Calarco, J. | Search bioRxiv

bioRxiv2022

Two Muscle-Specific and Direct Transcriptional Targets of DAF-16/FOXO Activated by Reduced Insulin/IGF-1 Signaling

C. elegans insulin/insulin-like growth factor 1 signaling, IIS, affects diverse physiological processes through the DAF-16/FOXO transcription factor. Despite its ubiquitous presence in somatic cells, DAF-16s effects exhibit prevalent tissue specificity as well as tissue crosstalk. This implies that tissue-specific DAF-16 transcriptional programs contribute to functional diversity of IIS. To further investigate this possibility, we sought muscle-cell-specific DAF-16 transcriptional targets. Using fluorescence-activated cell sorting to enrich for body wall muscle cells from young hermaphroditic adults, we compared the muscle cell mRNA transcriptomes under conditions of high and low IIS activity, with and without DAF-16. We further analyzed DAF-16as binding sites in muscle and intestine cells by chromatin-immunoprecipitation sequencing. Combined output of these analyses is 12 candidate DAF-16 targets enriched for muscle cells. Transcriptional and translational reporters for three out of the four top candidates - a secreted protein C54F6.5, a calcium-binding protein CEX-1/calexcitin, and a fatty acid metabolic enzyme MLCD-1/MCD - showed DAF-16-dependent activation specifically in body wall muscle cells. Notably, reporters for C54F6.5 and cex-1 exhibit DAF-16-independent, constitutive expression in non-muscle cells, explaining their low rank or absence from the DAF-16 target lists generated by whole-animal microarray or mRNA-sequencing analyses. These results highlight the need to examine FOXO targets in a cell-type-specific manner. Article SummaryThis study is relevant to those interested in functional specificity of signaling pathways. It describes a rigorous workflow to identify tissue-specific transcriptional changes activated by DAF-16/FOXO, a key effector of insulin signaling, its tissue-specific chromatin binding sites, and experimental validation of three previously unknown DAF-16 targets in body wall muscle cells. These findings highlight the intricacy of tissue-specific regulation exerted by a signaling pathway that is present and operates across tissues.

genomics↗

bioRxiv2018

The UBR-1 Ubiquitin Ligase Regulates Glutamate Metabolism to Generate Coordinated Motor Pattern in C. elegans

UBR1 is an E3 ubiquitin ligase best known for its ability to target protein degradation by the N-end rule. The physiological functions of UBR family proteins, however, remain not fully understood. We found that the functional loss of C. elegans UBR-1 leads to a specific motor deficit: when adult animals generate reversal movements, A-class motor neurons exhibit synchronized activation, preventing body bending. This motor deficit is rescued by removing GOT-1, a transaminase that converts aspartate to glutamate. Both UBR-1 and GOT-1 are expressed and critically required in premotor interneurons of the reversal motor circuit to regulate the motor pattern. ubr-1 and got-1 mutants exhibit elevated and decreased glutamate level, respectively. These results raise an intriguing possibility that UBR proteins regulate glutamate metabolism, which is critical for neuronal development and signaling.\n\nAuthor SummaryUbiquitin-mediated protein degradation is central to diverse biological processes. The selection of substrates for degradation is carried out by the E3 ubiquitin ligases, which target specific groups of proteins for ubiquitination. The human genome encodes hundreds of E3 ligases; many exhibit sequence conservation across animal species, including one such ligase called UBR1. Patients carrying mutations in UBR1 exhibit severe systemic defects, but the biology behinds UBR1s physiological function remains elusive. Here we found that the C. elegans UBR-1 regulates glutamate level. When UBR-1 is defective, C. elegans exhibits increased glutamate; this leads to synchronization of motor neuron activity, hence defective locomotion when animals reach adulthood. UBR1-mediated glutamate metabolism may contribute to the physiological defects of UBR1 mutations.

neuroscience↗