extfieldsofstudy: Biology Medicine

text:
Diet changes alter paternally inherited epigenetic pattern in male Wild guinea pigs

Abstract Epigenetic modifications, of which DNA methylation is the most stable, are a mechanism conveying environmental information to subsequent generations via parental germ lines. The paternal contribution to adaptive processes in the offspring might be crucial, but has been widely neglected in comparison to the maternal one. To address the paternal impact on the offspring’s adaptability to changes in diet composition, we investigated if low protein diet (LPD) in F0 males caused epigenetic alterations in their subsequently sired sons. We therefore fed F0 male Wild guinea pigs with a diet lowered in protein content (LPD) and investigated DNA methylation in sons sired before and after their father’s LPD treatment in both, liver and testis tissues. Our results point to a ‘heritable epigenetic response’ of the sons to the fathers’ dietary change. Because we detected methylation changes also in the testis tissue, they are likely to be transmitted to the F2 generation. Gene-network analyses of differentially methylated genes in liver identified main metabolic pathways indicating a metabolic reprogramming (‘metabolic shift’). Epigenetic mechanisms, allowing an immediate and inherited adaptation may thus be important for the survival of species in the context of a persistently changing environment, such as climate change.

Introduction
For (wild) species, environmental conditions are the pressures that define the ecological niche selecting for phenotypes with adaptive traits beneficial to survival and reproduction in that niche. Under different environmental conditions, a given genotype can give rise to different phenotypes [1,2]. We still understand little of the underlying mechanisms providing the genetic plasticity for this phenotypic diversity. Insights into these mechanisms are crucial for understanding the diversity of species as well as their ability to adapt to external changes. Known mechanisms realizing genetic plasticity are epigenetic modifications [3], which alter in response to intrinsic (e.g. stress reaction, disease) and extrinsic (e.g. environmental factors, toxins, temperature) changes without altering the DNA sequence and regulate gene expression [4,5]. Of the known epigenetic mechanisms such as histone modifications, non-coding RNAs and DNA methylation, the latter is the most stable one and can be transmitted to subsequent generations. Imprinted genes for example mediate paternal or maternal allelic transmission of specific DNA methylation patterns which is accompanied with a parent-of-origin gene expression [6].
In mammals, DNA methylation mostly occurs at cytosines in cytosine-phosphate-guanine dinucleotides context (CpG) [5]. CpG methylation at promoters [5] and enhancer regions [7] of genes silences gene transcription, whereas demethylation leads to transcriptional activation [5,8]. Intragenic methylation can have both functions-acitivation and repression [9,10]. Some DNA methylation patterns are set early in life and most of them remain stable throughout an individual's lifespan and across generations (epigenetic stability), others are highly plastic and change in response to a changing environment (epigenetic plasticity) [11].
Epigenetic alterations in response to changing environmental conditions have mainly been studied in isogenic lab strains, having the advantage of a large number of individuals providing statistical robustness and an insight into the mechanism, but lacking the functional understanding of these responses to ecological cues (e.g. climate change) in naturally occurring, genetically heterogeneous species [12,13]. Climate changes are changes in weather patterns such as temperature and rainfall, subsequently rearranging ecosystems by shifting the composition of floral communities, followed by changes in the composition of faunal communities. Surprisingly, epigenetic studies on wild species exposed to ecologically relevant impacts such as changes in ambient temperature and/or food quality are still rare (in plants [14], honey bees [15], fish [16], in baboons [17] and in guinea pig [18]), but mandatory to understand adaptation processes on a molecular level [12].
Medical reasons initiated studies on the epigenetic response to diet changes linking methylation changes to diabetes, obesity and other metabolic disorders [19][20][21][22][23]. However, such epigenetic modifications have mainly been studied with respect to maternal effects due to the close physical relationship of mother and offspring during and after pregnancy, and the reprogramming process in the blastocyst. In Agouti mice, e.g. fur colour and susceptibility to diabetes changed in offspring after feeding pregnant mice a diet rich in ascorbic and folic acid, which went along with a change in DNA methylation of the respective Agouti gene, and a change of its expression [20,22]. At the end of World War II the Dutch suffered of a famine during winter ('Dutch Hunger Winter'). Women exposed to the famine at an early stage of pregnancy gave birth to children with a stable, genome-wide change in methylation in genes important for growth and immunity (e.g. IGF2, INSIGF and IL10) [21]. Those children developed an increased rate of obesity altered lipid profiles, cardiovascular disease and schizophrenia [24,25].
More recently the paternal impact on DNA methylation patterns in the offspring has gained more attention, in response to cocaine consumption by fathers [26], traumata experience by fathers [27], and in response to changes in the diet of fathers [23,28,29].
In wild mammal species, males predominantly disperse from their natal site, while females are often philopatric. Therefore males (the paternal line) are more frequently exposed to changing environments and need to react quicker to rapid changes than philopatric females.
This also hold true in Wild guinea pigs, Cavia aperea. They are living in harem structures with one dominant male, who mateguards several females from other males [30]. The subordinate males disperse, and therefore need to adapt quite rapidly to novel habitats and available food sources in search of accepting female(s). Therefore, from an evolutionary point of view, we expect a paternal transmission of epigenetic responses to the offspring as it would contribute to a fitness increase of the offspring. Such fitness increase would be even more pronounced if the paternal response was transmitted over more than one generation.
As a small, mobile herbivore, the Wild guinea pig has high energy demands but only a small digestive tract. It compensates for its small gut capacity by (i) selection of high quality food, (ii) increased food intake, (iii) coprophagy and hindgut (caecum) fermentation [31][32][33] and (iv) an increased efficiency of digestion via symbiotic microorganisms fermenting even fibrous components [34][35][36].
Although dietary proteins are mandatory providers of nitrogen and essential amino acids for the animal's protein synthesis, needed in many vital functions (e.g. cell membrane components, enzymes, hormones, carriers in active transport systems etc. [37]), it is a scarce component in their vegetarian food. Severe nutritive protein deficiency causes muscle and organ degradation associated with body mass reduction [38]. Moderate protein deficiencies will lead to compensatory food intake, lower growth rates [39,40] and less reproductive success [41]. The compensatory increased food intake-known as 'protein leverage effect'-is usually associated with an elevated metabolic rate-which attenuates 'Luxus consumption', meaning that the surplus of energy is released by heat production [42,43].
Due to vegetation changes throughout seasons, herbivores regularly face varying and usually limited protein content in their food sources. Those effects may become more severe with global warming, as temperatures and reduced rainfall will cause long-term shifts in vegetation.
To test for a paternal contribution to an epigenetically adaptive response to diet changes, we temporally (2 months) exposed adult male Wild guinea pigs to a low protein diet (LPD) and allowed them to mate with the same females and sire offspring before and after LPD exposure. We then analysed and compared the two groups of 7-days old F1 sons-the 'control group' sired before (F1 C , C ¼ Control), and the 'diet group' sired after their fathers' LPD treatment (F1 D , D ¼ diet)-for altered DNA methylation patterns in response to their father's LPD (Fig. 1). We focussed on the liver (L), as the body's main metabolic organ (F1L C vs F1L D ), to assess the transmission of the father's epigenetic responses to the F1 generation. To investigate a potential transmission to the F2 generation, we also studied the DNA methylation patterns of the testes of the two F1 son groups (F1T C vs F1T D ).
About 70% of mammalian promoter regions are associated with 'CpG islands' (CGIs), genomic regions rich in CG content and main regulatory sites for gene silencing [10]. Therefore, we focussed our search for methylation changes on CGIs and other elements with regulatory functions, such as coding regions, promoters.

Animal Housing and Treatment
All husbandry and experimental procedures were approved of by the German Committee of Animal Welfare in Research (permit no. V3-2347-35-2011). Wild guinea pigs (C. aperea) originating from Argentina and Uruguay [30] were obtained from F. Trillmich (University of Bielefeld) and housed at the IZW field station in Niederfinow, Germany. The animals were kept in combined indoor-outdoor enclosures with free access to both compartments at all times. Indoor enclosures measured 0.75 Â 0.75 Â 1 m and the outdoor enclosures 0.75 Â 1 Â 1 m (length Â width Â height). Animals were housed under natural photoperiod and temperature. To avoid male competition, males were held as singles and were always separated by females kept in separate cages between them in such a way that social interaction but no direct contact was possible between male and female cavies. Although kept solitary, the opportunity to freely move between short-tunnelconnected inside and outside enclosures optimized their welfare (Schumann et al. 2014). Both cages were filled with a bedding of wood shavings and large opaque tubes for cover.

Diets
To assess the paternal effect of a LPD on male offspring, five male Wild guinea pigs (F0 fathers, n ¼ 5, labelled A-E) were fed a LPD with 42% less protein content (Supplementary Table S1), instead of standard maintenance diet (SMD). Pellets for both diets (Altromin Spezialfutter GmbH & Co. KG) were almost isoenergetic [dry matter (DM) energy], but differed in their protein to energy ratio and metabolizable energy, as protein content was partially made up with sucrose (Supplementary Table S1). Note that although the relevant dietary change in this experiment could be protein content, sucrose content, fat/protein ratio etc., for simplicity we refer to the diet as low protein henceforth.
LPD was fed for the duration of 62 days (April-June), which is the length of a full cycle of spermatogenesis in guinea pigs [44,45]. The experimental set-up is illustrated in Fig. 1. During SMD, animals received SMD pellets and hay ad libitum and a supplement of 50 g (each) of minced apple, carrot and cucumber per individual (listed in Table 1). During the LPD period, animals received exactly 100 g of LPD pellets and exactly 100 g of fresh hay per day and individual. At each morning during both diets, the cages were cleaned to avoid consumption of left-over hay or faeces from the previous day.

Analysis of Faecal Samples and Food Components
During LPD, food intake and digestibility was measured every 9-11 days (8-11 a.m.). The animals were weighted and the daily food consumption was determined by collecting all faecal samples and leftovers 24 h (630 min) after food supply. At measuring days, cages were cleaned and faeces and leftovers were collected. Leftovers were divided into the following components: dietary pellets and hay (in SMD and LPD), and apples, cucumbers and carrots (in SMD). Wet mass (60.01g) was determined using a scale (Sartorius BP 1200). For the analysis of energy and macronutrients (protein, fiber), faeces and samples of all food components were freeze-dried (Lyovac GT2, GEA Lyophil GmbH, Hü rth, Germany), weighted and ground (mill A11, IKA-Werke GmbH, Staufe, Germany). The dry mass of faeces was corrected for acid insoluble ash [46]. Ash content was determined by drying a subsample for 5 h at 525 C (Muffle kiln, Heraeus M110, Hanau, Germany). Energy content was determined using bomb calorimetry (IKA Kalorimetersystem C5000 control, IKA-Werke GmbH, Staufen, Germany), Protein content by using Dumas combustion (Elementar rapid N III; Elementar Analysesysteme GmbH, Hanau Germany). All analyses were performed in duplicates per food item and faecal sample.

Nutrition Data Calculations and Statistics
For nutrient content calculations, we used only DM corrected values. DM intake was calculated from the mass difference between dry mass of food offered and refused (leftovers). For net energy intake (NEI) and net protein intake (NPI), the energy and protein content of defaecated matter were subtracted from the respective content in ingested matter. Statistical analyses were performed within R (http://www.R-project.org). Consecutive values during treatment as well as values for maintenance diet were compared using a Wilcoxon rank sum test [47].

Parental Mating
Each F0 male (n ¼ 5) was mated twice with the same two F0 females (n ¼ 10), once before the LPD treatment (male 'control group'; F0 C ), and once after (male 'diet group'; F0 D ) ( Fig. 2A). The five F0 males (A-E) were born in mid-November and the ten F0 females in April-May the year after. In order to achieve mating, males were introduced to the female's cage, and after an observed mating, males were transferred back to their own cages. The 'control' mating took place from late December to early January and the mating after LPD treatment 6 months later in June. Because the males mated with the same two females before and after LPD exposure, the offspring produced (after gestation time of $62 days) per female were direct siblings and half-siblings.
Tissue Sampling F1 C sons were born in February and F1 D sons were born in August (Table 2). Seven days after their birth, we harvested whole livers (L) and testes (T) of all F1 sons from both groups (F1L C and F1T C D ¼ 15; F1L D and F1T D n ¼ 17). Livers and testes were homogenized, snapped frozen in liquid N 2 and stored at À80 C until DNA isolation (see below).

qPCR and Relative Quantification
Gene expression of two genes with key function in physiological pathways identified by the String network (Adcy9, Stat3) was measured by qPCR. Expression values were normalized by relative quantification of expression values using the three most stable expressed reference genes (Hmbs, Gapdh and B2m) as described previously (Weyrich et al. 2016b). RIN values were between 6.0 and 9.2. The run efficiency for Adcy9 was 2.03 and for Stat3 1.84. RNA of one sample of the control group was of low quality and thus excluded from statistical analysis. Amplicons were Sanger sequenced and verified by similarity control using BLAST (https:// blast.ncbi.nlm.nih.gov/Blast.cgi). Statistical analyses were performed on F1L C ¼ 17 and F1L D ¼ 16 with R (http://www.R-project. org) using a Wilcoxon rank sum test and an unpaired Ttest.

Physiological Effects of the LPD on Fathers
Surprisingly, with the start of the LPD, the male guinea pigs changed their feeding behaviour. They consumed 13.5% less pellets and 59.6% less protein (Table 1). This was also illustrated by their net protein ( Fig. 2A) and NEI (Fig. 2B), which dropped The table lists mean values of ingested food normalized per kg body mass during SMD and LPD period, and significant differences between both diets (*P < 0.05, **P < 0.001, ***P < 0.0001, unpaired T test).

Total DNA Methylation Changes in Sons
The overall single cytosine methylation level did not significantly vary between the compared control and treatment groups, neither for F1L C vs F1L D , nor for F1T C vs F1T D (schemata of experimental set-up, Fig. 1). On average, $10% of all cytosines were methylated in liver, out of which $70% were located in CpGs. In F1 (immature) testis of both son groups $9% of all cytosines occurred in a methylated stage, out of which $66% were in a CpG context. Only 1% was in CHGs (H can be C, A or T) and 1% a CHHs context, as expected from our former studies [18,50].

Epigenetic Inheritance
By comparing liver samples of sons sired before (F1L C ) and after their fathers' LPD (F1L D ), we identified 2796 DMRs of which 946 were located in annotated regions [promoters, coding sequences (CDSs), CGIs], hereafter referred to as 'annotated DMRs' (Fig. 3). These results indicated a 'heritable epigenetic response' (or 'epigenetic inheritance') to the LPD of the sires. With 4572 DMRs identified in the comparison of methylation patterns in testis (1420 of which were in annotated regions; Fig. 3), the F1T C vs F1T D comparison yielded a much larger number of DMRs than detected in the liver (F1L C vs F1L D ), which reveals from a greater coverage of CpG positions of testis data in comparison to liver. We verified the robustness of our results by performing a randomization approach, shuffling the methylation ratios of F1L C vs F1L D and F1T C vs F1T D , respectively, 100 times per father (total 500 times). For F1L C vs F1L D the approach resulted in only one DMR in 24 calculations and zero in the remaining 476 random calculations. No DMR was detected when shuffling F1T C vs F1T D . The observed DNA methylation changes in F1 sons clearly indicated an effect on the male offspring by the paternal epigenetic response to the diet change.

General Response
The LPD significantly affected the feeding behaviour of the adult male Wild guinea pig and thus their physiology (Table 1, Fig. 2). To evaluate if these physiological changes were reflected on the molecular level as a shift in the regulation of metabolic gene pathways, we further focussed on the liver as the main metabolic organ maintaining and regaining the body's homeostasis. In annotated regions in liver cells, we detected twice as many hypomethylated (n ¼ 655) than hypermethylated DMRs (n ¼ 330). Those annotated DMRs were overlapping with one or several annotated genomic regions, incl. promoters, transcription start sites (TSSs), CDSs and CGI (Fig. 4A). Of those hyper-and hypomethylated annotated DMRs, 493 were shared among the five son groups pooled by father (Fig. 4B). As these genomic regions are modified and thus react among the five son groups we interpret them as 'general response' to the environmental alteration (diet). Of the 493 annotated DMRs, 152 were overlapping with CDSs, 56 with promoter regions, 2 with TSSs and 283 with CGIs.
To evaluate which genes were affected by a change in composition of diet and to elucidate the physiological genepathways they were part of, we performed a gene-network analysis of the genes detected to have been epigenetically influenced in all F1 C /F1 N pairs (Fig. 4C). We therefore combined hypomethylated and hypermethylated DMRs with at least one annotated gene (total 210) and submitted them to the STRING database for analysis. Out of the 210 genes, 185 were recognized

Gene Expression
We tested two key genes (Stat3 and Adcy9, marked with stars in Fig. 4C and Supplementary Fig. S1) of the two major physiological pathways (development and growth, transcription, cell growth, communication, differentiation (highlighted in violet) and Calcium signalling pathway (highlighted in green)) for potential changes in gene expression by qPCR (Fig. 5). The Signal transducer and activator of transcription 3 (Stat3) expression was changed according to its methylation changes; Stat3 was hypermethylated in its CDS (in F1 N sons) and accordingly significantly changed its expression in liver samples of 15 F1 C vs 17 F1 N (foldchange ¼ 1.2, P ¼ 0.05). Adenylate cyclase (Adcy9) was hypomethylated in its CDS in sons. With a P ¼ 0.058 expression change of Adcy9 was slightly above the significance threshold of 5%.

Treatment-Dependent Response
Stat3 showed a response dependent to specific environmental change, because its expression decreased after a change in diet (Fig. 5, left), but increased after a change in temperature [55] ( Fig. 4). Due to a change in season between the first and the second mating, ambient temperature change may have acted as a cofounding environmental effect (Table 1 and Supplementary  Table S4). To further distinguish nutritional from temperature effects, we compared the 'annotated DMRs' obtained here to previously published results having a very similar experimental set-up. During the experiments we exposed five fathers of the same age to an increased temperature of 30 C (10 C more than ambient temperature) for also 2 months [generating F1 sons prior (control: F1L C ) and after heat (H) exposure of fathers (F1L H ); Supplementary Table S4] [18].
We identified overlapping annotated DMRs in livers of F1L D sons (this experiment) and F1L H sons (previous experiment; Weyrich et al. 2016b). Annotated DMRs were only partially overlapping in response to the two environmental effects (Table 3). Gene-network analysis of genes with methylation changes occurring only after paternal LPD resulted in the identification of same metabolic pathways (compare Supplementary Fig. S1 to Fig. 4), despite the removal of genes that overlapped in F1L D (this study) and F1L H (the 'heat experiment').

Discussion
Paternal nutrition alterations lead to treatment-specific changes in DNA methylation patterns, which were partially translated into changes in gene expression in the Wild guinea pig male offspring. This is the first study detecting a 'heritable epigenetic response' (or 'epigenetic inheritance') to diet changes in a wild, genetically heterogeneous mammal species. Epigenetic mechanisms, such as DNA methylation might therefore enable genetic plasticity in response to intrinsic and extrinsic factors, to better prepare the offspring for potential environmental changes and as such closes the gap between quick (and short-lasting) physiological responses and reaction shifts via very long-lasting mutational changes.

Diet-Related Paternal Epigenetic Effects
So far only a few studies investigated paternal epigenetic effects of DNA methylation changes studying highly inbred lab animals [23,26,28,29,56,57]. For example, a high fat diet of male rats  leads to changes in expression in the pancreatic ß-cells in their female offspring and hypomethylation of the Il13ra2 genes [28]. Although we did not detect hypomethylation of Il13ra2, similar pathways to ours were changed in their gene expression incl. Calcium-, Wnt-signalling pathways, apoptosis and the cell cycle. Pathway similarity between both studies point to a conserved evolutionary response to nutrition change.
In another study, male mice were fed a LPD from weaning up to 12 week of age. Their offspring had numerous DNA methylation changes in their livers. Among those loci, an enhancer of the lipid regulatory protein PPARa was increased in expression [23]. As this study's experimental set-up is similar to ours, we aimed to compare those results to ours, by investigating potential alterations of PPARa expression in F1 male Wild guinea pigs, but neither detected significant expression changes (data not shown) nor alterations in methylation.
In wild mammals, so far only one study investigated 'resource base' DNA methylation patterns in baboons (Lea et al. 2015) [58]. The authors compared the genome-wide methylation in both lodge and wild-feeding baboons, and also recorded physiological parameters (serum insulin, cholestrol and body fat). They detected feeding resource dependent genome-wide differential methylation patterns mainly in promoter and enhancer regions close to metabolism-related genes [58]. Our study is the first investigating inherited epigenetic effects of nutrition changes and the second study investigating DNA methylation changes to diet changes in a wild mammal.

Paternal Behavioural Change Due to Low Protein
We expected male Wild guinea pigs to ingest more pellets during the LPD than during the SMD to satisfy their daily supply of protein. This, however, was not the case. Instead they consumed less pellets (Table 1). With the ingestion of caecal faeces, caecotrophy guinea pigs can increase their protein uptake, due to ingestion of bacterial proteins [59,60]. By this they may have partially compensated for the lower protein levels in the LPD to perform amino acid synthesis. In addition, an alteration in regulation (type and amount) of protein-transporting channels of the gastro intestinal tract is essential for homeostasis [61] and might have enhanced protein uptake, allowing the male Wild guinea pigs to supplement their total protein intake. The latter is supported by the detected epigenetic change of the Calcium signalling and maintenance of homeostasis pathways in their male offspring (Fig. 4C and Supplementary Fig. S1).

Effects of Paternal Diet on DNA Methylation in Sons
Mounting evidence supports the hypothesis that diet influences DNA methylation patterns via three mechanisms: (i) the provision of substrates necessary for DNA methylation, and/or (ii) the provision of cofactors modulating the enzymatic activity of DNA methyltransferases, and/or (iii) a changing activity of the enzymes regulating the one-carbon cycle [62]. The universal methyl-donor for DNA and protein methyltransferases [63], S-adenosylmethionine (SAM) is synthesized in the methionine cycle from precursors present in the diet [64,65]. All these precursors, including methionine, folate, choline, betaine and vitamins B 2 , B 6 and B 12 , enter at different sites in the methionine pathway and contribute to the net synthesis of SAM.
We are uncertain whether the supply of methyl-donors is reduced in our studies, but as it is commonly accepted that undernutrition correlates with reduced methyl-donor availability (Zhang 2015), we assume that the protein reduction may have reduced the supply of methyl-donors such as Vitamin B and SAM, leading to an over-all hypomethylation (as shown in Fig. 4A) indicating a genome-wide activation.
Among all five son groups we detected changes in functional regions of the same genes, indicating a similar functional response to the paternal diet change despite the animals' genetic heterogeneity. We call this a 'general response'.

Metabolic Shift in Livers of Sons after LPD of Fathers
The gene-network analysis of 'general response' genes identified several main metabolic pathways in F1L C vs F1L D sons whose genes had been impacted in their methylation patterns by the diet change of their fathers. These genes were relevant in development and growth, transcription, cell growth, communication, differentiation, muscle contraction and energy synthesis, the canonical Wnt-signalling pathway, the Calcium signalling pathway, mitochondrial functions and the maintenance of homeostasis. The involvement of these pathways implies a general metabolic shift, which may change in the organisms' physiology in response to a longer-lasting environmental change. An environmental stimulus is sensed and initially processed by specific brain areal, releasing neurotransmitters and hormones. For example the Wnt-signalling pathway is important for reacting to outer signals by its Wnt (Wingless) and Integrator Complex Subunit 2 (Int-2) signal proteins [66]. These signals convey the information to the organs triggering metabolic processes, which stabilize or regain homeostasis [67]. The liver is the main metabolic organ, which also stores energy in form of glycogen, whose hydrolysis to glucose subsequently generates the energy required for a systemic response. The metabolic pathways detected here to be involved in the epigenetic response are part of this hepatic response. As this response was detected in F1L C vs F1L D sons, we hypothesize that the DNA-methylation patterns transmitted by the father to the sons pre-set a regulatory programme for the offspring to increase fitness by enabling to cope more rapidly with an environmental condition similar to the one the fathers had experienced (here LPD). If this holds true, the epigenetic response may enable the metabolic shift on the genetic level and thus a longterm memory set within a life-span and transmittable to the next generation(s). Further studies will be needed addressing this in the Wild guinea pig.

Epigenetic Response Depends on the Type of Environmental Change
Although Wild guinea pigs breed throughout the year, their body growth rate is somewhat greater in spring than in autumn, indicating seasonal dependency [68]. In our study control sons (F1L C ) were born in spring and sons sired after paternal diet change (F1L D , Table 2) and temperature change (F1L H , Supplementary  Table S4) were born in autumn. To identify potential seasonal changes in body weight we compared birth weights of sons born in February to that of sons born in August (F1 C vs F1 D/H ; Table 3  and Supplementary Table S4), but did not find significant weight differences at that early age (7 days). In addition, as both experiments ('diet change of fathers' and 'heat exposure of fathers') were run in the same season, 'season-based DMRs' would be identical in both experiments, resulting in overlapping DMRs (in the respective sons) between the two experiments. Assuming that 'season' had a great impact on the experiments, we would expect a large proportion of DMRs to overlap between the two experiments due to 'different season'. Even though the total number of overlapping DMRs may consist of DMRs both from (i) the different ambient temperatures the fathers were exposed to before and after their treatment (either LPD or heat) and (ii) from a general response to the changed season, the total number of overlapping DMRs between the two experiments was low (Table 1). In combination with the finding that the birth weights of sons in the two seasons did not significantly differ, we reject the hypothesis that the different seasons the sons had been born in had a significant impact on the epigenetic response measured.
Gene expression analysis of Stat3 supported the treatmentdependent response, because we detected a reverse pattern of response for diet and heat. Although in sons sired after F0 heat exposure Stat3 was hypermethylated and expression repressed compared with control sons (F1L C vs F1L H ) [55], in sons sired after LPD of fathers, Stat3 was hypermethylated and enhanced in expression compared with control sons (F1L C vs F1L D ).

DNA Methylation Regulation of Gene Expression
As visualized in the STRING network (Fig. 4C) Stat3 and Adcy9 are interacting with several other proteins, indicating their key function in those metabolic pathways. The Stat3 is a transcription factor which is activated by interleukins and growth factors. STAT3 mediates the expression of a variety of genes in response to cell stimuli and is essential for embryogenesis, cell survival and proliferation [69], the regulation of body weight [70] and many cellular processes [71] (GO terms listed in Supplementary Table S5).
Adenylyl cyclase 9 (Adcy9) is required for cellular signal transduction and has been associated with psychological diseases incl. bipolar disorder and depression [72]. Adcy9 gene encodes for an enzyme that catalyses Cyclic adenosine monophosphate (cAMP) to Adenosine triphosphate (ATP) and as such is crucial for cellular energy production, and as such necessary to cope with changing outer stressors (Supplementary Table S5) [73].
Even though Stat3 was changed in methylation in its CDS and not in its promoter regions, gene expression was detected to have changed. Although promoter methylation is strongly associated with gene repression and silencing [5,8] intragenic methylation can have both activating and repressive effects [74]. The methylation changes in the Adcy9 did not (yet) lead to changes in expression. Several hypotheses can be postulated to explain these findings: (i) the degree of methylation depends on the length of the period of environmental change and has (time-dependently) to reach a certain gene-specific level to become relevant for gene-expression, but here that level has not yet been reached because the length of the period of environmental change was too short (there were still non-methylated Cs), (ii) the genes are pleiotropically regulated and thus their regulation needs to be balanced among the different functions and pathways, (iii) [in conjunction with hypothesis i] methylation only serves a 'priming purpose' for a faster response should the sons experience a similar environment as their fathers.
To our knowledge, there is no prediction tool available at the moment to translate 'degree of methylation changes' into 'degree of gene expression changes'.

Tissues-Specific Methylation Effects
We measured the change in DNA methylation patterns in two organs of sons, the liver (F1L C vs F1L D ) and the testis (F1T C vs F1T D ). The presence of DMRs in both organs indicated a systemic, yet organ specific transmission of the epigenetic response to the environmental change the fathers had experienced. Such a response has a strong ecological relevance.
The changes we expected in testes of sons (F1T C vs F1T D ; Fig. 3) have the potential to be transmitted to the F2 generation if germ cells are affected. However, because we had to homogenize the testis tissue we could not distinguish between somatic cells and germ cells. In addition, DNA methylation is a regulator of cell differentiation [75] and at an age of 7-day post-partum testis cells were still differentiating [76]. Thus, what proportion of DMRs detected in testes was due to ongoing cellular differentiation and what proportion was due to paternal transmission of the epigenetic response could not be disentangled here. There is a growing evidence for tissue-specific epigenetic regulation, such in case of a-Actin (Acta1) increased in methylation within pancreas but in liver in mice fed a diet containing both daidzein and genistein [77]. Also the gene encoding the oestrogen receptor-a (Era), which show a defined methylation profile in liver, but is not methylated in pancreas [77].

Advantage and Limitation of Using Whole Liver as Sample
As for our research focus we were not interested in determining liver cell-type specific methylomes, we homogenised the whole liver of each son to avoid the results to be biased by unequal distribution of tissue cell type composition. This way we obtained liver-wide methylation patterns, which we then analysed, assuming that the cellular composition of the livers was very similar across individuals.
The differentiation of cells into functionally different cell types is regulated by methylation, wherefore different cell types have different methylomes [75,78]. Thus, a cell-type specific analysis would provide much more detailed results. However, with whole liver samples such detailed analysis is not possible. But because our aim was to investigate a liver-wide response (the liver being the main metabolic and thermo-regulating organ), and because cell-type specific analyses would have increased the costs drastically (without results adding equally to our research focus), we decided to use homogenized whole liver samples.

Epigenetic Plasticity during Puberty
Because in wild mammal species, males predominantly disperse from their natal site, they have to respond faster to outer changes. We hypothesise that the generally observed roaming behaviour of mammal males has led to a greater reactivity of epigenetic mechanisms in males during puberty, realized by a dynamic epigenetic-endocrine interplay. This is supported by the fact that the onset of puberty (and fertility) is accompanied by a major shift in endocrine levels as well as an overall genomic re-methylation [79]. Furthermore, because epigenetic factors are changed with altering environments roaming animals are exposed to, the mechanistic reactivity might be increased in comparison to females of the same age. To test this hypothesis, experiments in which both females and males are exposed to identical environmental factors would have to be set-up in parallel and samples would need to be collected along a time span before and after puberty to assess changes in hormone levels and epigenetic patterns.

Ecological Relevance
The experimentally applied reduction of 42% in protein content (LPD vs SMD) is a realistic scenario, caused by temporal resource limitations, for instance due to seasonal effects on the vegetation or dispersal to new habitats. Nevertheless, these changes are gradual. In our experiment, however, adult males were exposed not only to a sudden environmental change but also for a prolonged time span, mimicking global changes. Wild guinea pigs are generalists-they live in a wide range of habitats and across great heights and depths of South-America-and as such they are well equipped with an epigenetic 'tool box' to respond adequately to nutritional challenges [30,80]. Future research needs to focus on habitat specialists and their responses to environmental (e.g. dietary) changes.

Conclusion
Our results show that a changing environment procures a diet change-specific epigenetic response, which is transmitted to the next generation (and maybe beyond). The epigenetic regulation may therefore increase the genotypically encoded adaptive plasticity to a widened epigenetically modified phenotypic plasticity.
Dispersing male may not only increase genetic, but also epigenetic diversity. Those epigenetic processes in response to diet changes might be crucial for species health and survival. We conclude that DNA methylation is an essential epigenetic mechanism that provides (i) non-random and (ii) heritable genetic plasticity for wild species to quickly respond to environmental conditions (adaptability) which are likely to be beneficial for species survival and reproduction in a long term (adaptation).

Data Accessibility
Next generation-sequencing data were uploaded to the National Centre for Biotechnology Information Short Reads Archive (http://www.ncbi.nim.nih.gov/sra) and are publicly accessible under the SRA study accession number SRP048942 in .fastq file format. Raw data, coverage, methylation ratios (file names: F1L_and F1T_coverage_MethLevels) are accessible on Dryad doi:10.5061/dryad.7bg3t6s.

Supplementary data
Supplementary data are available at EnvEpig online.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Chemistry Medicine

text:
TRIM50 suppressed hepatocarcinoma progression through directly targeting SNAIL for ubiquitous degradation

Tripartite motif-containing 50 (TRIM50) belongs to the tripartite motif (TRIM) protein family, which has been implicated in the pathogenesis of multiple cancers. However, the role of TRIM50 in hepatocellular carcinoma (HCC) remains to be clarified. Here we showed that TRIM50 expression was significantly decreased in liver cancer tissues compared with corresponding non-cancerous liver tissues, and its decreased expression was significantly correlated with advanced disease progression. Gain-of-function assay by exogenous overexpression of TRIM50 in HCC cells showed that proliferation, colony formation, migration and invasion of HCC cells were significantly inhibited, whereas loss-of-function assay by TRIM50 knockdown showed that these malignant behaviors of HCC cells were significantly increased. Further investigation showed that TRIM50 could directly bind with SNAIL and induced K-48 linked poly-ubiquitous degradation of SNAIL protein, which further reversed SNAIL-mediated epithelial-to-mesenchymal transition (EMT) process of HCC cells. In vivo assay by xenograft tumor model verified the antitumor effect of TRIM50 on HCC. Taken together, these results showed that TRIM50 acted as a tumor suppressor in HCC cells by directly targeting SNAIL and reversing EMT, which further indicated that positive modulation of TRIM50 might be a novel therapeutic strategy for SNAIL overexpressed HCC cells.

Introduction
Hepatocellular carcinoma (HCC) is the primary malignancy of the liver and the third leading cause of cancerrelated death worldwide [1][2][3] . Most of the patients are diagnosed at late stages with limited therapeutic options. Identifying novel disease marker and clarifying the pathological mechanism will provide new insight into this disease and facilitate discovery of novel therapeutic strategies. In recent years, the role of tripartite motif (TRIM) proteins in the development of cancer has attracted much research interest, and novel tumor promoters and tumor suppressors have been identified in TRIM family members 4,5 . TRIM protein family includes >70 highly conserved proteins, which are usually composed of a RING (R) domain, one or two B-boxes (B) domain(s) and a predicted coiled coil (CC) domain 6,7 . TRIM proteins have been reported to play important roles in development, inflammation, anti-virus immunity and cancer 8 . Several TRIM family members were identified to play important roles in the development of liver cancer, which demonstrated that they might have potential applications as novel therapeutic targets or prognostic markers.
Tripartite motif-containing 50 (TRIM50) is a newly identified member of TRIM family and it was first identified as an E3 ubiquitin ligase in Williams-Beuren syndrome 9 . Later reports indicated that TRIM50 promoted the formation of sophisticated canaliculi and microvilli during acid secretion in parietal cells 10 . Another two reports suggested that TRIM50 interacted with HDAC6 and was involved in the regulation of P62 degradation 11,12 . Up to now, reports about the function of TRIM50 is very limited, and its biological function is far from being clarified. The role of TRIM50 in carcinogenesis has never been identified. Because of its recognized E3 ligase activity in diseases, we expected it might be involved in the regulation of the development of HCC.
In the study, we detected the expression of TRIM50 in clinical HCC specimen, analyzed the correlation of TRIM50 expression with disease progression, and further investigated its role in tumor growth, migration, and invasion of HCC cells. All these data revealed that TRIM50 acted as a tumor suppressor in HCC via directly targeting SNAIL and reversing epithelial-to-mesenchymal transition (EMT) process. Thus, this work provided a novel insight into the development of hepatocarcinoma and indicated a novel strategy for the treatment of SNAIL overexpressed HCC cells.

TRIM50 was downregulated in HCC tissues and its decreased expression was correlated with advanced disease progression
To explore whether expression of TRIM50 in HCC tissues was altered during the development of liver cancer, we detect the levels of TRIM50 in HCC tissues and corresponding non-cancerous liver tissues by immunohistochemistry (IHC), quantitative real-time polymerase chain reaction (qRT-PCR), and western blot. We first detected TRIM50 expression by IHC in HCC tissues and corresponding non-cancerous liver tissues from 79 clinical HCC patients, and our data showed that TRIM50 expression was significantly decreased in the liver cancer tissues compared with corresponding distal noncancerous liver tissues (Fig. 1a, Supplementary Table 1). To further clarify whether decreased expression of TRIM50 in HCC tissues contributed to disease progression, we further analyzed the correlation between TRIM50 expression and clinical disease status in these IHC staining data. Statistical analysis showed that patients with poorly differentiated tumors, as well as patients with metastasis were prone to have lower levels of TRIM50 expression (Fig. 1b, c).
Then, we did qRT-PCR assay in a cohort of 51 HCC patients and western blot assay in another cohort of 52 HCC patients. Both the qRT-PCR data (Fig. 1d) and western blot data (Fig. 1e) verified the IHC data, which showed that TRIM50 expression was significantly decreased in HCC tissues compared with corresponding non-cancerous liver tissues. Further assay of western blot data showed that patients with advanced Tumor Lymph Node Metastasis stages (TNM stages), Barcelona Clinic Liver Cancer stages (BCLC stages) and metastasis were prone to have lower levels of TRIM50 expression (Fig. 1f, h). Altogether, these data indicated that TRIM50 was downregulated in HCC tissues and its decreased expression contributed to HCC progression.

TRIM50 inhibited proliferation, colony formation, and invasion of HCC cells
To explore the effect of TRIM50 on the malignant behaviors of HCC cells, we constructed gain-of-function model by transfection of TRIM50 into HCC cells, and loss-of-function model by transfection of small interference RNA against TRIM50 into HCC cells. Western blot data showed that the protein levels of TRIM50 were lower in BEL7402 cells and HUH7 cells compared with those in HepG2 cells and SMMC7721 cells (Fig. 2a). Thus, BEL7402 and HUH7 cells were transfected with TRIM50 plasmid to construct the gain-of-function cellular model; and HepG2 cells and SMMC7721 cells were transfected with small interference RNA against TRIM50 (Si-TRIM50) to construct the loss-of-function model. These cellular models were investigated to define the effect of TRIM50 on the malignant behaviors of HCC cells.
Our data showed that after successful overexpression of TRIM50 in HCC cells (Fig. 2b), proliferation, colony formation, migration, and invasion capabilities of HCC cells were significantly inhibited ( Fig. 2c-f). After successful knockdown of TRIM50 expression by its specific siRNAs (Fig. 2g), the proliferation, colony formation, migration, and invasion capabilities of HCC cells were significantly increased ( Fig. 2h-k). Altogether, both of our gain-of-function model and loss-of-function model support the conclusion that TRIM50 could act as a tumor suppressor to inhibit malignant behaviors of HCC cells.

TRIM50 reversed resistance to anoikis of HCC cells
Resistance to anoikis is the hallmark of cancer and the prerequisite step for distant metastasis of HCC cells. Our previous data showed that HCC cells resisted to anoikis after anchorage deprival and acquired more malignant properties during its anoikis-resistant process 13,14 . In this study, we are interested to know whether TRIM50 also plays a role in the resistance to anoikis of HCC cells. Our data showed that TRIM50 overexpression significantly decreased cell viabilities in the anchorage-deprived HCC cells, which indicated that TRIM50 could reverse resistance to anoikis of HCC cells (Supplementary Figure 1A). Caspase cascade assay further verified that TRIM50 reversed anoikis resistance of HCC cells and induced apoptotic cell death after anchorage deprival (Supplementary Figure 1B and 1C).
Our previous data showed that during the process of anoikis resistance, the malignant behaviors of HCC cells were also significantly increased 13,14 . Thus, we are interested to verify whether these malignant behaviors of anoikis-resistant HCC cells could also be influenced by TRIM50. Our data showed that TRIM50 overexpression significantly inhibited the colony formation and invasion capabilities of anchorage-deprived HCC cells (Supplementary Figure 1D and E). These data further support the role of TRIM50 in HCC cells as a tumor suppressor.
TRIM50 exerted its antitumor effect through directly targeting SNAIL and reversing EMT TRIM family members usually take their effects by direct binding with target proteins and exert their function through modulation of target molecules. To further define the molecular mechanism of TRIM50 in the regulation of HCC progression, we tested a series of molecules, which might be involved in the process of carcinogenesis to define the target of TRIM50 by immunoprecipitation (data not shown). Our immunoprecipitation data showed that TRIM50 could bind with SNAIL protein (Fig. 3a). Further immunofluorescence (IF) data showed that TRIM50 and SNAIL could colocalized in HCC cells (Fig. 3b), which indicated the interaction between TRIM50 and SNAIL in HCC cells. To further define whether the interaction between TRIM50 and SNAIL is a direct binding effect, we did immunoprecipitation assay with an in vitro transcription and translation system as described before 15 . Our data showed that TRIM50 protein could directly interact with SNAIL protein as detected by the in vitro translation system (Fig. 3c), which indicated that TRIM50 could bind with SNAIL protein directly. Thus, these data indicated that TRIM50 might exert its function through its direct interaction and modulation of SNAIL. To further define whether TRIM50 could have any influence on the expression of SNAIL, we detected the protein levels of SNAIL in TRIM50 overexpressed cells and TRIM50 knockdown cells by western blot and mRNA level of TRIM50 by real-time PCR. Our data verified that TRIM50 could negatively regulate SNAIL expression at the protein level but not at the mRNA level (Fig. 3d, e). This negative regulation of SNAIL by TRIM50 was also verified in the clinical HCC patients (Supplementary Figure 2). Further cyclohexamide (CHX) chase assay showed that TRIM50 increased the degradation of SNAIL after de novo protein synthesis was blocked (Fig. 3f). When we put proteasome inhibitor MG132 in HCC cells, the negative regulation of SNAIL by TRIM50 was significantly rescued, which indicated that TRIM50 regulated SNAIL by proteasome mediated degradation (Fig. 3g). The presence of RING domain confers E3 ligase activity to TRIM family members, thus we were interested to know whether RING domain was responsible for the negative regulation of SNAIL by TRIM50. Therefore, we transfected HUH7 with TRIM50 RING domain mutant and analyzed its effect on the expression of SNAIL. Our data showed that deletion of RING domain in TRIM50 significantly rescued the negative regulation of SNAIL by TRIM50 (Fig. 3h). Thus, these data indicated that TRIM50 negatively regulated SNAIL expression via its RING domain. Fig. 1 TRIM50 was downregulated in HCC tissues and its expression was inversely correlated with advanced disease progression. a Immunohistochemical staining was used to determine the location and expression of TRIM50 in HCC tissues and corresponding non-cancerous liver tissues from 79 clinical HCC patients. The intensities of the IHC staining were quantitatively analyzed by IPP6 software and statistically analyzed (right panel). b, c TRIM50 protein levels in different pathology grades (b) and different metastasis stages (c) from 79 clinical HCC patients used for IHC staining were statistically analyzed and compared. d mRNA expression of TRIM50 was determined by qRT-PCR in HCC tissues and corresponding non-cancerous liver tissues from 51 HCC patients. e Western blot analysis of protein levels of TRIM50 in the liver cancer tissues and corresponding non-cancerous liver tissues from 52 HCC patients, with GAPDH expression as internal references. The presented images are representative blots from 24 HCC patients. Band intensities of all the investigated patients were measured by Image J software and statistically analyzed (right panel). f-h Statistical analysis of TRIM50 protein level in different TNM stages (f), different BCLC stages (g), and different metastasis stages (h) from HCC patients used for western blot assay. *P < 0.05, **P < 0.01, and ***P < 0.001 for statistical analysis of the indicated groups SNAIL is recognized as a transcription factor, which plays a critical role in the regulation of EMT process and further promotes the development of cancer. It is reported that suppression of E-cadherin is a key step in EMT process, whereas SNAIL is reported to direct repress E-cadherin 16 . Thus, SNAIL-mediated E-cadherin repression is a critical step in the EMT process of cancer 17 . As our data showed the effective suppression of SNAIL by TRIM50 in HCC cells, we are further interested to define whether TRIM50 acts as a tumor suppressor through its suppression of SNAIL-mediated EMT process. Our data showed that when HCC cells were transfected with TRIM50 plasmid, with the The basic protein levels of TRIM50 in BEL7402, SMMC7721, HepG2, and HUH7 cells were detected by western blot. b BEL7402 and HUH7 cells were transfected with TIRM50 expression plasmid or mock control, and western blot assay was performed to define the successful exogenous overexpression of TRIM50 in HCC cells. c BEL7402 and HUH7 cells were transfected with TRIM50 plasmid or mock control, and proliferation status of the transfected HCC cells was detected at 0 h, 12 h, 24 h, 36 h, and 48 h by CCK8 assay. d BEL7402, HUH7, and HepG2 cells were transfected with TRIM50 expression plasmid or mock control, and further cultured for 24 h before being transferred to six-well plates at the density of 1000 cells per well for colony formation assay. The clone formations were harvested after 14 days and the number of clone formation was counted. e, f After transfection with TRIM50 plasmid or mock control, transwell migration assay (e) and transwell invasion assay (f) were performed to investigate the migration and invasion capabilities of HCC cells. g HepG2 cells were transfected with siRNAs specifically targeting TRIM50 (Si-TRIM50-1&2, and Si-TRIM50-3), the cells transfected with random sequences (Si-NC) were used as mock control. The cells were further cultured for 24 h before being harvested and the block efficiency was measured by western blot. h HepG2 cells were transfected with siRNAs specifically targeting TRIM50 (Si-TRIM50-1&2) or its nonsense control (Si-NC), and proliferation status of transfected HCC cells were measured at 0 h,12 h, 24 h, 36 h, and 48 h by CCK8 assay. i HepG2 cells and SMMC7721 cells were transfected with Si-TRIM50-1&2 or Si-NC and further cultured for 24 h. The transfected cells were further transferred to six-well plates at the density of 1000 cells per well and allowed to grow for 14 days for colony formation assay. j, k After transfection of Si-TRIM50 or nonsense control (Si-NC) to HepG2 cells, transwell migratory assay (j) and invasion assay (k) of HCC cells were performed. Similar results were obtained in at least three independent experiments. *P < 0.05 and ***P < 0.001 for statistical analysis of the indicated groups suppression of SNAIL expression, expression of the epithelia marker E-cadherin was significantly upregulated, whereas expression of the mesenchyme marker vimentin was significantly downregulated (Fig. 3i). When TRIM50 expression was blocked by its specific interference RNAs, expression of E-cadherin was significantly downregulated, whereas vimentin level was significantly upregulated (Fig. 3j). Further IF assay confirmed the positive regulation of E-cadherin and β-catenin by TRIM50, and negative regulation of N-cadherin and SNAIL by TRIM50 (Fig. 3k). Besides, the phenotypic changes of HCC cells after overexpression of TIRM50 also indicated negative regulation of the EMT process by TRIM50 (Supplementary Figure 3). These data verified that TRIM50 acted as a tumor suppressor through its negative regulation of SNAIL and further reversing the EMT process.

TRIM50 induced ubiquitous degradation of SNAIL by K-48 linked poly-ubiquitination
Based on the presence of typical RING domain in TRIM50, we speculated that TRIM50 might exert its E3 ligase activities on SNAIL via its RING domain. Thus, we co-transfected the HCC cells with TRIM50 plasmid and HA-UB plasmid, followed by the immunoprecipitation assay to verify whether TRIM50 could put the polyubiquitin chain to SNAIL. Our data showed that at the presence of TRIM50, the poly-ubiquitin chain was successfully put to SNAIL protein, which indicated that TRIM50 regulated SNAIL by its poly-ubiquitous modification of SNAIL protein (Fig. 4a). Further assay showed that RING domain deleted TRIM50 mutant failed to put the poly-ubiquitin chain to SNAIL, which indicated that TRIM50 regulated ubiquitous modification of SNAIL via its RING domain (Fig. 4b). Lysine-48 (K-48)-linked polyubiquitous modification is mainly involved in targeting proteins for proteasomal degradation, whereas Lysine-63 (K-63)-linked poly-ubiquitous modification is coupled to mediate non-proteolytic signals, including those regulating subcellular localization, protein activation, and protein interactions 18 . By co-transfection of SNAIL and K-63only or K-48-only ubiquitin constructs into HCC cells, we found that TRIM50 could put the K-48 linked but not K- Fig. 3 TRIM50 exerted its antitumor effect through directly targeting SNAIL and reversing EMT. a BEL7402 and HepG2 cells were transfected with TRIM50 plasmid or mock control, the binding between TRIM50 and SNAIL protein were detected by immunoprecipitation. b HCC cells were cultured for 24 h before immunofluorescence assay to detect the expression and colocalization status of TRIM50 (green) and SNAIL (red). c TRIM50 and SNAIL proteins were separately expressed by the in vitro transcription and translation system, and the direct binding between TRIM50 and SNAIL were analyzed by co-IP assay. d, e BEL7402 cells were transfected with TRIM50 plasmid or mock control, and HepG2 cells were transfected Si-TRIM50 or Si-NC, the transfected cells were further cultured for 24 h. e The protein level of SNAIL was detected by western blot and further quantitatively analyzed (d); and the mRNA level of SNAIL was detected by qRT-PCR (e). f BEL7402 cells were transfected with TRIM50 plasmid or mock control and further cultured for 24 h before cyclohexamide (CHX) was put into the transfected cells. The cells were further cultured for 0 h, 2 h, and 4 h before being harvested for western blot assay of SNAIL expression. The band intensities were further quantitatively analyzed (right panel). g BEL7402 cells and HUH7 cells were transfected with TRIM50 plasmid or mock control, and further cultured for 24 h. Transfected cells were treated with MG132 (10 μM) for 4 h before protein lysates were isolated to detect the expression level of TRIM50 and SNAIL by western blot. h Wild-type Myc-tagged TRIM50 or its RING domain deleted mutant(△RING) was co-transfected with SNAIL into HUH cells, and further cultured for 24 h before being harvested for western blot assay of SNAIL. i BEL7402 cells were transfected with TRIM50 plasmid or mock control, and further cultured for 24 h. The expression of Ecadherin, vimentin, and SNAIL was detected by western blot and quantitatively analyzed. j HepG2 cells were transfected with Si-TRIM50 or Si-NC, and further cultured for 24 h. The expression of E-cadherin, vimentin, and SNAIL was detected by western blot and quantitatively analyzed. k BEL7402 cells were transfected with TRIM50 plasmid or mock control, and the cells were further cultured for 24 h before immunofluorescence assay to detect the expression of E-cadherin, β-catenin, N-cadherin, and SNAIL. Similar results were obtained in at least three independent experiments. **P < 0.01 and ***P < 0.001 for statistical analysis of the indicated groups 63 linked poly-ubiquitin chain to SNAIL protein (Fig. 4c). These data suggested that TRIM50 acted as an E3 ubiquitin ligase and mediated K-48 linked poly-ubiquitous degradation of SNAIL protein.
To further clarify whether TRIM50-mediated ubiquitous degradation of SNAIL occurred in the nuclear or cytoplasmic compartment, we isolated different compartments of the HCC cells for further analysis. Our immunoprecipitation data showed that TRIM50 could bind with SNAIL in both the nuclear and cytoplasm (Fig. 4d). When we co-transfected TRIM50 and HA-UB plasmid into HCC cells, our data showed that TRIM50 could successfully put the poly-ubiquitin chain to SNAIL in both the nucleic and cytoplasmic compartments. These data indicated that TRIM50 mediated ubiquitous degradation of SNAIL in the cytoplasmic, as well as in the nucleic compartment (Fig. 4e). Collectively, these above data also support our previous results showing that TRIM50 induced K-48 linked poly-ubiquitous degradation of SNAIL in HCC cells.

Exogenous overexpression of SNAIL rescued the antitumor effect of TRIM50
Our data showed that TRIM50 directly targeted SNAIL for degradation and further inhibited malignant behaviors of HCC cells. Thus, we are interested to know whether overexpression of SNAIL could rescue the tumorsuppressor role of TRIM50. We co-transfected TRIM50 and SNAIL plasmid into HCC cells, and western blot assay verified successful overexpression of both TRIM50 and SNAIL proteins (Fig. 5a, b). Further assay showed that inhibition of malignant behaviors of HCC cells by TRIM50 overexpression was significantly reversed after transfection with SNAIL plasmid (Fig. 5c, d). These data further confirmed that TRIM50 in these HCC cells prohibited cancer progression through directly targeting SNAIL for degradation.

Xenografted tumor model verified the antitumor effect of TRIM50
To further assess the antitumor effect of TRIM50 in vivo, we constructed xenograft tumor models by injection of BEL7402 cells to both flanks of nude mice. When visible tumor appeared, we injected TRIM50 expression plasmid to the left flanks and mock control to the right flanks of the mice. These plasmids were injected to the formed tumor every other day, and the sizes of the formed tumor were also measured until the mice were sacrificed on day 28 after the transplantation. The growth kinetics of the formed tumor showed that transfection of TRIM50 significantly inhibited tumor growth (Fig. 6a). The excised tumors from each group were compared, which showed that TRIM50 overexpressed tumors were much smaller than the mock group (Fig. 6b, c). The average size (d) and weight (e) of the TRIM50 transfected tumors were significantly decreased compared with the mock control group (Fig. 6d, e). qRT-PCR, IHC, and western blot assay further verified that TRIM50 was successfully overexpressed in the TRIM50 plasmid transfected group (Fig. 6f-h). Western blot assay confirmed that SNAIL expression was significantly suppressed in the TRIM50 transfected tumors (Fig. 6g). IHC assay confirmed the positive regulation of E-cadherin and β-catenin by TRIM50, and negative regulation of Ncadherin and SNAIL by TRIM50 (Fig. 6h). Thus, these in vivo data further verified our in vitro data that Fig. 4 TRIM50 induced ubiquitous degradation of SNAIL by K-48 linked poly-ubiquitination. a BEL7402 cells and HepG2 cells were transfected with TRIM50 plasmid and/or HA-UB plasmid as indicated, and further cultured for 24 h. The ubiquitous status of SNAIL was analyzed by co-IP assay. b HUH cells were co-transfected with Myc-tagged TRIM50 plasmid or TRIM50 truncation mutant (△RING) together with SNAIL expression plasmid, and further cultured for 24 h. The ubiquitous status of SNAIL was analyzed by co-IP assay. c BEL7402 cells and HepG2 cells were co-transfected with TRIM50 and HA-K-48-UB/HA-K-63-UB plasmids, and the ubiquitous status of SNAIL was analyzed by co-IP. d Cytoplasmic and nucleic fractions of HCC cells were prepared from HepG2 cells, and co-IP assay was used to detect the interaction between TRIM50 and SNAIL in different fractions of HCC cells. e The ubiquitous status of SNAIL protein in different fraction of HCC cells was also detected by co-IP. Lamin B1 was used as nuclear internal control, GAPDH was used as cytoplasm control, and β-actin was served as whole-cell loading control. Similar results were obtained in at least three independent experiments TRIM50 inhibited HCC growth through it suppression of SNAIL.

Discussion
Recent studies indicated that several members of the TRIM protein family were important regulators of carcinogenesis. Among these, TRIM24, TRIM26 were identified as tumor suppressors in the development of HCC, whereas TRIM31 was identified as a tumor promoter for HCC 5,15,19 . However, the role of TRIM50 in the progression of HCC was unknown. In this study, we identified the tumor-suppressor role of TRIM50 in the development of HCC and further clarified its involved molecular mechanism for the first time.
We first investigated the expression of TRIM50 in clinical specimen, and all of our data showed a significantly decreased expression of TRIM50 in HCC tissues, and its expression was inversely correlated with clinical stages and differentiation status of the patients.
These data indicated that decreased expression of TRIM50 may facilitate the development of liver cancer. Further cellular model data showed that proliferation, colony formation, and invasion capabilities of HCC cells were significantly inhibited after ectopic overexpression of TRIM50 in HCC cells, whereas these malignant behaviors were significantly enhanced after block of TRIM50 in HCC cells. These data indicated the tumor-suppressor role of TRIM50 in HCC cells, and further suggested that loss of TRIM50 in HCC tissues could lead to the progression of liver cancer.
Recent reports showed the function of TRIM proteins often depended on their interactions with other proteins, usually target proteins 20 . In this study, we identified SNAIL as a novel binding partner of TRIM50 in liver cancer cells. At the cellular level, we demonstrated that TRIM50 negatively regulated SNAIL expression. Further investigation showed that TRIM50 could directly bind with SNAIL and induce K-48 linked poly-ubiquitination were transfected with TRIM50 plasmid and SNAIL plasmid, and the expression of TRIM50 and SNAIL were detected by western blot. c BEL7402 and HUH7 cells were transfected with TRIM50 plasmid or SNAIL plasmid, and transwell invasive assay was performed to detect the invasive capability of these transfected HCC cells. d BEL7402 cells were transfected with TIRM50 expression plasmid or SNAIL expression plasmid, and the cells were transferred to six-well plates at the density of 1000 cells per well for colony formation assay. The colonies were stained after 14 days, and the number of colonies was counted and statistically analyzed. **P < 0.01 and ***P < 0.001 for statistical analysis of the indicated groups of SNAIL protein. To further clarify whether decreased expression of SNAIL by TRIM50 was required for TRIM50-induced antitumor effect on HCC cells, we reintroduced SNAIL into TRIM50 overexpressed cells and measured its influence on the malignant behaviors of HCC cells. Our data showed that ectopic overexpression of SNAIL significantly rescued the antitumor effect of TRIM50, which further verified that TRIM50 exerted its effect on HCC cells through its negative regulation of SNAIL.
SNAIL is a conserved transcription factor playing an essential role in EMT during cancer metastasis. EMT is a critical process involved in cancer progression. A hallmark for EMT is the loss of cell adhesion molecule Ecadherin, and it is reported that SNAIL could directly repress E-cadherin 16,21,22 . Our data showed that overexpression of TRIM50 in HCC cells could increase Ecadherin expression, which indicated that TRIM50 might exert its antitumor effect through reversing SNAILmediated EMT process.
It is reported that SNAIL is expressed in both the cytoplasm and nuclear in cancer cells 16,23 . To further clarify the interaction between TRIM50 and SNAIL, we separated different compartments of HCC cells and did the immunoprecipitation assay to identify where this interaction occurred. Our data showed that TRIM50 could bind with SNAIL in both the cytoplasmic and nucleic compartments of HCC cells (Fig. 7). These data indicated that TRIM50 could act as a tumor suppressor by directly targeting SNAIL in both cytoplasmic and nuclear compartments of cancer cells.
Like other TRIM family members, TRIM50 has a typical RING domain, which may confer it ubiquitous activation to its target proteins 24 . Ubiquitination is one of the most abundant and versatile post-translation modifications in cells where the ubiquitin is covalently added to lysine residues of target protein. There are several types of ubiquitin modifications with different effects on target proteins. For instance, the K-48 linked polyubiquitination could induce ubiquitous degradation of target proteins, whereas the K-63 linked polyubiquitination could modulate the activation of target proteins 18,25 . To better understand the posttranslational regulation of SNAIL by TRIM50, we performed the immunoprecipitation assay by co-transfection of SNAIL and ubiquitin expression plasmids into HCC cells. Our Fig. 6 Antitumor effect of TRIM50 was verified in xenografted tumor model. BEL7402 cells (10 7 cells) were subcutaneously injected to both flanks of the nude mice. When visible tumor appeared, TRIM50 expression plasmid was injected to the formed tumor in the left flank and mock control plasmid was injected to the tumor in the right flank every other day before the mice were sacrificed on day 28. a The growth curves of tumors with TRIM50 or mock plasmid transfection were analyzed every other day before the mice were sacrificed. b The formed tumors with TRIM50 or mock plasmid transfection were isolated and compared. c Images presented were the representative mice with subcutaneous xenograft tumor. d, e The volume (d) and weight (e) of the formed tumors transfected with TRIM50 plasmid or mock plasmid were analyzed and compared. f mRNA levels of TRIM50 in TRIM50 or mock control transfected tumors were analyzed by qRT-PCR. g Western blot assay was performed to detect the protein levels of TRIM50 and SNAIL in the formed tumors. h Immunohistochemical staining was used to detect the level of TRIM50, E-cadherin, β-catenin, Ncadherin, and SNAIL in formed tumors. *P < 0.05, **P < 0.01, and ***P < 0.001 for statistical analysis of the indicated groups data showed that TRIM50 could successfully put the polyubiquitin chain to SNAIL. Further analysis showed that TRIM50 could induce K-48 linked, but not K-63 linked poly-ubiquitination of SNAIL protein. Thus, we identified SNAIL as a novel important target for TRIM50-mediated poly-ubiquitination, and further analysis verified that TRIM50 induced K-48 linked ubiquitous degradation of SNAIL.
In conclusion, we investigated the role of TRIM50 in HCC progression in an integrate investigation system including clinical specimen, cellular model, and animal model. Our study showed that TRIM50 expression was significantly decreased in HCC tissues compared with corresponding distal non-cancerous tissues. Its downregulation was significantly inversely correlated with disease progression, which indicated its involvement in the development of cancer. Further in vitro and in vivo study verified the antitumor effect of TRIM50 on HCC cells was mediated by its K-48 linked poly-ubiquitous degradation of SNAIL protein. Altogether, this study provided clues to understand the pathogenesis of HCC, and it indicated that therapeutic strategy by upregulating TRIM50 in SNAIL overexpressed cancers may pave a new avenue for manipulating liver cancer.

Tissue samples
Paired samples of HCC tissues and corresponding non-cancerous liver tissues from the Department of Hepatobiliary Surgery of the Provincial Hospital Affiliated to Shandong University were used for detection of TRIM50 expression. Among them, 79 pairs of liver cancer and corresponding non-cancerous tissues were used for IHC assay, 52 pairs of matched specimens were used for western blot assay, and 51 pairs of matched specimens were used for qRT-PCR. These procedures dealing with human specimen were approved by Shandong University Research Ethics Committee and all the protocols dealing with the patients met the ethical guidelines of the Helsinki Declaration. Written informed consent was obtained from each patient before participation and approved by the ethics committee of Shandong University. Details of the clinicopathologic characteristics of these recruited HCC patients were shown in Table 1.

Immunohistochemistry
IHC was performed to detect the expression and location of TRIM50 on paraffin sections of HCC tissues and non-cancerous liver tissues. IHC staining and evaluation were performed according to the procedure described before 26,27 . Specific antibody against TRIM50 (ab174880) was from Abcam company (Cambridge, MA, USA). Immunohistochemical staining was evaluated using Image-Pro Plus v6.2 software (Media Cybernetics, Inc., Bethesda, MD, USA). For accurate reading of the staining, we used the same setting for all the analyzed fields. Integrated optical density (IOD) was measured in all investigated fields, and density of positive staining was evaluated as IOD/the total area of each field.

Quantitative real-time PCR
Total RNA was extracted from liver cancer tissues and qRT-PCR was performed as described before 15,27 . Primers for human TRIM50 gene were forward: 5′-CCCAT TTGCCTGGAGGTCTTC-3′, reverse: 5′-CAGGACAG CATAGCTCGGAG-3′. Relative gene expression levels were normalized to β-actin. Primers for β-actin gene were forward: 5′-GGCACCACACCTTCTACAATG-3′, reverse: 5′-TAGCACAGCCTGGATAGCAAC-3′. Fig. 7 Working model of the role of TRIM50 in HCC progression. TRIM50 was significantly downregulated in HCC cells and its decreased expression further promoted HCC progression. Further investigation showed that TRIM50 could target SNAIL for K-48 linked poly-ubiquitous degradation and thus reversed SNAIL-mediated epithelial-to-mesenchymal transition (EMT) transition. Altogether, loss of TRIM50 in HCC cells led to upregulation of EMT process and further promoted the malignant behaviors of HCC cells, including proliferation, colony formation, anoikis resistance, and invasion, thus promoted HCC progression The relative mRNA levels of target genes were obtained by using the 2 -ΔΔ Ct method with all assays performed in triplicate.

Cell culture, transfection and IF
All of the HCC cell lines, including BEL7402, SMMC7721, HepG2 and HUH7 cells, were obtained and cultured as previously described 15 . HCC cells grown on normal plates and poly-2-hydroxyethylmethacrylate (poly-HEMA) coated plates were established as attached cell and detached anchorage-deprived cells, respectively 13,14,28 . TRIM50 plasmid was synthesized by Ori-Gene (OriGene Technologies, Maryland, USA). RING domain deleted mutant of TRIM50 was generated using the KOD-Plus-Mutagenesis kit (Toyobo, Osaka, Japan) according to the manufacture's protocol. The small interfering RNAs targeting TRIM50 and SNAIL were synthesized by RIBOBIO (RIBOBIO, Guangzhou, China). Transfection and IF assay were performed as previously describe 15 .

In vitro binding assay
The direct interaction between TRIM50 protein and SNAIL protein were performed by a TNT Quick Coupled Transcription and Translation System (Promega, Madison, WI, USA) according to the manufacturer's protocol. The TRIM50 and SNAIL proteins were expressed, mixed together, and analyzed with immunoprecipitation by the TRIM50 antibody, followed by western blot assay by SNAIL antibody to determine the direct binding of TRIM50 and SNAIL proteins.

Subcellular fractionation
Extraction and isolation of nuclear and cytoplasmic protein from HCC cells were performed by the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Jiangsu, China) according to the manufacturer's protocol.

In vivo tumor growth assay
Five-week-old immunodeficient male BALB/c athymic nude mice (Huafukang Biotechnology Ltd, Beijing, China) were used for construction of xenograft tumor model as described before 15,26 . When visible tumors appeared, we injected 30 ug of pCMV-TRIM50 and empty pCMV vector control to the tumors in either flank once every other day before the mice were sacrificed by cervical dislocation. The isolated tumors in the TRIM50 transfected group and mock control group were further isolated and analyzed.

Statistical analysis
Statistical analysis was analyzed by SPSS 16.0 software (SPSS, IL, USA) and GraphPad Prism software (version 5.0). Χ2-test was employed to compare qualitative variables. Analysis of quantitative variables was performed using the Student's t-test or one-way analysis of variance (ANOVA). Data were presented as mean ± S.D. P-value < 0.05 was considered statistically significant for all tests and all statistical tests were two sided.