS/MS experiments that identified several proteins involved in pre-mRNA processing (Amelio, 2007). If the CRTCs form the basis for a protein scaffold that mediates RNA processing by recruiting other proteins, then the ability of CRTCs to recruit slightly different complexes (i.e., cell type or promoter specific) may contribute to these independent bipartite functions. Moreover, PKA may contribute to TORC-dependent alternative splicing through phosphorylation of components within the recruited splicing complex (Amelio, 2009).

There is now overwhelming evidence that the subcellular localization, and thus function, of the CRTC2 co-activator is mediated by several cues, including glucagon and insulin. CRTC2 increases fasting blood glucose levels and the induction of key genes directly involved in gluconeogenesis. Collectively, these studies showed that CRTC2 is the rate-limiting step of cAMP signalling and that it alone is sufficient to initiate the early stages of the gluconeogenic program. Interestingly, several of the genes within the gluconeogenic program such as Nurr1 (Nr4A2), as well as those encoding proteins that mediate later stages of glucose production such as PGC-1 (Ppargc1a), are alternatively spliced; this study shows that CRTC2 can promote alternative splicing of Nr4a2. Thus, CRTCs function as integrators of extracellular signals to influence transcript diversity by acting either as a conduit between components of the transcription and splicing machineries or as autonomous regulators of each process. However, although TORCs can selectively activate splicing in some promoter contexts without activating transcription, these promoters possess basal transcriptional activity, therefore, the effects of the TORCs on pre-mRNA splicing could still formally be considered co-transcriptional despite the absence of co-activator-induced transcription (Amelio, 2009).

There are two prevailing models of promoter-dependent coordination of transcription and pre-mRNA processing that could apply to CRTC-mediated alternative splicing. These include splicing factor recruitment to the carboxyl terminal domain (CTD) of RNA polymerase II (RNAP II) (recruitment model) and regulation of RNAP II elongation rates (kinetic model). These models embody a 'forward coupling' mechanism that links RNAP II transcription to pre-mRNA splicing. However, '‘reverse coupling' mechanisms have been described, whereby pre-mRNA splicing exerts an influence back on transcription. Indeed, promoter-proximal 5' splice sites seem to promote reverse coupling by increasing transcription initiation through recruitment of the PIC (Damgaard, 2008). CRTCs interact with TAFII130, a component of the PIC (Conkright, 2003). Therefore, the finding that CRTCs can induce splicing independent of their effects on transcription may reflect the failure of CRTCs to recruit components of the PIC to TATA-less promoters. Accordingly, although parts of both models help to explain mechanistically how the CRCT co-activators mediate alterative splicing, neither model can fully account for the current observations, suggesting that further refinement to the current working models may be necessary (Amelio, 2009).

Apart from CRTCs mechanistic role in pre-mRNA processing, CRTC-mediated splicing probably has physiological significance. This is supported by the vast number of genes that are alternatively spliced, the large cast of promoters that contain CRE elements (roughly 10%), and by the fact that the CRTC recruitment to promoters (e.g. through interactions with CREB) is sufficient to augment alternative splicing. The inability of the CRTC1-MAML2 oncoprotein to mediate CREB-dependent splice-site selection, despite retaining robust transcriptional activation properties, suggests that this fusion product may also promote cellular transformation through aberrant pre-mRNA splicing of CRE-containing genes. However, transcriptional activators have been shown to promote pre-mRNA splicing, therefore, it is possible that the MAML2 transactivation domain in the CRTC1-MAML2 fusion can mediate splicing in other promoter contexts. Furthermore, although the current data clearly show that the CRTC co-activators have the potential to regulate alternative splicing, this does not mandate that transcripts from CRE-containing genes will be alternatively spliced in a CRTC-dependent manner. Indeed, several studies have shown that the significance of the CREB co-activators, CBP/p300, is dictated by gene context and varies among tissues. Similar, cell-type specificity is also observed in CRTC-mediated alternative splicing of the endogenous Nr4a2 transcript. It is speculated that the generation of select transcript isoforms may differ between cell types, in part, due to the many diverse signalling events that converge on the CRTC co-activators (Amelio, 2009).

Traditional methods, such as expression arrays or TaqMan PCR focus on gene expression to examine gene regulation in response to signalling cascades. In these technologies, the probes and primer sets intentionally do not account for alternatively spliced products. As a result the canonical analysis of cAMP responsive genes has been restricted to the analysis of transcription induction effectively missing any affects of cAMP activation on pre-mRNA processing. The current findings establish that CRTCs direct alternative splicing independently or in coordination with transcriptional activation, thereby, linking the cAMP signalling cascade with the regulation of pre-mRNA processing. Future experiments will monitor alternative splicing in addition to transcriptional activation when identifying tissue-specific CRTC target promoters (Amelio, 2009).

Lifespan extension in C. elegans induced by AMPK and calcineurin is mediated by CRTC-1 and CREB

Activating AMPK or inactivating calcineurin slows ageing in C. elegans and both have been implicated as therapeutic targets for age-related pathology in mammals. However, the direct targets that mediate their effects on longevity remain unclear. In mammals, CREB-regulated transcriptional coactivators (CRTCs) are a family of cofactors involved in diverse physiological processes including energy homeostasis, cancer and endoplasmic reticulum stress. This study shows that both AMPK and calcineurin modulate longevity exclusively through post-translational modification of CRTC-1, the sole C. elegans CRTC. CRTC-1 is a direct AMPK target, and interacts with the CREB homologue-1 (CRH-1) transcription factor in vivo. The pro-longevity effects of activating AMPK or deactivating calcineurin decrease CRTC-1 and CRH-1 activity and induce transcriptional responses similar to those of CRH-1 null worms. Downregulation of crtc-1 increases lifespan in a crh-1-dependent manner and directly reducing crh-1 expression increases longevity, substantiating a role for CRTCs and CREB in ageing. Together, these findings indicate a novel role for CRTCs and CREB in determining lifespan downstream of AMPK and calcineurin, and illustrate the molecular mechanisms by which an evolutionarily conserved pathway responds to low energy to increase longevity (Mair, 2011).

These data indicate that CRTC-1 is the critical direct longevity target of both AMPK and calcineurin in C. elegans and identify a new role for CRTCs and CREB in modulating longevity. They also represent the first analysis of the transcriptional profiles of long-lived activated AMPK and deactivated calcineurin organisms and suggest the primary longevity-associated role of these perturbations is the modulation of CRTC-1 and CRH-1 transcriptional activity. Notably, both the FOXO transcription factor daf-16 and genes involved in autophagy have also been implicated in AMPK and calcineurin longevity, respectively. Further work to determine precisely where the AMPK-calcineurin-CRTC-1 pathway converges with FOXO and autophagy will be enlightening. It will also be interesting to determine if CRTC-1 mediates downstream effects of kinases other than AMPK. In mammals, CRTCs are regulated by multiple CAMKL kinase family members, and additive effects are seen of AMPK and related kinases on the localization of CRTC-1, in particular the MAP/microtubule affinity-regulating kinase (MARK) par-1, indicating that this kinase may also regulate CRTC-1 in vivo. At present, however, AMPK is the only CAMKL kinase shown to be a positive regulator of longevity (Mair, 2011).

Collectively, these data identify CRTC-1 as a central node linking the upstream lifespan modifiers AMPK and calcineurin to CREB activity via a shared signal-transduction pathway, and demonstrate that post-translational modification of CRTC-1 is required for their effects on longevity. Complementing the pro-longevity effects of inhibiting CRTC function in C. elegans, reducing components of the CRTC/CREB pathway has recently been shown to confer health benefits to mice. Given the evolutionary conservation of this pathway from C. elegans to mammals it will be fascinating to determine the role of CRTCs both as mammalian ageing modulators and as potential drug targets for patients with metabolic disorders and cancer (Mair, 2011).

The CRTC1-SIK1 pathway regulates entrainment of the circadian clock

Retinal photoreceptors entrain the circadian system to the solar day. This photic resetting involves cAMP response element binding protein (CREB)-mediated upregulation of Per genes within individual cells of the suprachiasmatic nuclei (SCN). Detailed understanding of this pathway is poor, and it remains unclear why entrainment to a new time zone takes several days. By analyzing the light-regulated transcriptome of the SCN, this study has identified a key role for salt inducible kinase 1 (SIK1) and CREB-regulated transcription coactivator 1 (CRTC1) in clock re-setting. An entrainment stimulus causes CRTC1 to coactivate CREB, inducing the expression of Per1 and Sik1. SIK1 then inhibits further shifts of the clock by phosphorylation and deactivation of CRTC1. Knockdown of Sik1 within the SCN results in increased behavioral phase shifts and rapid re-entrainment following experimental jet lag. Thus SIK1 provides negative feedback, acting to suppress the effects of light on the clock. This pathway provides a potential target for the regulation of circadian rhythms (Jagannath, 2013).

Clock and light regulation of the CREB coactivator CRTC1 in the suprachiasmatic circadian clock

The CREB/CRE transcriptional pathway has been implicated in circadian clock timing and light-evoked clock resetting. To date, much of the work on CREB in circadian physiology has focused on how changes in the phosphorylation state of CREB regulate the timing processes. However, beyond changes in phosphorylation, CREB-dependent transcription can also be regulated by the CREB coactivator CRTC (CREB-regulated transcription coactivator), also known as TORC (transducer of regulated CREB). This study profiled both the rhythmic and light-evoked regulation of CRTC1 and CRTC2 in the murine suprachiasmatic nucleus (SCN), the locus of the master mammalian clock. Immunohistochemical analysis revealed rhythmic expression of CRTC1 in the SCN. CRTC1 expression was detected throughout the dorsoventral extent of the SCN in the middle of the subjective day, with limited expression during early night, and late night expression levels intermediate between mid-day and early night levels. In contrast to CRTC1, robust expression of CRTC2 was detected during both the subjective day and night. During early and late subjective night, a brief light pulse induced strong nuclear accumulation of CRTC1 in the SCN. In contrast with CRTC1, photic stimulation did not affect the subcellular localization of CRTC2 in the SCN. Additionally, reporter gene profiling and chromatin immunoprecipitation analysis indicated that CRTC1 was associated with CREB in the 5' regulatory region of the period1 gene, and that overexpression of CRTC1 leads to a marked upregulation in period1 transcription. Together, these data raise the prospect that CRTC1 plays a role in fundamental aspects of SCN clock timing and entrainment (Sakamoto, 2013).

CRTC1 nuclear translocation following learning modulates memory strength via exchange of chromatin remodeling complexes on the Fgf1 gene

Memory is formed by synapse-to-nucleus communication that leads to regulation of gene transcription, but the identity and organizational logic of signaling pathways involved in this communication remain unclear. This study finds that the transcription cofactor CRTC1 (see Drosophila Crtc) is a critical determinant of sustained gene transcription and memory strength in the hippocampus. Following associative learning, synaptically localized CRTC1 is translocated to the nucleus and regulates Fgf1b transcription in an activity-dependent manner. After both weak and strong training, the HDAC3-N-CoR corepressor complex leaves the Fgf1b promoter and a complex involving the translocated CRTC1, phosphorylated CREB (see Drosophila CrebB), and histone acetyltransferase CBP (see Drosophila Nejire) induces transient transcription. Strong training later substitutes KAT5 (see Drosophila Tip60) for CBP, a process that is dependent on CRTC1, but not on CREB phosphorylation. This in turn leads to long-lasting Fgf1b transcription and memory enhancement. Thus, memory strength relies on activity-dependent changes in chromatin and temporal regulation of gene transcription on specific CREB/CRTC1 gene targets (Uchida, 2017).

Search PubMed for articles about Drosophila Crtc

Altarejos, J. Y. and Montminy, M. (2011). CREB and the CRTC co-activators: sensors for hormonal and metabolic signals. Nat Rev Mol Cell Biol 12: 141-151. PubMed ID: 21346730

Amelio, A. L., et al. (2007) A coactivator trap identifies NONO (p54nrb) as a component of the cAMP-signaling pathway. Proc. Natl. Acad. Sci. 104: 20314-20319. PubMed ID: 18077367

Amelio, A. L., Caputi, M. and Conkright, M. D. (2009). Bipartite functions of the CREB co-activators selectively direct alternative splicing or transcriptional activation. EMBO J. 28(18): 2733-47. PubMed ID: 19644446

Bittinger, M. A., et al. (2004). Activation of cAMP response element-mediated gene expression by regulated nuclear transport of TORC proteins. Curr. Biol. 14(23): 2156-61. PubMed ID: 15589160

Chen, L., Rio, D. C., Haddad, G. G. and Ma, E. (2004). Regulatory role of dADAR in ROS metabolism in Drosophila CNS. Brain Res. Mol. Brain Res. 131: 93-100. PubMed ID: 15530657

Choi, S., Kim, W. and Chung, J. (2011). Drosophila salt-inducible kinase (SIK) regulates starvation resistance through cAMP-response element-binding protein (CREB)-regulated transcription coactivator (CRTC). J. Biol. Chem. 286(4): 2658-64. PubMed ID: 21127058

Conkright, M. D., et al. (2003) TORCs: transducers of regulated CREB activity. Mol. Cell 12: 413-423. PubMed ID: 14536081

Damgaard, C. K., et al. (2008). A 5' splice site enhances the recruitment of basal transcription initiation factors in vivo. Mol. Cell 29: 271-278. PubMed ID: 18243121

Dentin, R., et al. (2007). Insulin modulates gluconeogenesis by inhibition of the coactivator TORC2. Nature 449: 366-369. PubMed ID: 1780530

Djawdan, M., et al. (1998). Metabolic reserves and evolved stress resistance in Drosophila melanogaster. Physiol. Zool. 71: 584-594. PubMed ID: 9754535

Fridell, Y. W., et al. (2005). Targeted expression of the human uncoupling protein 2 (hUCP2) to adult neurons extends life span in the fly. Cell Metab. 1: 145-152. PubMed ID: 16054055

Halder, R., Hennion, M., Vidal, R. O., Shomroni, O., Rahman, R. U., Rajput, A., Centeno, T. P., van Bebber, F., Capece, V., Garcia Vizcaino, J. C., Schuetz, A. L., Burkhardt, S., Benito, E., Navarro Sala, M., Javan, S. B., Haass, C., Schmid, B., Fischer, A. and Bonn, S. (2016). DNA methylation changes in plasticity genes accompany the formation and maintenance of memory. Nat Neurosci 19(1): 102-110. PubMed ID: 26656643

Hirano, Y., Masuda, T., Naganos, S., Matsuno, M., Ueno, K., Miyashita, T., Horiuchi, J. and Saitoe, M. (2013). Fasting launches CRTC to facilitate long-term memory formation in Drosophila. Science 339: 443-446. PubMed ID: 23349290

Hirano, Y., Ihara, K., Masuda, T., Yamamoto, T., Iwata, I., Takahashi, A., Awata, H., Nakamura, N., Takakura, M., Suzuki, Y., Horiuchi, J., Okuno, H. and Saitoe, M. (2016). Shifting transcriptional machinery is required for long-term memory maintenance and modification in Drosophila mushroom bodies. Nat Commun 7: 13471. PubMed ID: 27841260

Jagannath, A., Butler, R., Godinho, S. I., Couch, Y., Brown, L. A., Vasudevan, S. R., Flanagan, K. C., Anthony, D., Churchill, G. C., Wood, M. J., Steiner, G., Ebeling, M., Hossbach, M., Wettstein, J. G., Duffield, G. E., Gatti, S., Hankins, M. W., Foster, R. G. and Peirson, S. N. (2013). The CRTC1-SIK1 pathway regulates entrainment of the circadian clock. Cell 154: 1100-1111. PubMed ID: 23993098

Kim, M., Lee, H., Hur, J.H., Choe, J. and Lim, C. (2016). CRTC potentiates light-independent timeless transcription to sustain circadian rhythms in Drosophila. Sci Rep 6: 32113. PubMed ID: 27577611

Kim, S. K. and Rulifson, E. J. (2004). Conserved mechanisms of glucose sensing and regulation by Drosophila corpora cardiaca cells. Nature 431: 316-320. PubMed ID: 15372035

Koo, S. H., et al. (2005). The CREB coactivator TORC2 is a key regulator of fasting glucose metabolism. Nature 437: 1109-1111. PubMed ID: 16148943

Lee, G. and Park, J. H. (2004). Hemolymph sugar homeostasis and starvation-induced hyperactivity affected by genetic manipulations of the adipokinetic hormone-encoding gene in Drosophila melanogaster. Genetics 167: 311-323. PubMed ID: 15166157

Mair, W., Morantte, I., Rodrigues, A. P., Manning, G., Montminy, M., Shaw, R. J. and Dillin, A. (2011). Lifespan extension induced by AMPK and calcineurin is mediated by CRTC-1 and CREB. Nature 470: 404-408. PubMed ID: 21331044

Matsumoto, M., et al. (2007). Impaired regulation of hepatic glucose production in mice lacking the forkhead transcription factor foxo1 in liver. Cell Metab. 6: 208-216. PubMed ID: 17767907

Mockett, R. J., et al (2003). Ectopic expression of catalase in Drosophila mitochondria increases stress resistance but not longevity. Free Radic. Biol. Med. 34: 207-217. PubMed ID: 12521602

Placais, P. Y. and Preat, T. (2013). To favor survival under food shortage, the brain disables costly memory. Science 339: 440-442. PubMed ID: 23349289

Sakamoto, K., Norona, F. E., Alzate-Correa, D., Scarberry, D., Hoyt, K. R. and Obrietan, K. (2013). Clock and light regulation of the CREB coactivator CRTC1 in the suprachiasmatic circadian clock. J Neurosci 33: 9021-9027. PubMed ID: 23699513

Screaton, R. A., et al. (2004). The CREB coactivator TORC2 functions as a calcium- and cAMP-sensitive coincidence detector. Cell 119: 61-74. PubMed ID: 15454081

Shen, R., Wang, B., Giribaldi, M.G., Ayres, J., Thomas, J.B. and Montminy, M. (2016). Neuronal energy-sensing pathway promotes energy balance by modulating disease tolerance. Proc Natl Acad Sci U S A 113(23):E3307-14. PubMed ID: 27208092

Svensson, M. J. and Larsson, J. (2007). Thioredoxin-2 affects lifespan and oxidative stress in Drosophila. Hereditas 144: 25-32. PubMed ID: 17567437

Uchida, S., et al. (2017). CRTC1 nuclear translocation following learning modulates memory strength via exchange of chromatin remodeling complexes on the Fgf1 gene. J Cell Rep 18(2): 352-366. PubMed ID: 28076781

Wang, B., et al. (2008).The insulin-regulated CREB coactivator TORC promotes stress resistance in Drosophila. Cell Metab. 7(5): 434-44. PubMed ID: 18460334

date revised: 2 January 2023

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