The C-terminus of the retinal homeobox (rax) gene product modulates transcription in a context-dependent manner.

Purpose: The evolutionarily conserved retinal homeobox (Rax) transcription factor is essential for normal eye development in all vertebrates. Despite Rax's biologic significance, the molecular mechanisms underlying Rax molecular function as a transcriptional regulator are poorly defined. The rax gene encodes a conserved octapeptide motif (OP) near the N-terminus and several conserved regions in the C-terminus of unknown function, including the orthopedia, aristaless, rax (OAR) domain and the RX domain. The purpose of this study is to investigate the contribution of these conserved domains in Rax function. Methods: N-and C-terminal deletion and point mutations were generated in Xenopus laevis rax.L (previously known as Rx1A) using PCR-based methods. We examined the ability of mutated Rax to transactivate a reporter gene consisting of a portion of a rax target gene promoter (from the Xenopus rhodopsin gene) fused to a firefly luciferase coding region and transfected into human embryonic kidney 293T (HEK293T) cells. Portions of the Rax C-terminal region were also assayed for transactivation activity in the context of a heterologous DNA binding domain with an appropriate reporter gene. Results: Full-length Rax weakly activated the reporter. Deletion of the Rax C-terminus increased Rax activity, suggesting that the C-terminus functions to repress Rax activity. Further deletion eventually resulted in a decrease in activity, suggesting that the C-terminal region also can function to enhance Rax activity. Deletion or mutation of the OP motif resulted in a slight decrease in Rax activity. Mutation or deletion of the N-terminal OP motif resulted in a mild decrease in activity and dampened the activity levels of the C-terminal deletions. Further, fusion of the C-terminus of Rax to a heterologous DNA binding domain enhanced transactivation. Conclusions: The present data indicate that the C-terminus of Rax can function to repress or activate transcription in a context-dependent manner. These data support our hypothesis that the highly conserved OAR domain, in combination with other regulatory elements in the Rax C-terminus, coordinates Rax activity, perhaps through functional interaction with the N-terminal OP motif. Taken together, these data provide insight into the structural features that regulate Rax activity.

Evidence for transgenerational metabolic programming in Drosophila.

Worldwide epidemiologic studies have repeatedly demonstrated an association between prenatal nutritional environment, birth weight and susceptibility to adult diseases including obesity, cardiovascular disease and type 2 diabetes. Despite advances in mammalian model systems, the molecular mechanisms underlying this phenomenon are unclear, but might involve programming mechanisms such as epigenetics. Here we describe a new system for evaluating metabolic programming mechanisms using a simple, genetically tractable Drosophila model. We examined the effect of maternal caloric excess on offspring and found that a high-sugar maternal diet alters body composition of larval offspring for at least two generations, augments an obese-like phenotype under suboptimal (high-calorie) feeding conditions in adult offspring, and modifies expression of metabolic genes. Our data indicate that nutritional programming mechanisms could be highly conserved and support the use of Drosophila as a model for evaluating the underlying genetic and epigenetic contributions to this phenomenon.

A novel interaction between Glycogen Synthase Kinase-3α (GSK-3α) and the scaffold protein Receptor for Activated C-Kinase 1 (RACK1) regulates the circadian clock.

Glycogen synthase kinase-3α (GSK-3α) and GSK-3ß are intracellular kinases with largely redundant functions. However, the deletion of each GSK-3 isoform in the mouse has distinct consequences, suggesting that these related enzymes also have non-overlapping isoform-specific functions. A yeast two-hybrid screen for GSK-3α interacting partners revealed an interaction with the Receptor for Activated C-Kinase 1 (RACK1). We confirm this interaction in mammalian cells, and provide evidence that RACK1 does not interact with GSK-3ß. Structure-function analyses revealed that WD repeats 5-6 are required to interact with GSK-3α. Furthermore, this interaction is independent of GSK-3α activity. Finally, our data show that the GSK-3α-RACK1 interaction is necessary for regulating the circadian clock in mammalian cells. In summary, our data provides a mechanistic link between GSK-3 and RACK-1 in the regulation of the circadian clock, and demonstrates that this effect is specific to the GSK-3α isoform.

A noncatalytic domain of glycogen synthase kinase-3 (GSK-3) is essential for activity.

Glycogen synthase kinase-3 (GSK-3) isoforms, GSK-3alpha and GSK-3beta, are serine/threonine kinases involved in numerous cellular processes and diverse diseases, including Alzheimer disease, cancer, and diabetes. GSK-3 isoforms function redundantly in some settings, while, in others, they exhibit distinct activities. Despite intensive investigation into the physiological roles of GSK-3 isoforms, the basis for their differential activities remains unresolved. A more comprehensive understanding of the mechanistic basis for GSK-3 isoform-specific functions could lead to the development of isoform-specific inhibitors. Here, we describe a structure-function analysis of GSK-3alpha and GSK-3beta in mammalian cells. We deleted the noncatalytic N and C termini in both GSK-3 isoforms and generated point mutations of key regulatory residues. We examined the effect of these mutations on GSK-3 activity toward Tau, activity in Wnt signaling, interaction with Axin, and GSK-3alpha/beta Tyr(279/216) phosphorylation. We found that the N termini of both GSK-3 isoforms were dispensable, whereas progressive C-terminal deletions resulted in protein misfolding exhibited by deficient activity, impaired ability to interact with Axin, and a loss of Tyr(279/216) phosphorylation. Our data predict that small molecules targeting the divergent C terminus may lead to isoform-specific GSK-3 inhibition through destabilization of the GSK-3 structure.

Metformin therapy in a transgenic mouse model of Huntington's disease.

Huntington's disease (HD) is a hereditary neurodegenerative disease that leads to striatal degeneration and a severe movement disorder. We used a transgenic mouse model of HD (the R6/2 line with approximately 150 glutamine repeats) to test a new therapy for this disease. We treated HD mice with metformin, a widely used anti-diabetes drug, in the drinking water (0, 2 or 5mg/ml) starting at 5 weeks of age. Metformin treatment significantly prolonged the survival time of male HD mice at the 2mg/ml dose (20.1% increase in lifespan) without affecting fasting blood glucose levels. This dose of metformin also decreased hind limb clasping time in 11-week-old mice. The higher dose did not prolong survival, and neither dose of metformin was effective in female HD mice. Collectively, our results suggest that metformin may be worth further investigation in additional HD models.