Effects of temperature on biological processes are complex. Diffusion is less affected than the diverse enzymatic reactions that have distinct individual temperature profiles. Hence thermal fluctuations pose a formidable challenge to ectothermic organisms in which body temperature is largely dictated by the ambient temperature. How cells in ectotherms cope with the myriad disruptive effects of temperature variation is poorly understood at the molecular level. Here we show that nucleocytoplasmic posttranslational modification of proteins with O-linked GlcNAc (O-GlcNAc) is closely correlated with ambient temperature during development of distantly related ectotherms ranging from the insect Drosophila melanogaster to the nematode Caenorhabditis elegans to the fish Danio rerio. Regulation seems to occur at the level of activity of the only two enzymes, O-GlcNAc transferase and O-GlcNAcase, that add and remove, respectively, this posttranslational modification in nucleus and cytoplasm. With genetic approaches in D. melanogaster and C. elegans, we demonstrate the importance of high levels of this posttranslational modification for successful development at elevated temperatures. Because many cytoplasmic and nuclear proteins in diverse pathways are O-GlcNAc targets, temperature-dependent regulation of this modification might contribute to an efficient coordinate adjustment of cellular processes in response to thermal change.O-GlcNAcylation | temperature acclimation A mbient temperature can fluctuate over various time scales and degrees in different ecological niches. Organisms cope with thermal fluctuations using alternative strategies. Endotherms like humans rely primarily on internally generated heat in combination with intricate regulation for maintenance of a relatively high and constant core body temperature. In contrast, the majority of organisms are ectotherms that produce far less heat. Their internal temperature is primarily dictated by the environment. Cells in some ectotherms are able to acclimate over a remarkable range of ambient temperatures even though temperature change has pervasive effects. All biological processes depend on temperature, but notably not in a uniform manner. Compensation of the myriad disruptive effects of temperature change at the cellular level necessitates extremely complex regulation. Our understanding of the responsible molecular mechanisms is still remarkably poor, even though ambient temperature is often the major ecological determinant of species range.Beyond advanced genetics, Drosophila melanogaster embryos provide additional advantages for studies at the cellular and molecular level, because behavioral responses to temperature change do not yet occur during the immotile early stages. Our characterization of temperature effects on early D. melanogaster development has revealed a unique temperature sensitivity of posttranslational modification of nucleocytoplasmic proteins with O-linked GlcNAc (O-GlcNAc). O-GlcNAc modification is known to occur on thousands of proteins involve...

Cells in ectotherms function normally within an often wide temperature range. As temperature dependence is not uniform across all the distinct biological processes, acclimation presumably requires complex regulation. The molecular mechanisms that cope with the disruptive effects of temperature variation are still poorly understood. Interestingly, one of five different β-tubulin paralogs, , was among the genes upregulated at low temperature in cultured cells. As microtubules are known to be cold sensitive, we analyzed whether protects microtubules at low temperatures. During development at the optimal temperature (25°C), was expressed in a tissue-specific pattern primarily in the gut. There, as well as in hemocytes, expression was increased at low temperature (14°C). Although mutants were viable and fertile at 25°C, their sensitivity within the well-tolerated range was slightly enhanced during embryogenesis specifically at low temperatures. Changing β-tubulin isoform ratios in hemocytes demonstrated that β-Tubulin 97EF has a pronounced microtubule stabilizing effect. Moreover, is required for normal microtubule stability in the gut. These results suggest that upregulation at low temperature contributes to acclimation by stabilizing microtubules.

Background Temperature change affects the myriad of concurrent cellular processes in a non-uniform, disruptive manner. While endothermic organisms minimize the challenge of ambient temperature variation by keeping the core body temperature constant, cells of many ectothermic species maintain homeostatic function within a considerable temperature range. The cellular mechanisms enabling temperature acclimation in ectotherms are still poorly understood. At the transcriptional level, the heat shock response has been analyzed extensively. The opposite, the response to sub-optimal temperature, has received lesser attention in particular in animal species. The tissue specificity of transcriptional responses to cool temperature has not been addressed and it is not clear whether a prominent general response occurs. Cis-regulatory elements (CREs), which mediate increased transcription at cool temperature, and responsible transcription factors are largely unknown. Results The ectotherm Drosophila melanogaster with a presumed temperature optimum around 25 °C was used for transcriptomic analyses of effects of temperatures at the lower end of the readily tolerated range (14–29 °C). Comparative analyses with adult flies and cell culture lines indicated a striking degree of cell-type specificity in the transcriptional response to cool. To identify potential cis-regulatory elements (CREs) for transcriptional upregulation at cool temperature, we analyzed temperature effects on DNA accessibility in chromatin of S2R+ cells. Candidate cis-regulatory elements (CREs) were evaluated with a novel reporter assay for accurate assessment of their temperature-dependency. Robust transcriptional upregulation at low temperature could be demonstrated for a fragment from the pastrel gene, which expresses more transcript and protein at reduced temperatures. This CRE is controlled by the JAK/STAT signaling pathway and antagonizing activities of the transcription factors Pointed and Ets97D. Conclusion Beyond a rich data resource for future analyses of transcriptional control within the readily tolerated range of an ectothermic animal, a novel reporter assay permitting quantitative characterization of CRE temperature dependence was developed. Our identification and functional dissection of the pst_E1 enhancer demonstrate the utility of resources and assay. The functional characterization of this CoolUp enhancer provides initial mechanistic insights into transcriptional upregulation induced by a shift to temperatures at the lower end of the readily tolerated range.

9 3) Author for correspondence (christian.lehner@imls.uzh.ch) 10 11 12 Running title: Drosophila beta-Tubulin 97EF 13 14 KEY WORDS: microtubules, tubulin isoform, boundary cells, low temperature, ectotherm 15 16Summary statement 17 Ectotherms thrive within an often remarkable temperature range. At low temperature, 18 betaTub97EF, a beta-tubulin paralog stabilizing microtubules, is upregulated in a tissue-19 specific manner in the fly Drosophila melanogaster. Abstract 22Cells in ectotherms function normally within an often wide temperature range. As 23 temperature dependence is not uniform across all the distinct biological processes, 24 acclimation presumably requires complex regulation. The molecular mechanisms coping 25 with the disruptive effects of temperature variation are still poorly understood. 26 Interestingly, one of five different beta-tubulin paralogs, betaTub97EF, was among the 27 genes up-regulated at low temperature in cultured Drosophila cells. As microtubules are 28 known to be cold-sensitive, we analyzed whether betaTub97EF protects microtubules at low 29 temperatures. During development at the optimal temperature (25°C), betaTub97EF was 30 expressed in a tissue-specific pattern primarily in the gut. There, as well as in hemocytes, 31 expression was increased at low temperature (14°C). While betaTub97EF mutants were 32 viable and fertile at 25°C, their sensitivity within the well-tolerated range was slightly 33 enhanced during embryogenesis specifically at low temperatures. Changing beta-tubulin 34 isoform ratios in hemocytes demonstrated that beta-Tubulin 97EF has a pronounced 35 microtubule stabilizing effect. Moreover, betaTub97EF is required for normal microtubule 36 stability in the gut. These results suggest that betaTub97EF up-regulation at low 37 temperature contributes to acclimation by stabilizing microtubules. 38 39 45 the environment. But even in endotherms, the exposed peripheral cells need to function 46 over a range of temperatures. 47 77 posttranslational modifications and indirectly via microtubule-associated proteins (MAPs) 78 and motors. Paralogs encoding distinct alpha-and beta-tubulins have evolved in many 79 lineages (Findeisen et al., 2014). Sequence differences are found predominantly in the C-80 terminal tails, where also most posttranslational modifications occur. These modifications 81 are thought to generate a 'tubulin-code' controlling binding and function of many MAPs and 82 motors (Gadadhar et al., 2017;Janke, 2014; Sirajuddin et al., 2014; Yu et al., 2015). 83 In mammals, MAP6 adapts its conformation according to temperature to maintain 84 microtubule networks in cells exposed low temperature (Delphin et al., 2012). Moreover, 85 the recent production of pure tubulin isoforms has definitely established inherent isoform-86 specific differences in microtubule dynamics in vitro (Pamula et al., 2016; Ti et al., 2016; 87 Vemu et al., 2016). 88 Genetic analyses in Drosophila melanogaster have provided the first unequivocal evidence 89 for para...

Cells of many ectothermic species, including Drosophila melanogaster, maintain homeostatic function within a considerable temperature range. The cellular mechanisms enabling temperature acclimation are still poorly understood. At the transcriptional level, the heat shock response has been extensively analyzed. The opposite has received less attention. Here, using cultured Drosophila cells, we have identified genes with increased transcript levels at the lower end of the readily tolerated temperature range, as well as chromatin regions with increased DNA accessibility. Candidate cis-regulatory elements (CREs) for transcriptional upregulation at low temperature were selected and evaluated with a novel reporter assay for accurate assessment of their temperature-dependency. Robust transcriptional upregulation at low temperature could be demonstrated for a fragment from the pastrel gene, which expresses more transcript and protein at reduced temperatures. The CRE is controlled by the JAK/STAT signaling pathway and antagonizing activities of the transcription factors Pointed and Ets97D.

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