The Rab GTPase activating protein TBC-2 regulates endosomal localization of DAF-16 FOXO and lifespan.

FOXO transcription factors have been shown to regulate longevity in model organisms and are associated with longevity in humans. To gain insight into how FOXO functions to increase lifespan, we examined the subcellular localization of DAF-16 in C. elegans. We show that DAF-16 is localized to endosomes and that this endosomal localization is increased by the insulin-IGF signaling (IIS) pathway. Endosomal localization of DAF-16 is modulated by endosomal trafficking proteins. Disruption of the Rab GTPase activating protein TBC-2 increases endosomal localization of DAF-16, while inhibition of TBC-2 targets, RAB-5 or RAB-7 GTPases, decreases endosomal localization of DAF-16. Importantly, the amount of DAF-16 that is localized to endosomes has functional consequences as increasing endosomal localization through mutations in tbc-2 reduced the lifespan of long-lived daf-2 IGFR mutants, depleted their fat stores, and DAF-16 target gene expression. Overall, this work identifies endosomal localization as a mechanism regulating DAF-16 FOXO, which is important for its functions in metabolism and aging.

Identification of Plagl1/Zac1 binding sites and target genes establishes its role in the regulation of extracellular matrix genes and the imprinted gene network.

PLAGL1/ZAC1 undergoes parental genomic imprinting, is paternally expressed, and is a member of the imprinted gene network (IGN). It encodes a zinc finger transcription factor with anti-proliferative activity and is a candidate tumor suppressor gene on 6q24 whose expression is frequently lost in various neoplasms. Conversely, gain of PLAGL1 function is responsible for transient neonatal diabetes mellitus, a rare genetic disease that results from defective pancreas development. In the present work, we showed that Plagl1 up-regulation was not associated with DNA damage-induced cell cycle arrest. It was rather associated with physiological cell cycle exit that occurred with contact inhibition, growth factor withdrawal, or cell differentiation. To gain insights into Plagl1 mechanism of action, we identified Plagl1 target genes by combining chromatin immunoprecipitation and genome-wide transcriptomics in transfected cell lines. Plagl1-elicited gene regulation correlated with multiple binding to the proximal promoter region through a GC-rich motif. Plagl1 target genes included numerous genes involved in signaling, cell adhesion, and extracellular matrix composition, including collagens. Plagl1 targets also included 22% of the 409 genes that make up the IGN. Altogether, this work identified Plagl1 as a transcription factor that coordinated the regulation of a subset of IGN genes and controlled extracellular matrix composition.

The tumor suppressor folliculin regulates AMPK-dependent metabolic transformation.

The Warburg effect is a tumorigenic metabolic adaptation process characterized by augmented aerobic glycolysis, which enhances cellular bioenergetics. In normal cells, energy homeostasis is controlled by AMPK; however, its role in cancer is not understood, as both AMPK-dependent tumor-promoting and -inhibiting functions were reported. Upon stress, energy levels are maintained by increased mitochondrial biogenesis and glycolysis, controlled by transcriptional coactivator PGC-1α and HIF, respectively. In normoxia, AMPK induces PGC-1α, but how HIF is activated is unclear. Germline mutations in the gene encoding the tumor suppressor folliculin (FLCN) lead to Birt-Hogg-Dubé (BHD) syndrome, which is associated with an increased cancer risk. FLCN was identified as an AMPK binding partner, and we evaluated its role with respect to AMPK-dependent energy functions. We revealed that loss of FLCN constitutively activates AMPK, resulting in PGC-1α-mediated mitochondrial biogenesis and increased ROS production. ROS induced HIF transcriptional activity and drove Warburg metabolic reprogramming, coupling AMPK-dependent mitochondrial biogenesis to HIF-dependent metabolic changes. This reprogramming stimulated cellular bioenergetics and conferred a HIF-dependent tumorigenic advantage in FLCN-negative cancer cells. Moreover, this pathway is conserved in a BHD-derived tumor. These results indicate that FLCN inhibits tumorigenesis by preventing AMPK-dependent HIF activation and the subsequent Warburg metabolic transformation.

Folliculin regulates ampk-dependent autophagy and metabolic stress survival.

Dysregulation of AMPK signaling has been implicated in many human diseases, which emphasizes the importance of characterizing AMPK regulators. The tumor suppressor FLCN, responsible for the Birt-Hogg Dubé renal neoplasia syndrome (BHD), is an AMPK-binding partner but the genetic and functional links between FLCN and AMPK have not been established. Strikingly, the majority of naturally occurring FLCN mutations predisposing to BHD are predicted to produce truncated proteins unable to bind AMPK, pointing to the critical role of this interaction in the tumor suppression mechanism. Here, we demonstrate that FLCN is an evolutionarily conserved negative regulator of AMPK. Using Caenorhabditis elegans and mammalian cells, we show that loss of FLCN results in constitutive activation of AMPK which induces autophagy, inhibits apoptosis, improves cellular bioenergetics, and confers resistance to energy-depleting stresses including oxidative stress, heat, anoxia, and serum deprivation. We further show that AMPK activation conferred by FLCN loss is independent of the cellular energy state suggesting that FLCN controls the AMPK energy sensing ability. Together, our data suggest that FLCN is an evolutionarily conserved regulator of AMPK signaling that may act as a tumor suppressor by negatively regulating AMPK function.

TBC-2 regulates RAB-5/RAB-7-mediated endosomal trafficking in Caenorhabditis elegans.

During endosome maturation the early endosomal Rab5 GTPase is replaced with the late endosomal Rab7 GTPase. It has been proposed that active Rab5 can recruit and activate Rab7, which in turn could inactivate and remove Rab5. However, many of the Rab5 and Rab7 regulators that mediate endosome maturation are not known. Here, we identify Caenorhabditis elegans TBC-2, a conserved putative Rab GTPase-activating protein (GAP), as a regulator of endosome to lysosome trafficking in several tissues. We show that tbc-2 mutant animals accumulate enormous RAB-7-positive late endosomes in the intestine containing refractile material. RAB-5, RAB-7, and components of the homotypic fusion and vacuole protein sorting (HOPS) complex, a RAB-7 effector/putative guanine nucleotide exchange factor (GEF), are required for the tbc-2(-) intestinal phenotype. Expression of activated RAB-5 Q78L in the intestine phenocopies the tbc-2(-) large late endosome phenotype in a RAB-7 and HOPS complex-dependent manner. TBC-2 requires the catalytic arginine-finger for function in vivo and displays the strongest GAP activity on RAB-5 in vitro. However, TBC-2 colocalizes primarily with RAB-7 on late endosomes and requires RAB-7 for membrane localization. Our data suggest that TBC-2 functions on late endosomes to inactivate RAB-5 during endosome maturation.

TBC-2 is required for embryonic yolk protein storage and larval survival during L1 diapause in Caenorhabditis elegans.

C. elegans first stage (L1) larvae hatched in the absence of food, arrest development and enter an L1 diapause, whereby they can survive starvation for several weeks. The physiological and metabolic requirements for survival during L1 diapause are poorly understood. However, yolk, a cholesterol binding/transport protein, has been suggested to serve as an energy source. Here, we demonstrate that C. elegans TBC-2, a RAB-5 GTPase Activating Protein (GAP) involved in early-to-late endosome transition, is important for yolk protein storage during embryogenesis and for L1 survival during starvation. We found during embryogenesis, that a yolk::green fluorescent protein fusion (YP170::GFP), disappeared much more quickly in tbc-2 mutant embryos as compared with wild-type control embryos. The premature disappearance of YP170::GFP in tbc-2 mutants is likely due to premature degradation in the lysosomes as we found that YP170::GFP showed increased colocalization with Lysotracker Red, a marker for acidic compartments. Furthermore, YP170::GFP disappearance in tbc-2 mutants required RAB-7, a regulator of endosome to lysosome trafficking. Although tbc-2 is not essential in fed animals, we discovered that tbc-2 mutant L1 larvae have strongly reduced survival when hatched in the absence of food. We show that tbc-2 mutant larvae are not defective in maintaining L1 diapause and that mutants defective in yolk uptake, rme-1 and rme-6, also had strongly reduced L1 survival when hatched in the absence of food. Our findings demonstrate that TBC-2 is required for yolk protein storage during embryonic development and provide strong correlative data indicating that yolk constitutes an important energy source for larval survival during L1 diapause.

Zac1 regulates an imprinted gene network critically involved in the control of embryonic growth.

Genomic imprinting is an epigenetic mechanism of regulation that restrains the expression of a small subset of mammalian genes to one parental allele. The reason for the targeting of these approximately 80 genes by imprinting remains uncertain. We show that inactivation of the maternally repressed Zac1 transcription factor results in intrauterine growth restriction, altered bone formation, and neonatal lethality. A meta-analysis of microarray data reveals that Zac1 is a member of a network of coregulated genes comprising other imprinted genes involved in the control of embryonic growth. Zac1 alters the expression of several of these imprinted genes, including Igf2, H19, Cdkn1c, and Dlk1, and it directly regulates the Igf2/H19 locus through binding to a shared enhancer. Accordingly, these data identify a network of imprinted genes, including Zac1, which controls embryonic growth and which may be the basis for the implementation of a common mechanism of gene regulation during mammalian evolution.