Chromatin-associated effectors of energy-sensing pathways mediate intergenerational effects

Environmental stimuli experienced by the parental generation influence the phenotype of subsequent generations. The effects of these stimuli on the parental generation may be passed through the germline, but the mechanisms of this non-Mendelian type of inheritance are poorly known. Here we show that modulation of nutrient-sensing pathways in the parental generation of a nematode (Auanema freiburgensis) regulates phenotypic plasticity of its offspring. In response to pheromones, AMP-activated protein kinase (AMPK), mechanistic target of rapamycin complex 1 (mTORC1) and insulin signaling regulate stress resistance and sex determination across a generation. The effectors of these pathways are closely associated with the chromatin and their regulation affects the acetylation chromatin status in the germline. These results suggest that highly conserved metabolic sensors regulate phenotypic plasticity by changing the epigenetic status of the germline.


INTRODUCTION
The phenotype of an individual is the result of the interactions between its genome 27 and the environment. However, the phenotype may also be influenced by 28 experiences of the parents: parental environment, such as diet, may result in 29 epigenetic changes in the germline that cause non-adaptive phenotypes in the 30 offspring (Chen et al., 2016, Sharma et al., 2016). An example case in humans 31 suggests that famine increases the risk of metabolic defects in one or more 32 generations (Kaati et al., 2007). 33 34 However, there are also mechanisms for passing information about the maternal 35 environment to the offspring that increase fitness (Burton et al., 2017, Dantzer et al., 36 2013, Jablonka, 2013). This is referred to as adaptive phenotypic plasticity, which 37 allows parents to match the phenotype of their offspring to changes in the local . This is because the 52 assays for studying the mechanisms of inheritance of parental effects in A. 53 freiburgensis are fast and easy to perform due its short generation time (~4 days at 54 20 °C) and easy-to-distinguish morphologies in the offspring.

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A. freiburgensis produces three sexes, consisting of males, females and 57 hermaphrodites (Kanzaki et al., 2017). The male of A. freiburgensis is determined 58 genetically (XO), by mechanisms that will be addressed in a separate report. The 59 hermaphrodite versus female sex (both XX) is determined by the environment 60 experienced by the mother. Hermaphrodite individuals kept in isolation produce 61 mostly female offspring, whereas hermaphrodites exposed to high population density 62 conditions produce mostly hermaphrodite offspring (Fig. 1A). 63

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Here we show that high-density population conditions experienced by the A. 65 freiburgensis mother, a signal for imminent starvation, triggers the formation of F1 66 dauer larvae. These dauers develop into hermaphrodite adults, while non-dauer 67 larvae develop into female adults (or males). Pharmacological assays indicate that 68 energy-sensing signaling mediated by AMP-activated protein kinase (AMPK), 69 mechanistic target of rapamycin complex 1 (mTORC1), and insulin signaling are 70 involved in intergenerational inheritance in A. freiburgensis. Effectors of these 71 pathways are associated with chromatin, which changes the histone acetylation 72 status in the germline chromatin to produce F1 dauers, which then develop into 73 hermaphrodite adults. 74

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A crucial factor in the development of Auanema hermaphrodites is the passage 77 through the stress-resistant dauer stage (Félix, 2004, Chaudhuri et   (10 broods, with a total of 199 F1s scored). The data in colored dots represent the 97 percentage of F1 hermaphrodites in each brood and is plotted on the upper axes. The 98 colored vertical lines indicate ± SD and the mean is represented as a gap in the lines. N= 99 sample size. B. In C. elegans, the L1 larvae respond to environmental signals to facultatively 100 form stress-resistant dauers. In A. freiburgensis, it is the mother and not the L1s that 101 respond to environmental signals. A. freiburgensis dauers obligatorily develop into 102 hermaphrodite adults. C. In the experimental setup (top), the same individual mother 103 hermaphrodite was transferred every 24 hours to a new environmental condition. Initially, it 104 was placed in a plate without conditioned medium (-) CM, followed by the transfer to a (+) 105 CM plate and then to a new (-) CM plate. The plot representation is the same as for Fig. 1A  To functionally test the role of AMPK in mediating intergenerational inheritance in A. 194 freiburgensis, we used pharmacological compounds that modulate AMPK activity. 195 We measured the effects of these compounds on intergenerational inheritance by 196 scoring hermaphrodite and female sexes in the offspring. As mentioned previously, 197 high population densities induce the production of dauer larvae in the F1, which 198 mature to become hermaphrodite adults. Consistent with a role of AMPK in 199 mediating this effect, we found that AMPK activators induce the production of   (Solomon et al., 2006). In all cases, more hermaphrodites than females were 348 produced relative to control (Fig. 5D). By contrast, incubating the mothers in high-349 density conditions together with the inhibitor of acetylation 4-tert-butylbenzoic acid 350 (Chen et al., 2014) resulted in less hermaphrodite offspring (Fig. 5D).  To ameliorate the possibility of lack of specificity for AMPK activation, for instance, 425 we used compounds that act through several mechanisms (high production of AMP, 426 allosteric binding, protection against dephosphorylation, activation of 427 phosphorylation). Genetic approaches using loss-and gain-of-function mutants will 428 help to address some of the above-mentioned concerns (Adams et al., 2019).

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Strain and culture 468 We used the Caenorhabditis elegans N2 strain, and the Auanema freiburgensis