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Early life diet conditions the molecular response to post-weaning protein restriction in the mouse

Early life diet conditions the molecular response to post-weaning protein restriction in the mouse Background: Environmental influences fluctuate throughout the life course of an organism. It is therefore important to understand how the timing of exposure impacts molecular responses. Herein, we examine the responses of two key molecular markers of dietary stress, namely variant-specific methylation at ribosomal DNA (rDNA) and small RNA distribution, including tRNA fragments, in a mouse model of protein restriction (PR) with exposure at pre- and/or post-weaning. Results: We first confirm that pre-weaning PR exposure modulates the methylation state of rDNA in a genotype- dependent manner, whereas post-weaning PR exposure has no such effect. Conversely, post-weaning PR induces a shift in small RNA distribution, but there is no effect in the pre-weaning PR model. Intriguingly, mice exposed to PR throughout their lives show neither of these two dietary stress markers, similar to controls. Conclusions: The results show that the timing of the insult affects the nature of the molecular response but also, critically, that ‘matching’ diet exposure either side of weaning eliminates the stress response at the level of rDNA methylation and small RNA in sperm. Keywords: Nutrition, protein restriction, ribosomal DNA, DNA methylation, small RNA, mismatch Background promoter [2]. rDNA codes for the ribosomal RNA that Various environmental factors, such as levels of physical contributes to the structure of the ribosome and exists activity or poor diet, can potentially influence health and in the genome in tandem repeats at multiple loci (Fig. disease states in mammals. As environmental stressors 1a)[3]. We found a significant negative relationship can operate at any point during the life course [1], it is between mice weaning weight and methylation status of necessary to understand how the timing of exposure to a specific functional CpG, 133 bp upstream of the tran- these factors influences molecular responses. Within this scriptional start site of the rDNA locus (CpG −133), the context, understanding the dynamics of molecular methylation of which is associated with suppression of markers of the stress response would greatly enhance transcription of that particular rDNA gene copy [2, 4]. the ability to monitor the impact of environmental However, this relationship was only observed in mice stressors in mammals and, ultimately, to gain mechanis- exposed to in utero PR, and not in controls [2]. This tic insight into the stress pathways involved. epigenetic response remained into adulthood, even after Recently, we reported that protein restriction (PR) in the PR mice were put on a control diet after weaning. mice from conception until weaning induced a linear Crucially, this response only occurred at a subset of the correlation between growth in early life and DNA rDNA copies within the mouse genome, specifically methylation within the ribosomal DNA (rDNA) those containing an ‘A’ base at position 104 bp upstream of the TSS (Fig. 1a). rDNA copies with ‘C’ at this position did not show environment-induced methylation * Correspondence: michelle.holland@kcl.ac.uk; v.rakyan@qmul.ac.uk Department of Medical and Molecular Genetics, King’s College London, dynamics at CpG −133. Furthermore, the epigenetic Guys Hospital, London SE1 9RT, UK state correlated with both transcriptional and phenotypic The Blizard Institute, Queen Mary University of London, 4 Newark Street, outcomes, and hypermethylation of rDNA was also London E1 2AT, UK © Rakyan et al. 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Danson et al. BMC Biology (2018) 16:51 Page 2 of 10 Fig. 1 A C57BL/6 J protein restriction mouse model that combines two previously studied models with contradictory molecular consequences. a BisPCR-seq was used to simultaneously analyse methylation at CpG −133 (methylation = black circles) and genetic variation at the A/C SNP at position −104 bp in the promoter region of the 45S rDNA tandem repeats. b Small RNA-seq was used to analyse tRNA fragments derived from cleaved mature tRNAs. c Breeding and experimental scheme. Pregnant female C57BL/6 J mice were put on either a control (CT, 20% protein) or protein restricted (PR, 8% protein) diet from conception to the end of lactation. Male offspring in each litter were put on either a CT or PR diet from weaning to give four diet combinations – CTCT (black), CTPR (blue), PRCT (orange), PRPR (red). As in previously studied models, phenotype, rDNA methylation and tRNA frag- ment analyses were performed on sperm and other tissues taken at 84 days of age identified after exposure to both high-fat and obesogenic [2] and Shea et al. [6] examined inbred C57BL/6 J mice diets from conception to weaning [2]. Collectively, these and used similar PR exposures. Although part of the rea- results identified a mammalian example of epigenetic son for the discrepant observations between these two dynamics induced by an interaction between the geno- studies could be that Shea et al. [6] did not discriminate type and the early life environment. Mouse rDNA hyper- between the A or C genetic variants, another potential methylation in response to in utero PR exposure was explanation lies in the differences in the timing of PR also independently demonstrated by Denisenko et al. [5]. exposure. Interestingly, using a similar post-weaning PR On the other hand, Shea et al. [6] reported a mouse exposure mouse model (albeit in a different mouse model in which they exposed mice to a PR diet from strain), the authors subsequently reported a marked in- weaning onwards, and although they observed substan- crease in transfer RNA (tRNA) fragments that result tial genetic and epigenetic heterogeneity at rDNA, there from the cleavage of mature tRNAs at specific sites [7] were no observable diet-specific effects. Both our group (Fig. 1b). Separately, Chen et al. [8] reported tRNA Danson et al. BMC Biology (2018) 16:51 Page 3 of 10 fragments to be increased in the sperm of male mice Results after being fed a high-fat diet post-weaning. Pregnant inbred C57BL/6 J mice were fed either a con- Although valuable insights have been gained by previ- trol (CT, 20% protein, 11 litters) diet or a PR (8% pro- ous genomic and epigenomic analyses performed in the tein, 10 litters) diet throughout pregnancy and lactation context of dietary models in rodents [9–13], what is (Additional file 1: Table S1). All litters were derived from noteworthy about the findings related to rDNA and independent females, i.e., no females were used to gener- tRNA fragments is that they have been reported by ate more than one litter. At weaning (3 weeks), the male independent groups in different dietary exposure models offspring from each litter were assigned to either the CT [2, 5, 7, 8, 14]. Indeed, both rDNA and tRNA fragments or PR diet until they were killed at 11–13 weeks of age have been implicated in cellular stress response mecha- (Fig. 1c). Four diet combinations were therefore studied, nisms that are conserved amongst species [15–20]. namely (1) CT throughout life (CTCT, n = 10), (2) Litters Given the potential of rDNA and tRNA fragments to be CT pre-weaning followed by PR post-weaning (CTPR, as robust ‘molecular barometers’ of dietary stress in differ- in Shea et al. [6], n = 11), (3) PR pre-weaning Litters ent experimental models, including rodents, we set out followed by CT post-weaning (PRCT, as in Holland et al. to address three key inter-related questions raised by the [2], 2016, n = 9) and (4) PR throughout life (PRPR, Litters recent studies described above. Firstly, is the difference n = 10). Litters in rDNA responses between our previous model [2] and We previously demonstrated that body weight at that of Shea et al. [6] due to the timing of the PR expos- weaning was significantly lower in offspring of mothers ure? Secondly, are tRNA fragments upregulated when fed a PR diet [2]. Our data in the present study replicates the environmental challenge is experienced during early this finding, as offspring exposed to pre-weaning PR life? And finally, what happens when the animal is were, on average, 34% lighter than the offspring of CT- − 16 exposed to poor nutrition throughout the life-course? fed mothers (Fig. 2a dotted line, P = 2.2 × 10 ). After Answers to these questions would, in a more general weaning, the growth rate of mice was determined by a sense, provide an enhanced understanding of how the post-weaning diet (Fig. 2a, Additional file 1: Figure S1a). timing of environmental exposures impacts the dynamic Despite this, the absolute weight of the PRCT group did molecular responses of a mammalian genome. not catch up to that of the CTCT group, indicating that Fig. 2 A C57BL/6 J protein restriction mouse model that replicates phenotypes seen previously. a Weight progression of male offspring between 7 and 84 days of age. Grey shading = 95% confidence interval. Dotted line = time of weaning. Weaning weights of male offspring exposed to PR diet (n =33, − 16 n = 10) were 34% lower than those exposed to CT diet (n = 34, n = 11). P =2×10 ,Welch’s t test using litter means. b Fasting weight loss during Litters Litters 16 h fast as a proportion of pre-fast weight was lower in CTPR (mean = 8.87%, n = 17, n = 11) than CTCT (mean = 10.6%, n = 17, n = 10) (P =0.048) Litters Litters and the same in PRCT (mean = 11.99%, n = 18, n = 9) and PRPR (mean = 9.37%, n = 15, n = 10) as CTCT (P =0.26 and P = 0.37). A linear model was Litters Litters run for each phenotype against diet and sibling relatedness was accounted for using robust standard errors. P values were adjusted for n = 3 tests using Bonferroni’smethod. c Weightatdeath wasdistinctbetween thefourdietgroups. CTPR mice were significantly lighter (mean = 26.05 g) than CTCT mice − 6 (mean = 28.53 g) at death (P = 0.0003). PRCT mice were lighter than CTCT mice (mean = 24.06 g, P =4.65×10 ) and PRPR mice were the lightest (mean − 12 = 21.76 g, P =7.5 ×10 ). A linear model was run for each phenotype against diet and sibling relatedness was accounted for using robust standard errors. P values were adjusted for n = 3 tests using Bonferroni’s method Danson et al. BMC Biology (2018) 16:51 Page 4 of 10 the post-weaning diet could not compensate for the genetic variant frequency at position −104 bp in the pro- growth retardation induced by the pre-weaning diet. moter region of rDNA and of the frequency of methyla- Mice were killed at 11–13 weeks of age, after over- tion at the functional CpG site at −133 bp. night fasting. The death weights of the four diet groups In our previous work, only the CTCT and PRCT were significantly different from each other (Fig. 2c; groups were examined. The key finding was that mater- − 6 CTPR P = 0.0003, PRCT P = 4.65 × 10 , PRPR P = 7.5 × nal PR induced a correlation between the relative num- − 12 10 ) and this was because CTPR mice lost less weight ber of rDNA copies with an ‘A’ at position −104 bp in an than CTCT mice (Fig. 2b; P = 0.048), whilst weight loss individual (%A) and the frequency of methylation of A in PRCT and PRPR mice was similar to the controls variants at CpG −133 (CpG –133 A meth%, abbreviated (P =0.26 and P = 0.37, respectively). Interestingly, to A ). The present study replicates these findings, meth% despite their differences in size, relative organ and fat with a positive correlation between %A and A in meth% deposit weights were the same between the CTCT the PRCT group (Fig. 3c; PRCT orange, cor = 0.50; linear and the PRPR groups (Additional file 1:Figures S2 model with robust standard errors, P = 0.0008) but not lin and S3), except for the relative kidney weight, which in the CTCT group (Fig. 3a; CTCT black, cor = −0.25; was lower in the PRPR group (Additional file 1: linear model with robust standard errors, P = 0.99). lin Figure S2e). We used all mice in this analysis (rather than litter aver- Overall, the PR model reported here replicates the ages) as well as a linear model with robust standard phenotypes seen previously by our group [2] and others errors to correct for relatedness among littermates (see in the PRCT [2, 21] and CTPR branches [22, 23]. We Methods). Expanding upon our previous work, we ex- have extended the model to include the PRPR group, amined this relationship in the other dietary regimes in which, despite being the lightest in body mass, appears the present study. We observed no correlation between to differ little from the CTCT group in terms of pheno- %A and A in the CTPR group (Fig. 3b; CTPR blue, meth% type, at least as measured between 11 and 13 weeks. cor = −0.59; linear model with robust standard errors, To study the molecular responses to differences in P = 0.15), suggesting that exposure to PR pre-weaning lin timing of PR exposure, we focussed on mature sperm, as is necessary for the induction of this particular DNA this was the common tissue between the previous methylation response at rDNA. Surprisingly, there was no models from our group [2] and the model from Sharma relationship between %A and A in the PRPR group meth% et al. [7], thus permitting direct comparison. High sperm (Fig. 3d;PRPRred,cor= −0.14; linear model with robust purity was consistently obtained (Additional file 1: standard errors, P =0.99), suggesting that post-weaning lin Figure S4). Extracted DNA was sequenced by multiplex PR may reverse the effect of pre-weaning PR. We conclude bisulfite PCR sequencing (‘BisPCR-Seq’), as in Holland from these findings that rDNA epigenetic responses to mal- et al. [2]. This method allowed quantification of the A/C nutrition may also be modulated by a post-weaning diet. Fig. 3 Molecular changes to variant-specific rDNA methylation response to protein restriction are restricted to pre-weaning exposure. a In sperm, percentage of rDNA copies with an A at position −104 bp (%A) does not correlate with percentage of those A copies with methylation at CpG −133 (CpG –133 A meth %) in CTCT (black, n = 17, n = 10, cor = −0.25, adjusted P = 0.99). b %A in CTPR (blue, n = 17, n = 11, cor = −0.59, adjusted P = 0.15). c %A correlates Litters lin Litters lin positively with CpG –133 A meth % in PRCT (orange, n = 16, n =9, cor =0.5, adjusted P = 0.0008). d There was no correlation between %A and CpG – Litters lin 133 A meth % in PRPR (red, n = 14, n = 10, cor = −0.14, adjusted P = 0.99). Pearson’s product moment correlation coefficients are given and a linear Litters lin model was run to assess the relationship in group with sibling relatedness being accounted for using robust standard errors. P values are adjusted for n =4 tests using Bonferroni’s method Danson et al. BMC Biology (2018) 16:51 Page 5 of 10 Having established that the rDNA variant-specific nucleolar RNA. Length distribution analyses indicated methylation changes in response to protein restriction that the majority of reads aligning to tRNA were occur during early life only, we next sought to analyse between 28 and 34 nucleotides in length and were there- tRNA fragment and small RNA profiles in the four dif- fore considered to be tRNA fragments (Additional file 1: ferent groups. Small RNA was extracted from the same Figure S5). Differential expression analysis of reads that sperm samples and RNA-seq was performed. Reads were mapped to tRNA showed that there were more differ- aligned to the whole genome and then to databases for ences in tRNA fragments between the controls and the tRNA, Piwi-interacting RNA (piRNA), microRNA and CTPR and PRCT groups (Fig. 4b, nominally significantly other small RNA categories. Figure 4a shows the per- different tRNA fragments lie above the dotted line and centage of mappable reads falling into each small RNA are labelled) than the PRPR group but none of these category in the four groups. The distribution of reads changes reached genome-wide significance. Overall, across the different small RNA classes was significantly although statistical analysis of individual small RNA different in the CTPR group from the distribution in the groups, such as tRNA fragments, did not show the CTCT group (Fisher’s exact test, P = 0.001) but not dif- differences between groups seen by Sharma et al. [7], it ferent in the PRCT (P = 0.09) or PRPR groups (P = 0.99). is clear that there is a change in small RNA composition In particular, the differences in the CTPR group ap- in response to post-weaning PR, which is not seen in peared to be driven by differences in reads aligning to response to pre-weaning PR. Interestingly, these changes cellular tRNA, mitochondrial tRNA, piRNA and small are also not seen in the PR throughout life group Fig. 4 Post-weaning protein restriction leads to an altered small RNA profile in sperm but only when there has been no previous PR exposure. a The percentage of mappable reads falling into each class of small RNA, including transfer RNA (tRNA) fragments (tRF), small nuclear RNA (snRNA), small nucleolar RNA, ribosomal RNA (rRNA), Piwi-interacting RNA (piRNA), mitochondrial tRNA (Mt tRNA), mitochondrial ribosomal RNA (Mt rRNA), microRNA and long intergenic non-coding RNA (lincRNA). The percentage of unannotated reads are marked in grey and all other reads (proc- essed transcripts and mRNA) are marked in beige. Fisher’s exact test for count data was used to assess the differences in distribution across the classes between the CTCT group and the CTPR group (adjusted P = 0.001), PRCT group (adjusted P = 0.09), and PRPR group (adjusted P = 0.99). P values were adjusted for n = 3 tests using Bonferroni’s method. b Differential expression analysis showed no genome-wide significant changes in abundances of tRNA-derived fragments between the CTCT group and any of the other groups (CTPR = blue, PRCT = orange, PRPR = red). The dotted line in each plot represents the nominal significance level (P = 0.05) Danson et al. BMC Biology (2018) 16:51 Page 6 of 10 (PRPR), suggesting that small RNA changes are an acute response and that early-life PR may be ‘protective’ of further changes on continued exposure. Discussion Genetic–epigenetic interactions at rDNA and an in- crease in tRNA fragments have been shown to represent molecular markers of dietary stress (such as PR) in different mouse models [2, 5, 7, 24]. The model used herein includes a re-examination of previously published pre- or post-weaning only PR exposures (PRCT and CTPR, respectively), and reveals the following key obser- vations. Firstly, we confirm that rDNA variant-specific methylation effects are induced when PR exposure occurs pre-weaning only but not when it occurs post- weaning only and, secondly, that the CTPR group was the only one of the three different exposure groups that displayed a significant redistribution of the relative pro- portion of mapped tRNA fragments, small nucleolar RNAs and piRNAs. We were unable to reproduce the relative increase in specific tRNA fragments in the CTPR group as reported by Sharma et al. [7], but this may simply reflect natural experimental variation between our iteration of the model versus theirs, or sub- tle but relevant differences between our C57BL/6 J mice and the FVB/NJ mice used therein. However, the data do support the broader conclusion that post-weaning dietary stress induces perturbation of small RNAs (at least in sperm), whereas this is not observed in the pre- weaning PR exposure. Collectively, these results address the first two aims of our study, and further underline the robustness of rDNA and tRNA as ‘molecular barome- ters’ of dietary stress in mouse models. The more remarkable finding relates to the molecular responses to persistent poor nutrition throughout life (PRPR group). Across both the rDNA and small RNA responses, the PRPR group is indistinguishable from the CTCT group (Fig. 5). Variant-specific rDNA methylation effects are absent in the PRPR group, all of whom are siblings of the mice in the PRCT group in which we see Fig. 5 Early life diet conditions the response of rDNA and tRNA to the distinctive relationship between %A and A . This meth% post-weaning diet in a pre- and post-weaning protein restriction either suggests that the rDNA methylation response is mouse model established at weaning and later ‘reversed’ in the PRPR group during post-weaning life, or that the response is established in adulthood in the PRCT group only. In ‘protective’ against a small RNA stress response on ex- either case, the variant-specific rDNA methylation effect posure to further PR during adulthood. Taken together, is a reflection of the diet combinations across the whole we conclude that the rDNA and small RNA responses life-course of the animal; that is to say, it is the result of observed herein are a consequence of the combined adulthood exposure to a control diet given that the effects of control and PR diets during particular time animal was exposed to PR during early life (PRCT). windows across the entire life-course. Our results are Furthermore, changes in the relative proportions of reminiscent of observations made in human studies, in small RNAs are seen in response to post-weaning PR which mismatch between early life and adulthood diets only (CTPR), but are largely absent from the PRPR lead to adverse metabolic outcomes in later life [25, 26]. group (Fig. 5). Pre-weaning PR may therefore be It will therefore be interesting to test whether such Danson et al. BMC Biology (2018) 16:51 Page 7 of 10 perturbations at rDNA and small RNAs are also found Conclusions in these affected human populations. To conclude, we show that the nature of the molecular There are some limitations to our work that can be response to PR is different depending on the timing of addressed in future studies. First, it is well established the exposure and that ‘matching’ diets either side of that the birth-to-weaning time window is another critical weaning eliminates responses measured at rDNA and period in the life-course of the mouse [27], with growth small RNA. It is important to emphasise that both rDNA and development of many neuroendocrine systems and small RNA stress responses are, broadly speaking, occurring in this period in mice as would occur in utero conserved amongst different species [15–20] and in dif- in humans [28]. No single model can capture the nu- ferent nutritional stress models [2, 8, 32, 33]. Our work ances of mismatch between every critical developmental supports the idea that genetic–epigenetic interactions at stage so the birth-to-weaning period will be an import- rDNA and small RNA could have utility as biomarkers ant model to address in the future, and this may also to study key aspects of human biology and disease and shed light on why the relationship between %A and how environmental pressures during the entire life- A is not observed in PRPR mice. Second, it will be course could impact outcomes. meth% important to analyse the role that rDNA and small RNA perturbations play in the development of disease pheno- Methods types using dedicated models in which one would specif- Breeding and housing conditions ically modulate, for example, specific small RNAs in vivo All animal procedures were conducted in accordance or alter the methylation of specific rDNA copies. These with the Home Office Animals (Scientific Procedures) experiments would also need to be performed in tissues Act 1986 (Project License number: 70/6693). Female potentially more relevant to the phenotype under con- and male C57BL/6 J mice were obtained from Charles sideration, for example, in liver or adipose tissues, to Rivers UK, aged 6–8 weeks and 10 weeks, respectively. investigate the potential downstream metabolic out- Mice were maintained on a 12 h light/dark cycle (07:00– comes. We focused on sperm in this study allowing us 19:00) and housed at a constant temperature and to directly compare our previous model [2] and that humidity. After 1 week of acclimatisation in the mouse from Sharma et al. [7], and because sperm can be iso- facility on standard chow (control diet, 20% protein), lated to very high degrees of purity, thus reducing the matings were set up by transferring one, or sometimes differential cell composition biases that can potentially two, females into a male’s cage in the late afternoon. On arise when using more complex tissues (although it the discovery of a vaginal plug the next morning (desig- should be noted that we also observed the %A vs. A meth% nated 0.5 days post coitum), pregnant females were sin- relationship in livers of PRCT mice [2]). Indeed, the gly housed and given ad libitum access to either a PR mechanisms by which rDNA and small RNAs act as diet (8% protein) or maintained on the CT diet. Breeding stress responses may be interconnected – it was recently males were housed individually for the duration of the found that certain tRNA fragments can modulate the breeding period. Females were maintained on the expression of ribosomal proteins and therefore ribosome respective diet until offspring were weaned. Whole litters biogenesis [29]. In this case, we suggest that different were weighed at 7 and 14 days. Upon weaning at 21 days, molecular mechanisms could operate to bring about the male offspring from each litter were put on either a CT same outcome (changes to ribosome biogenesis) when or PR diet until death. Only litters with 5–10 pups were the stressor occurs at different times in the life-course. It included. Litter sizes had no impact on the conclusions will be interesting to investigate whether there are other reported here (Additional file 1: Figures S8 and S9). Male genomic perturbations conserved across different diet- offspring were housed in cages containing 3–5mice from ary mouse models that may differ in their nature de- weaning and weighed individually every week from wean- pending on the timing of the stress exposure [30]. ing until they were killed at 11–13 weeks of age. Finally, it has been shown that tRNA fragments in sperm can cause gene expression changes in the livers of the offspring of sires exposed to PR during adult- Diets hood only [7, 8], which raises the question of whether The CT diet was PicoLab Mouse Diet 20 Extruded inter-generational effects would be seen in the off- (5R58*), consisting of a standard chow containing spring of the PRPR group where no tRNA or other roughly 20% of calories from protein. The PR diet was a small RNA response is seen. The result supports the custom diet obtained from Special Diet Services and was idea that small RNA in sperm are reflective of the isocaloric with the control diet but contained only 8% of paternal state [31], whichinthiscaseisthe absence calories from protein (code: 829277, name: RB 8% CP of an acute stress response due to long-term exposure ISO E (P)) (Diet compositions outlined in to the stressor. Additional file 1: Table S1). Danson et al. BMC Biology (2018) 16:51 Page 8 of 10 Adult male dissection and phenotyping resuspended in 200 μL of TE buffer and incubated at Mice were fasted for 16 h before being killed by CO as- 50 °C for 3 h before storage at 4 °C. DNA was quantified phyxiation. After weighing the whole animal and meas- using the High Sensitivity Qubit kit (Thermo Fish uring its length from nose to base of the tail, cardiac Scientific, Cat. Q32851) as per the protocol. Sperm puncture was performed using a 23 G needle and 1 mL purity was confirmed by Bis-PCR-Seq of imprinting con- syringe. Between 100 and 500 μL of blood was collected. trol regions associated with MEST, MCTS2, NESP and A drop of blood from the syringe (0.6 μL) was placed on IGF2/H19. a glucose measurement strip and blood glucose concen- tration was measured using a Bayer NEXT Contour Glu- RNA extraction and small RNA library preparation cose meter. The remaining blood was decanted into a 1. For RNA extraction, three-quarters of the extracted 5 mL Eppendorf tube and allowed to clot at room sperm were incubated at 60 °C for 15 min with slow ro- temperature before being placed on ice. In male mice, tation in 33.3 μL of sperm lysis buffer (6.4 M Guanidine the epididymis was next dissected from the base of the HCl, 5% Tween 20, 5% Triton, 120 mM EDTA, 120 mM testes and transferred to a 2 mL Eppendorf tube con- Tris; pH 8.0) with 3.3 μL of Proteinase K (19 mg/mL) taining pre-made sperm motility medium warmed to and 3.3 μL of 0.1 M DTT. After the incubation, one vol- 37 °C in a water bath (sperm motility medium: 1 M ume (100 μL) of ultra-pure water was added, followed NaCl, 100 mM KCl, 25 mM KH PO , 20 mM MgSO ,0. by 700 μL of Qiazol Lysis reagent (QIAGEN, Cat. 2 4 4 6% sodium lactate, 500 mM NaHCO , 25 mM sodium 79,306) and samples were vortexed for 5 min. Chloro- pyruvate, 25 mM CaCl, 500 mM HEPES, 34.5 mg/mL of form (140 μL) was added and samples were shaken BSA). Epididymides were homogenised using a fine pair vigorously for 30 s before 3 min incubation at room of scissors in the tube for 5 min and placed in a water temperature. Samples were centrifuged at 12,000x g at bath for 30 min at 37 °C, with regular inversion, to allow 4 °C for 15 min then the upper aqueous phase was the sperm to swim out. After incubation, the tube was transferred to a new reaction tube. One volume of 70% briefly spun down using a nanofuge to collect debris, EtOH was added and mixed thoroughly. Samples were then the supernatant containing free-swimming sperm transferred to a RNeasy mini spin column and the was removed and placed in a new 1.5 mL Eppendorf protocol from the miRNeasy Mini Kit (Qiagen, Cat. tube and stored on ice for the rest of the dissection. 217,004) was then followed, including the separation of Liver, kidneys and visceral gonadal white adipose tissue the small RNA and large RNA fractions using an RNA deposits were dissected out, weighed and flash frozen in MinElute spin column (Qiagen, Cat. 74,204). The small liquid nitrogen. A small amount of pancreas and small RNA fraction was eluted in 14 μL of RNase-free water intestine were also removed and flash frozen. Next, the and quantified using the microRNA kit from Qubit subcutaneous inguinal white adipose tissue deposits on (Cat. Q32880). Them, 6 μL of the small RNA fraction each side of the mouse and the interscapular brown was used for small RNA library preparation using the adipose tissue deposits on the back of the mouse were NEBNext Small RNA library prep set for Illumina (Cat. removed, weighed and flash frozen. Finally, a small sec- E7330S) as per the protocol. Each sample was uniquely tion of ear was flash frozen. barcoded using one of the NEBNext Index Primers for Illumina (Cat. E7300S, E7580S, E7710S, E7730S). For Phenol:chloroform DNA extraction PCR amplification, 15 cycles were used. Libraries were For DNA extraction, one-quarter of the extracted sperm purified using the QIAQuick PCR purification kit was incubated overnight in 600 μL of PK buffer (10 mM (Qiagen, Cat. 28,104) and DNA was eluted into 32 μLof Tris-HCl, 100 mM NaCl, 25 mM EDTA, 1% SDS) with nuclease-free water. An aliquot of each library was di- 2 μL of Proteinase K enzyme (19 mg/mL) and 0.1 M luted and 1 μL was run on an Agilent 2100 Bioanalyzer DTT at 55 °C with slow rotation. Phenol (750 μL) was using the Agilent High Sensitivity DNA kit (Cat. 5067- added to the samples and agitated for several minutes 4626) to assess the size distribution of the library. The before spinning at 17,000x g for 5 min at 4 °C. The libraries were pooled using equal volumes and size upper aqueous phase was transferred to a new tube and selected for between 140 and 200 bp (corresponding to the process repeated with phenol:chloroform, then an insert size of 13–73 bp) using a BluePippin machine chloroform alone. After the final spin, 5 μL of Rnase was (Sage Science) with 3% agarose cassettes (Sage Science, added and samples were incubated at 37 °C for 60 min. Cat. BDF3010). Then, 0.1 volumes of 3 M sodium acetate (pH 5.2) and 2.5 volumes of 100% EtOH were added and samples Sequencing and data analysis incubated at −20 °C for 1 h. Samples were spun at DNA from sperm was diluted to a concentration of 17,000x g for 10 min at 4 °C and the pellet was washed 11 ng/μL and 45 μL of each sample was sent for sequen- with 75% EtOH. Finally, the pellet was air-dried at 37 °C, cing to the Genome Centre Facility at Charterhouse Danson et al. BMC Biology (2018) 16:51 Page 9 of 10 Square, QMUL. Bis-PCR-Seq was performed using the and linear models with robust standard errors were used ® ® 48.48 layout on the Fluidigm C1 system (Fluidigm , to correct for any biases due to sibling relatedness (a full USA), coupled with Illumina MiSeq sequencing using justification is provided in Additional file 1: Supplemen- version2 chemistry (150 bp, paired-end). See Add- tary methods). Fisher’s exact test was used to assess the itional file 1: Table S3 for the primer sequences. Small differences in distribution of the small RNA compositions RNA libraries were initially sequenced using Illumina compared to CTCT, using percentages of mapped reads MiSeq Nano sequencing (75 bp, single-end) and read corresponding to each species in each sample and these counts for each samples were used to re-balance the were rounded to the nearest integer (corrected for n =3 library pool. The final pool was then sequenced using tests using Bonferroni’s correction). ANOVA was used to Illumina NextSeq sequencing (75 bp, single-end). assess whether the %A or %C and CpG –133 A meth % or Bismark (v0.7.12) was used to align Bis-PCR-Seq data CpG –133 C meth % were different between the diet to the mm10 reference genome (imprinting control re- groups. gion data; Additional file 1: Figure S4) or to the adjusted consensus rDNA reference, using Bowtie2 (v2.1.0). Only Additional file reads that mapped to the correct starting position and perfectly matched the consensus were used for further Additional file 1: Table S1. Compositions of the control (CT) diet and the protein restricted (PR) diet. Table S2. List of all male mice studied analysis. For rDNA analysis, the R package RSamtools with each litter represented by the first two numbers and letter of each was used to identify each read as either having an A or a ID. Table S3. Sequences of primers used for targeted analysis of DNA C at position −104 bp and to determine the methylation methylation at rDNA CpG −133 and the imprinted regions MEST, MCTS2, NESP and IGF2/H19. Figure S1. Growth rates of mice and lengths at status at position −133 bp of each read. Reads could death in each group. Figure S2. Absolute and relative organ weights. m u m u therefore be assigned to either A ,A ,C ,orC . The Figure S3. Absolute and relative adipose tissue deposit weights. Figure total number of reads in each group was summed and S4. Sperm small RNA size distribution analysis and sperm purity analysis. m u m u Figure S5. Read length distribution of reads mapping to the genome %A ((A +A )/(C +C )) and CpG –133 A meth % that also map to different classes of small RNA, normalised by total m m u (A /(A +A )) calculated for each sample. Methylation number of reads mapping to the genome. Figure S6. Maternal weight, of imprinting control regions were assessed using a food intake and litter size data. Figure S7. %A/C and CpG –133 meth % distribution in four diet groups. Figure S8. Growth trajectories plotted by custom program (https://bitbucket.org/lowelabqmul/ pre-weaning litter size (including females). Figure S9. Pre-weaning litter methylation-extractor). size (including females) has no impact on CpG –133 A meth % in any of Small RNA sequencing data was mapped to the whole the groups. Supplementary methods. Rationale and explanation of the use of a linear model and robust standard errors to analyse the relationship genome (UCSC, mm10), piRNA, tRNA, miRNA and between %A and CpG –133 A meth % instead of using litter averages or rRNA databases using the SPORTS 1.0 pipeline (https:// individuals from the same litter without correction for relatedness. R script github.com/junchaoshi/sports1.0.git). Total read counts used to perform the analysis is also included. (DOCX 1338 kb) for each small RNA class were expressed as percentages of number of reads mapped to the genome for each Acknowledgements sample in the composition analysis (Fig. 4a). Differential We thank the BSU technicians for their help with the animal work and the Bart’s and the London Genome Centre staff for performing the high expression analysis of tRNA fragments was performed through-put sequencing. We also thank Féaron Cassidy, Philip Howard and using edgeR (glmQLFTest) using the number of reads Gabriel Rosser for their help and advice. mapping to the genome as the library sizes for normalisation. Funding AFD is funded by an MRC Studentship (MR/K501372/1) and a Life Sciences Initiative Small Grant and the work was supported by a Biotechnology and Statistics Biological Sciences Research Council, UK (BB/M012494/1) grant awarded to VKR. All statistical analysis and plotting were performed using R (v3.2.3). For all phenotype plots, a linear model was Availability of data and materials The datasets supporting the conclusions of this article are available in the run on individuals and P values were derived by using GEO repository, Series GSE107541. robust standard errors to account for the relatedness be- tween siblings in each diet group (R packages plm [34] Authors’ contributions and lmtest [35]) and corrected for n = 3 tests using AFD, MLH and VKR conceived the project and carried out the experiments. AFD analysed the data with the help of RL and SJM advised on statistical Bonferroni’s correction. A Pearson’s product moment analyses. All authors discussed the results and interpretation and approved correlation coefficient was calculated to describe the the final manuscript. relationship between %A and CpG –133 A methylation percentage in each diet group (cor) and a linear model Ethics approval All animal procedures were conducted in accordance with the Home Office with robust standard errors was then run to obtain Animals (Scientific Procedures) Act 1986 (Project License number: 70/6693). P values, which were then corrected for n =4 com- parisons using Bonferroni’s correction (P ). All mice lin Competing interests were used in the analyses instead of litter averages The authors declare that they have no competing interests. Danson et al. BMC Biology (2018) 16:51 Page 10 of 10 Publisher’sNote 22. Solon-Biet SM, McMahon AC, Ballard JWO, Ruohonen K, Wu LE, Cogger VC, Springer Nature remains neutral with regard to jurisdictional claims in et al. 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Nutr Metab Cardiovasc Dis. 2012;22:1067–74. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BMC Biology Springer Journals

Early life diet conditions the molecular response to post-weaning protein restriction in the mouse

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Springer Journals
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Copyright © 2018 by Rakyan et al.
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Life Sciences; Life Sciences, general
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1741-7007
DOI
10.1186/s12915-018-0516-5
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Abstract

Background: Environmental influences fluctuate throughout the life course of an organism. It is therefore important to understand how the timing of exposure impacts molecular responses. Herein, we examine the responses of two key molecular markers of dietary stress, namely variant-specific methylation at ribosomal DNA (rDNA) and small RNA distribution, including tRNA fragments, in a mouse model of protein restriction (PR) with exposure at pre- and/or post-weaning. Results: We first confirm that pre-weaning PR exposure modulates the methylation state of rDNA in a genotype- dependent manner, whereas post-weaning PR exposure has no such effect. Conversely, post-weaning PR induces a shift in small RNA distribution, but there is no effect in the pre-weaning PR model. Intriguingly, mice exposed to PR throughout their lives show neither of these two dietary stress markers, similar to controls. Conclusions: The results show that the timing of the insult affects the nature of the molecular response but also, critically, that ‘matching’ diet exposure either side of weaning eliminates the stress response at the level of rDNA methylation and small RNA in sperm. Keywords: Nutrition, protein restriction, ribosomal DNA, DNA methylation, small RNA, mismatch Background promoter [2]. rDNA codes for the ribosomal RNA that Various environmental factors, such as levels of physical contributes to the structure of the ribosome and exists activity or poor diet, can potentially influence health and in the genome in tandem repeats at multiple loci (Fig. disease states in mammals. As environmental stressors 1a)[3]. We found a significant negative relationship can operate at any point during the life course [1], it is between mice weaning weight and methylation status of necessary to understand how the timing of exposure to a specific functional CpG, 133 bp upstream of the tran- these factors influences molecular responses. Within this scriptional start site of the rDNA locus (CpG −133), the context, understanding the dynamics of molecular methylation of which is associated with suppression of markers of the stress response would greatly enhance transcription of that particular rDNA gene copy [2, 4]. the ability to monitor the impact of environmental However, this relationship was only observed in mice stressors in mammals and, ultimately, to gain mechanis- exposed to in utero PR, and not in controls [2]. This tic insight into the stress pathways involved. epigenetic response remained into adulthood, even after Recently, we reported that protein restriction (PR) in the PR mice were put on a control diet after weaning. mice from conception until weaning induced a linear Crucially, this response only occurred at a subset of the correlation between growth in early life and DNA rDNA copies within the mouse genome, specifically methylation within the ribosomal DNA (rDNA) those containing an ‘A’ base at position 104 bp upstream of the TSS (Fig. 1a). rDNA copies with ‘C’ at this position did not show environment-induced methylation * Correspondence: michelle.holland@kcl.ac.uk; v.rakyan@qmul.ac.uk Department of Medical and Molecular Genetics, King’s College London, dynamics at CpG −133. Furthermore, the epigenetic Guys Hospital, London SE1 9RT, UK state correlated with both transcriptional and phenotypic The Blizard Institute, Queen Mary University of London, 4 Newark Street, outcomes, and hypermethylation of rDNA was also London E1 2AT, UK © Rakyan et al. 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Danson et al. BMC Biology (2018) 16:51 Page 2 of 10 Fig. 1 A C57BL/6 J protein restriction mouse model that combines two previously studied models with contradictory molecular consequences. a BisPCR-seq was used to simultaneously analyse methylation at CpG −133 (methylation = black circles) and genetic variation at the A/C SNP at position −104 bp in the promoter region of the 45S rDNA tandem repeats. b Small RNA-seq was used to analyse tRNA fragments derived from cleaved mature tRNAs. c Breeding and experimental scheme. Pregnant female C57BL/6 J mice were put on either a control (CT, 20% protein) or protein restricted (PR, 8% protein) diet from conception to the end of lactation. Male offspring in each litter were put on either a CT or PR diet from weaning to give four diet combinations – CTCT (black), CTPR (blue), PRCT (orange), PRPR (red). As in previously studied models, phenotype, rDNA methylation and tRNA frag- ment analyses were performed on sperm and other tissues taken at 84 days of age identified after exposure to both high-fat and obesogenic [2] and Shea et al. [6] examined inbred C57BL/6 J mice diets from conception to weaning [2]. Collectively, these and used similar PR exposures. Although part of the rea- results identified a mammalian example of epigenetic son for the discrepant observations between these two dynamics induced by an interaction between the geno- studies could be that Shea et al. [6] did not discriminate type and the early life environment. Mouse rDNA hyper- between the A or C genetic variants, another potential methylation in response to in utero PR exposure was explanation lies in the differences in the timing of PR also independently demonstrated by Denisenko et al. [5]. exposure. Interestingly, using a similar post-weaning PR On the other hand, Shea et al. [6] reported a mouse exposure mouse model (albeit in a different mouse model in which they exposed mice to a PR diet from strain), the authors subsequently reported a marked in- weaning onwards, and although they observed substan- crease in transfer RNA (tRNA) fragments that result tial genetic and epigenetic heterogeneity at rDNA, there from the cleavage of mature tRNAs at specific sites [7] were no observable diet-specific effects. Both our group (Fig. 1b). Separately, Chen et al. [8] reported tRNA Danson et al. BMC Biology (2018) 16:51 Page 3 of 10 fragments to be increased in the sperm of male mice Results after being fed a high-fat diet post-weaning. Pregnant inbred C57BL/6 J mice were fed either a con- Although valuable insights have been gained by previ- trol (CT, 20% protein, 11 litters) diet or a PR (8% pro- ous genomic and epigenomic analyses performed in the tein, 10 litters) diet throughout pregnancy and lactation context of dietary models in rodents [9–13], what is (Additional file 1: Table S1). All litters were derived from noteworthy about the findings related to rDNA and independent females, i.e., no females were used to gener- tRNA fragments is that they have been reported by ate more than one litter. At weaning (3 weeks), the male independent groups in different dietary exposure models offspring from each litter were assigned to either the CT [2, 5, 7, 8, 14]. Indeed, both rDNA and tRNA fragments or PR diet until they were killed at 11–13 weeks of age have been implicated in cellular stress response mecha- (Fig. 1c). Four diet combinations were therefore studied, nisms that are conserved amongst species [15–20]. namely (1) CT throughout life (CTCT, n = 10), (2) Litters Given the potential of rDNA and tRNA fragments to be CT pre-weaning followed by PR post-weaning (CTPR, as robust ‘molecular barometers’ of dietary stress in differ- in Shea et al. [6], n = 11), (3) PR pre-weaning Litters ent experimental models, including rodents, we set out followed by CT post-weaning (PRCT, as in Holland et al. to address three key inter-related questions raised by the [2], 2016, n = 9) and (4) PR throughout life (PRPR, Litters recent studies described above. Firstly, is the difference n = 10). Litters in rDNA responses between our previous model [2] and We previously demonstrated that body weight at that of Shea et al. [6] due to the timing of the PR expos- weaning was significantly lower in offspring of mothers ure? Secondly, are tRNA fragments upregulated when fed a PR diet [2]. Our data in the present study replicates the environmental challenge is experienced during early this finding, as offspring exposed to pre-weaning PR life? And finally, what happens when the animal is were, on average, 34% lighter than the offspring of CT- − 16 exposed to poor nutrition throughout the life-course? fed mothers (Fig. 2a dotted line, P = 2.2 × 10 ). After Answers to these questions would, in a more general weaning, the growth rate of mice was determined by a sense, provide an enhanced understanding of how the post-weaning diet (Fig. 2a, Additional file 1: Figure S1a). timing of environmental exposures impacts the dynamic Despite this, the absolute weight of the PRCT group did molecular responses of a mammalian genome. not catch up to that of the CTCT group, indicating that Fig. 2 A C57BL/6 J protein restriction mouse model that replicates phenotypes seen previously. a Weight progression of male offspring between 7 and 84 days of age. Grey shading = 95% confidence interval. Dotted line = time of weaning. Weaning weights of male offspring exposed to PR diet (n =33, − 16 n = 10) were 34% lower than those exposed to CT diet (n = 34, n = 11). P =2×10 ,Welch’s t test using litter means. b Fasting weight loss during Litters Litters 16 h fast as a proportion of pre-fast weight was lower in CTPR (mean = 8.87%, n = 17, n = 11) than CTCT (mean = 10.6%, n = 17, n = 10) (P =0.048) Litters Litters and the same in PRCT (mean = 11.99%, n = 18, n = 9) and PRPR (mean = 9.37%, n = 15, n = 10) as CTCT (P =0.26 and P = 0.37). A linear model was Litters Litters run for each phenotype against diet and sibling relatedness was accounted for using robust standard errors. P values were adjusted for n = 3 tests using Bonferroni’smethod. c Weightatdeath wasdistinctbetween thefourdietgroups. CTPR mice were significantly lighter (mean = 26.05 g) than CTCT mice − 6 (mean = 28.53 g) at death (P = 0.0003). PRCT mice were lighter than CTCT mice (mean = 24.06 g, P =4.65×10 ) and PRPR mice were the lightest (mean − 12 = 21.76 g, P =7.5 ×10 ). A linear model was run for each phenotype against diet and sibling relatedness was accounted for using robust standard errors. P values were adjusted for n = 3 tests using Bonferroni’s method Danson et al. BMC Biology (2018) 16:51 Page 4 of 10 the post-weaning diet could not compensate for the genetic variant frequency at position −104 bp in the pro- growth retardation induced by the pre-weaning diet. moter region of rDNA and of the frequency of methyla- Mice were killed at 11–13 weeks of age, after over- tion at the functional CpG site at −133 bp. night fasting. The death weights of the four diet groups In our previous work, only the CTCT and PRCT were significantly different from each other (Fig. 2c; groups were examined. The key finding was that mater- − 6 CTPR P = 0.0003, PRCT P = 4.65 × 10 , PRPR P = 7.5 × nal PR induced a correlation between the relative num- − 12 10 ) and this was because CTPR mice lost less weight ber of rDNA copies with an ‘A’ at position −104 bp in an than CTCT mice (Fig. 2b; P = 0.048), whilst weight loss individual (%A) and the frequency of methylation of A in PRCT and PRPR mice was similar to the controls variants at CpG −133 (CpG –133 A meth%, abbreviated (P =0.26 and P = 0.37, respectively). Interestingly, to A ). The present study replicates these findings, meth% despite their differences in size, relative organ and fat with a positive correlation between %A and A in meth% deposit weights were the same between the CTCT the PRCT group (Fig. 3c; PRCT orange, cor = 0.50; linear and the PRPR groups (Additional file 1:Figures S2 model with robust standard errors, P = 0.0008) but not lin and S3), except for the relative kidney weight, which in the CTCT group (Fig. 3a; CTCT black, cor = −0.25; was lower in the PRPR group (Additional file 1: linear model with robust standard errors, P = 0.99). lin Figure S2e). We used all mice in this analysis (rather than litter aver- Overall, the PR model reported here replicates the ages) as well as a linear model with robust standard phenotypes seen previously by our group [2] and others errors to correct for relatedness among littermates (see in the PRCT [2, 21] and CTPR branches [22, 23]. We Methods). Expanding upon our previous work, we ex- have extended the model to include the PRPR group, amined this relationship in the other dietary regimes in which, despite being the lightest in body mass, appears the present study. We observed no correlation between to differ little from the CTCT group in terms of pheno- %A and A in the CTPR group (Fig. 3b; CTPR blue, meth% type, at least as measured between 11 and 13 weeks. cor = −0.59; linear model with robust standard errors, To study the molecular responses to differences in P = 0.15), suggesting that exposure to PR pre-weaning lin timing of PR exposure, we focussed on mature sperm, as is necessary for the induction of this particular DNA this was the common tissue between the previous methylation response at rDNA. Surprisingly, there was no models from our group [2] and the model from Sharma relationship between %A and A in the PRPR group meth% et al. [7], thus permitting direct comparison. High sperm (Fig. 3d;PRPRred,cor= −0.14; linear model with robust purity was consistently obtained (Additional file 1: standard errors, P =0.99), suggesting that post-weaning lin Figure S4). Extracted DNA was sequenced by multiplex PR may reverse the effect of pre-weaning PR. We conclude bisulfite PCR sequencing (‘BisPCR-Seq’), as in Holland from these findings that rDNA epigenetic responses to mal- et al. [2]. This method allowed quantification of the A/C nutrition may also be modulated by a post-weaning diet. Fig. 3 Molecular changes to variant-specific rDNA methylation response to protein restriction are restricted to pre-weaning exposure. a In sperm, percentage of rDNA copies with an A at position −104 bp (%A) does not correlate with percentage of those A copies with methylation at CpG −133 (CpG –133 A meth %) in CTCT (black, n = 17, n = 10, cor = −0.25, adjusted P = 0.99). b %A in CTPR (blue, n = 17, n = 11, cor = −0.59, adjusted P = 0.15). c %A correlates Litters lin Litters lin positively with CpG –133 A meth % in PRCT (orange, n = 16, n =9, cor =0.5, adjusted P = 0.0008). d There was no correlation between %A and CpG – Litters lin 133 A meth % in PRPR (red, n = 14, n = 10, cor = −0.14, adjusted P = 0.99). Pearson’s product moment correlation coefficients are given and a linear Litters lin model was run to assess the relationship in group with sibling relatedness being accounted for using robust standard errors. P values are adjusted for n =4 tests using Bonferroni’s method Danson et al. BMC Biology (2018) 16:51 Page 5 of 10 Having established that the rDNA variant-specific nucleolar RNA. Length distribution analyses indicated methylation changes in response to protein restriction that the majority of reads aligning to tRNA were occur during early life only, we next sought to analyse between 28 and 34 nucleotides in length and were there- tRNA fragment and small RNA profiles in the four dif- fore considered to be tRNA fragments (Additional file 1: ferent groups. Small RNA was extracted from the same Figure S5). Differential expression analysis of reads that sperm samples and RNA-seq was performed. Reads were mapped to tRNA showed that there were more differ- aligned to the whole genome and then to databases for ences in tRNA fragments between the controls and the tRNA, Piwi-interacting RNA (piRNA), microRNA and CTPR and PRCT groups (Fig. 4b, nominally significantly other small RNA categories. Figure 4a shows the per- different tRNA fragments lie above the dotted line and centage of mappable reads falling into each small RNA are labelled) than the PRPR group but none of these category in the four groups. The distribution of reads changes reached genome-wide significance. Overall, across the different small RNA classes was significantly although statistical analysis of individual small RNA different in the CTPR group from the distribution in the groups, such as tRNA fragments, did not show the CTCT group (Fisher’s exact test, P = 0.001) but not dif- differences between groups seen by Sharma et al. [7], it ferent in the PRCT (P = 0.09) or PRPR groups (P = 0.99). is clear that there is a change in small RNA composition In particular, the differences in the CTPR group ap- in response to post-weaning PR, which is not seen in peared to be driven by differences in reads aligning to response to pre-weaning PR. Interestingly, these changes cellular tRNA, mitochondrial tRNA, piRNA and small are also not seen in the PR throughout life group Fig. 4 Post-weaning protein restriction leads to an altered small RNA profile in sperm but only when there has been no previous PR exposure. a The percentage of mappable reads falling into each class of small RNA, including transfer RNA (tRNA) fragments (tRF), small nuclear RNA (snRNA), small nucleolar RNA, ribosomal RNA (rRNA), Piwi-interacting RNA (piRNA), mitochondrial tRNA (Mt tRNA), mitochondrial ribosomal RNA (Mt rRNA), microRNA and long intergenic non-coding RNA (lincRNA). The percentage of unannotated reads are marked in grey and all other reads (proc- essed transcripts and mRNA) are marked in beige. Fisher’s exact test for count data was used to assess the differences in distribution across the classes between the CTCT group and the CTPR group (adjusted P = 0.001), PRCT group (adjusted P = 0.09), and PRPR group (adjusted P = 0.99). P values were adjusted for n = 3 tests using Bonferroni’s method. b Differential expression analysis showed no genome-wide significant changes in abundances of tRNA-derived fragments between the CTCT group and any of the other groups (CTPR = blue, PRCT = orange, PRPR = red). The dotted line in each plot represents the nominal significance level (P = 0.05) Danson et al. BMC Biology (2018) 16:51 Page 6 of 10 (PRPR), suggesting that small RNA changes are an acute response and that early-life PR may be ‘protective’ of further changes on continued exposure. Discussion Genetic–epigenetic interactions at rDNA and an in- crease in tRNA fragments have been shown to represent molecular markers of dietary stress (such as PR) in different mouse models [2, 5, 7, 24]. The model used herein includes a re-examination of previously published pre- or post-weaning only PR exposures (PRCT and CTPR, respectively), and reveals the following key obser- vations. Firstly, we confirm that rDNA variant-specific methylation effects are induced when PR exposure occurs pre-weaning only but not when it occurs post- weaning only and, secondly, that the CTPR group was the only one of the three different exposure groups that displayed a significant redistribution of the relative pro- portion of mapped tRNA fragments, small nucleolar RNAs and piRNAs. We were unable to reproduce the relative increase in specific tRNA fragments in the CTPR group as reported by Sharma et al. [7], but this may simply reflect natural experimental variation between our iteration of the model versus theirs, or sub- tle but relevant differences between our C57BL/6 J mice and the FVB/NJ mice used therein. However, the data do support the broader conclusion that post-weaning dietary stress induces perturbation of small RNAs (at least in sperm), whereas this is not observed in the pre- weaning PR exposure. Collectively, these results address the first two aims of our study, and further underline the robustness of rDNA and tRNA as ‘molecular barome- ters’ of dietary stress in mouse models. The more remarkable finding relates to the molecular responses to persistent poor nutrition throughout life (PRPR group). Across both the rDNA and small RNA responses, the PRPR group is indistinguishable from the CTCT group (Fig. 5). Variant-specific rDNA methylation effects are absent in the PRPR group, all of whom are siblings of the mice in the PRCT group in which we see Fig. 5 Early life diet conditions the response of rDNA and tRNA to the distinctive relationship between %A and A . This meth% post-weaning diet in a pre- and post-weaning protein restriction either suggests that the rDNA methylation response is mouse model established at weaning and later ‘reversed’ in the PRPR group during post-weaning life, or that the response is established in adulthood in the PRCT group only. In ‘protective’ against a small RNA stress response on ex- either case, the variant-specific rDNA methylation effect posure to further PR during adulthood. Taken together, is a reflection of the diet combinations across the whole we conclude that the rDNA and small RNA responses life-course of the animal; that is to say, it is the result of observed herein are a consequence of the combined adulthood exposure to a control diet given that the effects of control and PR diets during particular time animal was exposed to PR during early life (PRCT). windows across the entire life-course. Our results are Furthermore, changes in the relative proportions of reminiscent of observations made in human studies, in small RNAs are seen in response to post-weaning PR which mismatch between early life and adulthood diets only (CTPR), but are largely absent from the PRPR lead to adverse metabolic outcomes in later life [25, 26]. group (Fig. 5). Pre-weaning PR may therefore be It will therefore be interesting to test whether such Danson et al. BMC Biology (2018) 16:51 Page 7 of 10 perturbations at rDNA and small RNAs are also found Conclusions in these affected human populations. To conclude, we show that the nature of the molecular There are some limitations to our work that can be response to PR is different depending on the timing of addressed in future studies. First, it is well established the exposure and that ‘matching’ diets either side of that the birth-to-weaning time window is another critical weaning eliminates responses measured at rDNA and period in the life-course of the mouse [27], with growth small RNA. It is important to emphasise that both rDNA and development of many neuroendocrine systems and small RNA stress responses are, broadly speaking, occurring in this period in mice as would occur in utero conserved amongst different species [15–20] and in dif- in humans [28]. No single model can capture the nu- ferent nutritional stress models [2, 8, 32, 33]. Our work ances of mismatch between every critical developmental supports the idea that genetic–epigenetic interactions at stage so the birth-to-weaning period will be an import- rDNA and small RNA could have utility as biomarkers ant model to address in the future, and this may also to study key aspects of human biology and disease and shed light on why the relationship between %A and how environmental pressures during the entire life- A is not observed in PRPR mice. Second, it will be course could impact outcomes. meth% important to analyse the role that rDNA and small RNA perturbations play in the development of disease pheno- Methods types using dedicated models in which one would specif- Breeding and housing conditions ically modulate, for example, specific small RNAs in vivo All animal procedures were conducted in accordance or alter the methylation of specific rDNA copies. These with the Home Office Animals (Scientific Procedures) experiments would also need to be performed in tissues Act 1986 (Project License number: 70/6693). Female potentially more relevant to the phenotype under con- and male C57BL/6 J mice were obtained from Charles sideration, for example, in liver or adipose tissues, to Rivers UK, aged 6–8 weeks and 10 weeks, respectively. investigate the potential downstream metabolic out- Mice were maintained on a 12 h light/dark cycle (07:00– comes. We focused on sperm in this study allowing us 19:00) and housed at a constant temperature and to directly compare our previous model [2] and that humidity. After 1 week of acclimatisation in the mouse from Sharma et al. [7], and because sperm can be iso- facility on standard chow (control diet, 20% protein), lated to very high degrees of purity, thus reducing the matings were set up by transferring one, or sometimes differential cell composition biases that can potentially two, females into a male’s cage in the late afternoon. On arise when using more complex tissues (although it the discovery of a vaginal plug the next morning (desig- should be noted that we also observed the %A vs. A meth% nated 0.5 days post coitum), pregnant females were sin- relationship in livers of PRCT mice [2]). Indeed, the gly housed and given ad libitum access to either a PR mechanisms by which rDNA and small RNAs act as diet (8% protein) or maintained on the CT diet. Breeding stress responses may be interconnected – it was recently males were housed individually for the duration of the found that certain tRNA fragments can modulate the breeding period. Females were maintained on the expression of ribosomal proteins and therefore ribosome respective diet until offspring were weaned. Whole litters biogenesis [29]. In this case, we suggest that different were weighed at 7 and 14 days. Upon weaning at 21 days, molecular mechanisms could operate to bring about the male offspring from each litter were put on either a CT same outcome (changes to ribosome biogenesis) when or PR diet until death. Only litters with 5–10 pups were the stressor occurs at different times in the life-course. It included. Litter sizes had no impact on the conclusions will be interesting to investigate whether there are other reported here (Additional file 1: Figures S8 and S9). Male genomic perturbations conserved across different diet- offspring were housed in cages containing 3–5mice from ary mouse models that may differ in their nature de- weaning and weighed individually every week from wean- pending on the timing of the stress exposure [30]. ing until they were killed at 11–13 weeks of age. Finally, it has been shown that tRNA fragments in sperm can cause gene expression changes in the livers of the offspring of sires exposed to PR during adult- Diets hood only [7, 8], which raises the question of whether The CT diet was PicoLab Mouse Diet 20 Extruded inter-generational effects would be seen in the off- (5R58*), consisting of a standard chow containing spring of the PRPR group where no tRNA or other roughly 20% of calories from protein. The PR diet was a small RNA response is seen. The result supports the custom diet obtained from Special Diet Services and was idea that small RNA in sperm are reflective of the isocaloric with the control diet but contained only 8% of paternal state [31], whichinthiscaseisthe absence calories from protein (code: 829277, name: RB 8% CP of an acute stress response due to long-term exposure ISO E (P)) (Diet compositions outlined in to the stressor. Additional file 1: Table S1). Danson et al. BMC Biology (2018) 16:51 Page 8 of 10 Adult male dissection and phenotyping resuspended in 200 μL of TE buffer and incubated at Mice were fasted for 16 h before being killed by CO as- 50 °C for 3 h before storage at 4 °C. DNA was quantified phyxiation. After weighing the whole animal and meas- using the High Sensitivity Qubit kit (Thermo Fish uring its length from nose to base of the tail, cardiac Scientific, Cat. Q32851) as per the protocol. Sperm puncture was performed using a 23 G needle and 1 mL purity was confirmed by Bis-PCR-Seq of imprinting con- syringe. Between 100 and 500 μL of blood was collected. trol regions associated with MEST, MCTS2, NESP and A drop of blood from the syringe (0.6 μL) was placed on IGF2/H19. a glucose measurement strip and blood glucose concen- tration was measured using a Bayer NEXT Contour Glu- RNA extraction and small RNA library preparation cose meter. The remaining blood was decanted into a 1. For RNA extraction, three-quarters of the extracted 5 mL Eppendorf tube and allowed to clot at room sperm were incubated at 60 °C for 15 min with slow ro- temperature before being placed on ice. In male mice, tation in 33.3 μL of sperm lysis buffer (6.4 M Guanidine the epididymis was next dissected from the base of the HCl, 5% Tween 20, 5% Triton, 120 mM EDTA, 120 mM testes and transferred to a 2 mL Eppendorf tube con- Tris; pH 8.0) with 3.3 μL of Proteinase K (19 mg/mL) taining pre-made sperm motility medium warmed to and 3.3 μL of 0.1 M DTT. After the incubation, one vol- 37 °C in a water bath (sperm motility medium: 1 M ume (100 μL) of ultra-pure water was added, followed NaCl, 100 mM KCl, 25 mM KH PO , 20 mM MgSO ,0. by 700 μL of Qiazol Lysis reagent (QIAGEN, Cat. 2 4 4 6% sodium lactate, 500 mM NaHCO , 25 mM sodium 79,306) and samples were vortexed for 5 min. Chloro- pyruvate, 25 mM CaCl, 500 mM HEPES, 34.5 mg/mL of form (140 μL) was added and samples were shaken BSA). Epididymides were homogenised using a fine pair vigorously for 30 s before 3 min incubation at room of scissors in the tube for 5 min and placed in a water temperature. Samples were centrifuged at 12,000x g at bath for 30 min at 37 °C, with regular inversion, to allow 4 °C for 15 min then the upper aqueous phase was the sperm to swim out. After incubation, the tube was transferred to a new reaction tube. One volume of 70% briefly spun down using a nanofuge to collect debris, EtOH was added and mixed thoroughly. Samples were then the supernatant containing free-swimming sperm transferred to a RNeasy mini spin column and the was removed and placed in a new 1.5 mL Eppendorf protocol from the miRNeasy Mini Kit (Qiagen, Cat. tube and stored on ice for the rest of the dissection. 217,004) was then followed, including the separation of Liver, kidneys and visceral gonadal white adipose tissue the small RNA and large RNA fractions using an RNA deposits were dissected out, weighed and flash frozen in MinElute spin column (Qiagen, Cat. 74,204). The small liquid nitrogen. A small amount of pancreas and small RNA fraction was eluted in 14 μL of RNase-free water intestine were also removed and flash frozen. Next, the and quantified using the microRNA kit from Qubit subcutaneous inguinal white adipose tissue deposits on (Cat. Q32880). Them, 6 μL of the small RNA fraction each side of the mouse and the interscapular brown was used for small RNA library preparation using the adipose tissue deposits on the back of the mouse were NEBNext Small RNA library prep set for Illumina (Cat. removed, weighed and flash frozen. Finally, a small sec- E7330S) as per the protocol. Each sample was uniquely tion of ear was flash frozen. barcoded using one of the NEBNext Index Primers for Illumina (Cat. E7300S, E7580S, E7710S, E7730S). For Phenol:chloroform DNA extraction PCR amplification, 15 cycles were used. Libraries were For DNA extraction, one-quarter of the extracted sperm purified using the QIAQuick PCR purification kit was incubated overnight in 600 μL of PK buffer (10 mM (Qiagen, Cat. 28,104) and DNA was eluted into 32 μLof Tris-HCl, 100 mM NaCl, 25 mM EDTA, 1% SDS) with nuclease-free water. An aliquot of each library was di- 2 μL of Proteinase K enzyme (19 mg/mL) and 0.1 M luted and 1 μL was run on an Agilent 2100 Bioanalyzer DTT at 55 °C with slow rotation. Phenol (750 μL) was using the Agilent High Sensitivity DNA kit (Cat. 5067- added to the samples and agitated for several minutes 4626) to assess the size distribution of the library. The before spinning at 17,000x g for 5 min at 4 °C. The libraries were pooled using equal volumes and size upper aqueous phase was transferred to a new tube and selected for between 140 and 200 bp (corresponding to the process repeated with phenol:chloroform, then an insert size of 13–73 bp) using a BluePippin machine chloroform alone. After the final spin, 5 μL of Rnase was (Sage Science) with 3% agarose cassettes (Sage Science, added and samples were incubated at 37 °C for 60 min. Cat. BDF3010). Then, 0.1 volumes of 3 M sodium acetate (pH 5.2) and 2.5 volumes of 100% EtOH were added and samples Sequencing and data analysis incubated at −20 °C for 1 h. Samples were spun at DNA from sperm was diluted to a concentration of 17,000x g for 10 min at 4 °C and the pellet was washed 11 ng/μL and 45 μL of each sample was sent for sequen- with 75% EtOH. Finally, the pellet was air-dried at 37 °C, cing to the Genome Centre Facility at Charterhouse Danson et al. BMC Biology (2018) 16:51 Page 9 of 10 Square, QMUL. Bis-PCR-Seq was performed using the and linear models with robust standard errors were used ® ® 48.48 layout on the Fluidigm C1 system (Fluidigm , to correct for any biases due to sibling relatedness (a full USA), coupled with Illumina MiSeq sequencing using justification is provided in Additional file 1: Supplemen- version2 chemistry (150 bp, paired-end). See Add- tary methods). Fisher’s exact test was used to assess the itional file 1: Table S3 for the primer sequences. Small differences in distribution of the small RNA compositions RNA libraries were initially sequenced using Illumina compared to CTCT, using percentages of mapped reads MiSeq Nano sequencing (75 bp, single-end) and read corresponding to each species in each sample and these counts for each samples were used to re-balance the were rounded to the nearest integer (corrected for n =3 library pool. The final pool was then sequenced using tests using Bonferroni’s correction). ANOVA was used to Illumina NextSeq sequencing (75 bp, single-end). assess whether the %A or %C and CpG –133 A meth % or Bismark (v0.7.12) was used to align Bis-PCR-Seq data CpG –133 C meth % were different between the diet to the mm10 reference genome (imprinting control re- groups. gion data; Additional file 1: Figure S4) or to the adjusted consensus rDNA reference, using Bowtie2 (v2.1.0). Only Additional file reads that mapped to the correct starting position and perfectly matched the consensus were used for further Additional file 1: Table S1. Compositions of the control (CT) diet and the protein restricted (PR) diet. Table S2. List of all male mice studied analysis. For rDNA analysis, the R package RSamtools with each litter represented by the first two numbers and letter of each was used to identify each read as either having an A or a ID. Table S3. Sequences of primers used for targeted analysis of DNA C at position −104 bp and to determine the methylation methylation at rDNA CpG −133 and the imprinted regions MEST, MCTS2, NESP and IGF2/H19. Figure S1. Growth rates of mice and lengths at status at position −133 bp of each read. Reads could death in each group. Figure S2. Absolute and relative organ weights. m u m u therefore be assigned to either A ,A ,C ,orC . The Figure S3. Absolute and relative adipose tissue deposit weights. Figure total number of reads in each group was summed and S4. Sperm small RNA size distribution analysis and sperm purity analysis. m u m u Figure S5. Read length distribution of reads mapping to the genome %A ((A +A )/(C +C )) and CpG –133 A meth % that also map to different classes of small RNA, normalised by total m m u (A /(A +A )) calculated for each sample. Methylation number of reads mapping to the genome. Figure S6. Maternal weight, of imprinting control regions were assessed using a food intake and litter size data. Figure S7. %A/C and CpG –133 meth % distribution in four diet groups. Figure S8. Growth trajectories plotted by custom program (https://bitbucket.org/lowelabqmul/ pre-weaning litter size (including females). Figure S9. Pre-weaning litter methylation-extractor). size (including females) has no impact on CpG –133 A meth % in any of Small RNA sequencing data was mapped to the whole the groups. Supplementary methods. Rationale and explanation of the use of a linear model and robust standard errors to analyse the relationship genome (UCSC, mm10), piRNA, tRNA, miRNA and between %A and CpG –133 A meth % instead of using litter averages or rRNA databases using the SPORTS 1.0 pipeline (https:// individuals from the same litter without correction for relatedness. R script github.com/junchaoshi/sports1.0.git). Total read counts used to perform the analysis is also included. (DOCX 1338 kb) for each small RNA class were expressed as percentages of number of reads mapped to the genome for each Acknowledgements sample in the composition analysis (Fig. 4a). Differential We thank the BSU technicians for their help with the animal work and the Bart’s and the London Genome Centre staff for performing the high expression analysis of tRNA fragments was performed through-put sequencing. We also thank Féaron Cassidy, Philip Howard and using edgeR (glmQLFTest) using the number of reads Gabriel Rosser for their help and advice. mapping to the genome as the library sizes for normalisation. Funding AFD is funded by an MRC Studentship (MR/K501372/1) and a Life Sciences Initiative Small Grant and the work was supported by a Biotechnology and Statistics Biological Sciences Research Council, UK (BB/M012494/1) grant awarded to VKR. All statistical analysis and plotting were performed using R (v3.2.3). For all phenotype plots, a linear model was Availability of data and materials The datasets supporting the conclusions of this article are available in the run on individuals and P values were derived by using GEO repository, Series GSE107541. robust standard errors to account for the relatedness be- tween siblings in each diet group (R packages plm [34] Authors’ contributions and lmtest [35]) and corrected for n = 3 tests using AFD, MLH and VKR conceived the project and carried out the experiments. AFD analysed the data with the help of RL and SJM advised on statistical Bonferroni’s correction. A Pearson’s product moment analyses. All authors discussed the results and interpretation and approved correlation coefficient was calculated to describe the the final manuscript. relationship between %A and CpG –133 A methylation percentage in each diet group (cor) and a linear model Ethics approval All animal procedures were conducted in accordance with the Home Office with robust standard errors was then run to obtain Animals (Scientific Procedures) Act 1986 (Project License number: 70/6693). P values, which were then corrected for n =4 com- parisons using Bonferroni’s correction (P ). All mice lin Competing interests were used in the analyses instead of litter averages The authors declare that they have no competing interests. Danson et al. BMC Biology (2018) 16:51 Page 10 of 10 Publisher’sNote 22. Solon-Biet SM, McMahon AC, Ballard JWO, Ruohonen K, Wu LE, Cogger VC, Springer Nature remains neutral with regard to jurisdictional claims in et al. 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Published: May 2, 2018

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