Loss of the RNA polymerase III repressor MAF1 confers obesity resistance

MAF1 is a global repressor of RNA polymerase III transcription that regulates the expression of highly abundant noncoding RNAs in response to nutrient availability and cellular stress. Thus, MAF1 function is thought to be important for metabolic economy. Here we show that a whole-body knockout of Maf1 in mice confers resistance to diet-induced obesity and nonalcoholic fatty liver disease by reducing food intake and increasing metabolic inefficiency. Energy expenditure in Maf1(-/-) mice is increased by several mechanisms. Precursor tRNA synthesis was increased in multiple tissues without significant effects on mature tRNA levels, implying increased turnover in a futile tRNA cycle. Elevated futile cycling of hepatic lipids was also observed. Metabolite profiling of the liver and skeletal muscle revealed elevated levels of many amino acids and spermidine, which links the induction of autophagy in Maf1(-/-) mice with their extended life span. The increase in spermidine was accompanied by reduced levels of nicotinamide N-methyltransferase, which promotes polyamine synthesis, enables nicotinamide salvage to regenerate NAD(+), and is associated with obesity resistance. Consistent with this, NAD(+) levels were increased in muscle. The importance of MAF1 for metabolic economy reveals the potential for MAF1 modulators to protect against obesity and its harmful consequences.

Genomic targets and molecular partners of the chromatin regulator KAP1

Annamaria Kauzlaric

KAP1 is an enigmatic regulatory protein, first described some twenty years ago, shown to be involved in multiple and diverse cellular functions. Specifically, it mediates tasks critical to cell growth and differentiation, pluripotency, apoptosis, gene silencing, DNA repair and genomic integrity maintenance. In line with these findings, Kap1 deletion results in embryonic lethality, and its conditional ablation impairs crucial processes such as erythropoiesis, hepatic metabolism homeostasis and stress response. Initially named also TIF1ðœ, transcriptional intermediary factor 1ðœ, KAP1 was thought to mediate mostly chromatin-related functions. Accordingly, global maps of genomic recruitment of KAP1, generated with the advent of high-throughput technologies, display binding of this protein to thousands of loci in a multiplicity of cell types. Coupling such maps with data of transcriptional perturbations detected upon Kap1 depletion, and with the protein complexes associated with KAP1, proved to be a highly efficient approach to elucidate the molecular functions of KAP1. In particular, such studies unveiled the mechanisms of KAP1-mediated repression of different classes of transposable elements (TEs), and led to important evolutionary considerations on the strategies evolved by higher organisms to restrict the activity of these entities. Through its multiple functional domains, KAP1 interacts with a broad range of proteins, and our first goal was to establish the complete set of such potential cofactors. We found marked enrichment of KAP1 in the chromatin fraction of the nucleus, and accordingly, among its associated proteins, a strong preponderance of nucleic-acid contacting proteins. We revealed that KAP1 is part of a variety of relatively low molecular weight complexes, co-existing at comparable abundances. Study of the molecular functions of the hits suggested that KAP1 could partake in processes thus far unexplored in this context, such as DNA replication and active transcription. We then pursued with the thorough characterization of genome-wide KAP1 binding sites, and found significant fractions of RNA polymerase II (PolII)-dependent promoters and RNA polymerase III (PolIII)-transcribed elements, for the majority enriched with euchromatic marks, to be targeted by KAP1. Using high-throughput techniques, we uncovered that KAP1 variably affected the PolII pausing index of defined sets of genes, and downregulated transcription of tRNA genes. Our analyses of KAP1 genomic recruitments further led us to the discovery of novel genetic entities in the mouse genome, highly related to KRAB zinc finger protein (KZFP) genes. We described possible mechanisms underlying the evolution and amplification of these sequences and more broadly of the KZFP genes family. Furthermore, our data supported the existence of a KAP1-dependent regulatory circuit taming TE-contained enhancers located in the vicinity of these genetic units, thereby regulating their expression. We explored the proposed models of KAP1 functions in euchromatic regions of the genome, and with our results we provided the most complete analysis of the interactome of KAP1 thus far reported. Moreover, we annotated new potentially protein-coding transcripts belonging to the biggest single family of transcription factors encoded in the murine and human genomes. In sum, this work brings novel pieces of information that complement the current knowledge in the field, opening the way to further molecular studies clarifying the physiological roles of KAP1 in higher vertebrates.

EPFL2017

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

Robust landscapes of ribosome dwell times and aminoacyl-tRNAs in response to nutrient stress in liver

Frédéric Bruno Martin Gachon, Cédric Gobet, Felix Naef, Nagammal Neelagandan, Benjamin Dieter Weger

Translation depends on messenger RNA (mRNA)-specific initiation, elongation, and termination rates. While translation elongation is well studied in bacteria and yeast, less is known in higher eukaryotes. Here we combined ribosome and transfer RNA (tRNA) profiling to investigate the relations between translation elongation rates, (aminoacyl-) tRNA levels, and codon usage in mammals. We modeled codon-specific ribosome dwell times from ribosome profiling, considering codon pair interactions between ribosome sites. In mouse liver, the model revealed site- and codon-specific dwell times that differed from those in yeast, as well as pairs of adjacent codons in the P and A site that markedly slow down or speed up elongation. While translation efficiencies vary across diurnal time and feeding regimen, codon dwell times were highly stable and conserved in human. Measured tRNA levels correlated with codon usage and several tRNAs showed reduced aminoacylation, which was conserved in fasted mice. Finally, we uncovered that the longest codon dwell times could be explained by aminoacylation levels or high codon usage relative to tRNA abundance.