The metabolic footprint of aging in mice

Aging is characterized by a general decline in cellular function, which ultimately will affect whole body homeostasis. Although DNA damage and oxidative stress all contribute to aging, metabolic dysfunction is a common hallmark of aging at least in invertebrates. Since a comprehensive overview of metabolic changes in otherwise healthy aging mammals is lacking, we here compared metabolic parameters of young and 2 year old mice. We systemically integrated in vivo phenotyping with gene expression, biochemical analysis, and metabolomics, thereby identifying a distinguishing metabolic footprint of aging. Among the affected pathways in both liver and muscle we found glucose and fatty acid metabolism, and redox homeostasis. These alterations translated in decreased long chain acylcarnitines and increased free fatty acid levels and a marked reduction in various amino acids in the plasma of aged mice. As such, these metabolites serve as biomarkers for aging and healthspan.

Circadian clock- and feeding-dependent regulation of translation initiation and elongation in the liver

Cédric Gobet

Circadian rhythms allow organisms to anticipate and adapt to periodic environmental changes related to the day-night cycle. These rhythms originate from a genetic network ticking in almost every cell of our body and the coordination between different organs, thus allowing to generate physiological outputs at the right time of the day. In the liver, which is one of the major metabolic organ of the body, gene expression has to be highly regulated by the circadian clock and feeding rhythms to generate such a timely behavior. Indeed, at every step from transcription to post-translational modifications, the circadian clock and metabolism interplay. In this context, we quantified transcription, mRNA accumulation and translation in liver from wild-type and clock-deficient mice fed ad libitum or only during the night period. RNA-Sequencing and ribosome profiling combined with mathematical modeling were used to assess rhythmic regulation along the gene expression process. Transcription was found to be the main driver of temporal mRNA accumulation and its subsequent translation. We observed a diurnal translation efficiency in genes with 5'-Terminal Oligo Pyrimidine tract (5'-TOP) sequences or with Translation Initiator of Short 5'-UTR (TISU) motifs. Remarkably, the rhythmic translation efficiency was mainly driven by feeding cues although clock-deficiency slightly affected the amplitude and phase of the oscillations. In a subsequent work, we emphasized the above analysis of translation regulation with a focus on the ribosome elongation process. A statistical model and bioinformatics pipeline were developed to infer codon dwell times from ribosome profiling data. The method has the particularity of integrating pairs of codons between the sites of the ribosome and to fit every sequencing read on an individual basis. We validated our approach on a yeast dataset and then studied extensively the mouse liver translation elongation landscape. A large dynamic range between the fastest and longest codons was uncovered in the ribosome A and P sites with strong synergic interaction of pairs of codons clustering by amino acids. In order to challenge our system, we performed drug free ribosome profiling, modified to reduce library biases, in liver of mice fed ad libitum or starved. Unexpectedly, codon dwell times were conserved between the two conditions despite a gene expression landscape reflecting low energy state of the mice and subsequent amino acid shortage. Finally, to understand how the ribosome elongation velocity is regulated by (aminoacyl)-tRNA levels, we adapted and modified a published DNA hybridization-based tRNA profiling method in liver of mice fed ad libitum or starved. While the quantified tRNAs abundance showed a large dynamic range and specific isoacceptors patterns, no differences in (aminoacyl)-tRNAs were found between the two feeding conditions. Nevertheless, tRNAs for isoleucine, asparagine, aspartate and arginine exhibited a relative low loading state compared to other tRNA in both conditions. Altogether, correlation between codon usage, codon dwell times and tRNA abundances provided insights of how translation elongation is regulated in mammals. Together, this thesis aimed to dissect the mechanisms regulating gene expression by the interplay of the circadian clock and metabolism, as well as understand how translation elongation is regulated in mammals.

EPFL2018

In Vivo Studies on the Control of Gene Expression and Protein Degradation

Karolina Bojkowska

Cellular homeostasis is maintained by tightly regulated gene networks, controlled notably at the levels of transcription, mRNA processing, and protein post-translational modification and turnover. This thesis focuses on two of these regulatory steps, namely gene transcription and protein stability. I first explored the impact of the KRAB-ZFP/KAP1 gene regulation system on liver function. KRAB-ZFPs constitute the largest family of transcription factors encoded by mouse and humans, yet their physiological roles and gene targets remain mostly elusive. Conversely, their molecular mechanism of action has been rather well characterized, as they can recognize DNA in a sequence-specific manner and recruit the universal co-factor KAP1 (also known as TRIM28), which in turn serves as a scaffold for various heterochromatin-inducing effectors. Several studies have shed some light on the roles played by this system in embryonic tissues, but its functions in the adult remain almost completely unknown. As chromatin regulation has been shown to be a major factor in the control of metabolism, we decided to investigate the role of KRAB-ZFP/KAP1 in the liver, an organ central to this process. For this, we generated conditional liver-specific Kap1 KO mice, the study of which revealed a strikingly sex-specific phenotype, with early-onset steatosis and age-related tumorigenesis restricted to males, contrasting with the mild metabolic disturbances recorded in Kap1-deleted females. Underlying this phenotype, our combined transcriptome and chromatin analyses revealed that KAP1 controls sexually dimorphic genes notably involved in hormone, drug and xenobiotic metabolism. Finally, by using a recently developed technique for direct RNA quantification, we established the range of KRAB-ZFPs expressed in the liver and likely responsible for at least part of the observed effects. This study thus demonstrates the central role of the KRAB/KAP1 system in control of sexual dimorphism, responses to xenobiotic stress and metabolic control in the liver, and further links disturbances in these processes with hepatic carcinogenesis. In parallel, in an effort to pursue a multidisciplinary training combining skills in biology and chemistry, I worked towards the development of a method to study the half-life of proteins in vivo. Protein turnover critically influences many biological functions, yet methods have been lacking to assess this parameter in vivo. Capitalizing on an in vitro technology previously developed in Kai Johnsson's laboratory, I demonstrated how chemical labeling can be used to measure the half-life of resident intracellular and extracellular proteins in living mice. Our approach is based on labeling of SNAP-tag-fusion proteins with fluorescent probes. First, we verified that SNAP-tag substrates have wide bio-availability in mice and can be used for the specific in vivo labeling of SNAP-tag fusion proteins. We then applied near-infrared probes to perform non-invasive imaging of in vivo labeled tumors. Finally, we used SNAP-mediated chemical pulse-chase labeling to measuring in vivo the half-life of different extra- and intracellular proteins, including KAP1. Importantly, this tagging technology can be applied to any protein amenable to expression as a fusion partner, whether cell-associated or secreted. Furthermore, because the SNAP-tag chemical labeling allows for a large array of probes also suited for experiments aimed at manipulating and monitoring protein function in vivo, such as cross-linking, pull-down or FRET, this methodology opens broad perspectives for studying protein function in living animals.

EPFL2011

An evolutionarily conserved role for the aryl hydrocarbon receptor in the regulation of movement

Pénélope Andreux, Johan Auwerx, Bart Deplancke, Michael Vincent Frochaux, Laurent Mouchiroud, Evan Graehl Williams

The BXD genetic reference population is a recombinant inbred panel descended from crosses between the C57BL/6 (B6) and DBA/2 (D2) strains of mice, which segregate for about 5 million sequence variants. Recently, some of these variants have been established with effects on general metabolic phenotypes such as glucose response and bone strength. Here we phenotype 43 BXD strains and observe they have large variation (∼5-fold) in their spontaneous activity during waking hours. QTL analyses indicate that ∼40% of this variance is attributable to a narrow locus containing the aryl hydrocarbon receptor (Ahr), a basic helix-loop-helix transcription factor with well-established roles in development and xenobiotic metabolism. Strains with the D2 allele of Ahr have reduced gene expression compared to those with the B6 allele, and have significantly higher spontaneous activity. This effect was also observed in B6 mice with a congenic D2 Ahr interval, and in B6 mice with a humanized AHR allele which, like the D2 allele, is expressed much less and has less enzymatic activity than the B6 allele. Ahr is highly conserved in invertebrates, and strikingly inhibition of its orthologs in D. melanogaster and C. elegans (spineless and ahr-1) leads to marked increases in basal activity. In mammals, Ahr has numerous ligands, but most are either non-selective (e.g. resveratrol) or highly toxic (e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)). Thus, we chose to examine a major environmental influence-long term feeding with high fat diet (HFD)-to see if the effects of Ahr are dependent on major metabolic differences. Interestingly, while HFD robustly halved movement across all strains, the QTL position and effects of Ahr remained unchanged, indicating that the effects are independent. The highly consistent effects of Ahr on movement indicate that changes in its constitutive activity have a role on spontaneous movement and may influence human behavior.

Public Library of Science2014