Changes in Global DNA Methylation Intensity and DNMT1 Transcription During the Aging Process of Scallop Chlamys farreri

LIAN Shanshan, HE Yan, LI Xue, ZHAO Bosong, HOU Rui, HU Xiaoli, ZHANG Lingling, and BAO Zhenmin

Key Laboratory of Marine Genetics and Breeding (MGB) of Ministry of Education,College of Marine Life Sciences,Ocean University of China,Qingdao266003,P. R. China

Changes in Global DNA Methylation Intensity and DNMT1 Transcription During the Aging Process of Scallop Chlamys farreri

LIAN Shanshan, HE Yan, LI Xue, ZHAO Bosong, HOU Rui, HU Xiaoli, ZHANG Lingling, and BAO Zhenmin*

Key Laboratory of Marine Genetics and Breeding (MGB) of Ministry of Education,College of Marine Life Sciences,Ocean University of China,Qingdao266003,P. R. China

DNA methylation is an important epigenetic regulatory mechanism that influences genomic stability, gene activation, X-chromosome inactivation and other factors. A change in DNA methylation is usually associated with aging and cellular senescence. DNA methyltransferase 1 (DNMT1) is the most abundant DNA methyltransferase, and it plays an important role in maintaining the established methylation pattern during DNA replication in vertebrates. Although the effect of aging on DNA methylation has been well studied in vertebrates, little research has been conducted in invertebrates, especially in marine bivalves. In this study, we examined global DNA methylation levels in four groups of adult Zhikong scallopChlamys farreriat different ages. The results showed that both the age and tissue type had a strong effect on the DNA methylation. In addition, a significant decrease in DNA methylation with aging (1-4 years) can be detected in mantle, kidney and hepatopancreas. We further measured the change inDNMT1transcript abundance using quantitative reverse transcription PCR (qRT-PCR), which revealed thatDNMT1transcription significantly decreased with aging in mantle and hepatopancreas and strongly correlated with DNA methylation (R= 0.72). Our data provided greater insight into the aging-related decline of DNA methylation, which could aid in gaining a better understanding of the relationship between DNA methylation and the aging process in bivalve mollusks.

aging; DNA methylation;DNMT1transcription;Chlamys farreri

1 Introduction

Epigenetic changes in DNA play an essential role in determining gene transcription. As an important epigenetic modification, DNA methylation is involved in the regulation of development, aging and carcinogenesis in mammals (Liuet al., 2003; Klose and Bird, 2006; Calvanese, 2009). Approximately 70%-80% of cytosines in CpG dinucleotides are methylated in vertebrates (Bird and Taggart, 1980). However, with the aging of animals, DNA demethylation increases (Wilsonet al., 1987; Mazin, 1993, 1994; Kresset al., 2001, 2006; Richardson, 2002; Rodriguezet al., 2008), and the total 5 mC loss could be as high as 91% in cows, 93% in mice, and even 99% in rats for old animals (Mazin, 1993). In humans, DNA methylation associates with chronological age over long time scales and is linked to complex aging-related diseases. The global loss in DNA methylation during aging and in tumor cells was also found in human beings, which could mainlybe the result of the progressive loss of DNMT1 efficacy (Fraga and Esteller, 2007). In a recent study, Hannumet al. (2013) measured more than 450000 CpG markers in hundreds of people from 19 to 101 years of age and found that 70387 (15%) of the markers had significant associations with the aging rate; the genome-wide methylation pattern represents a strong and reproducible biomarker of the biological aging rate. The genome-wide loss of DNA methylation during aging could be relevant to genome instability, the risk of carcinogenesis, aging disorders or other complex age-associated diseases, and the retardation of cell proliferation in tissues of aging organisms (Mazin, 1993; Barres and Zierath, 2011; Lao and Grady, 2011; Tappet al., 2013).

Maintenance of the DNA methylation pattern in vertebrates is mediated by DNA methyltransferases (DNMTs), which catalyze the transfer of a methyl moiety from S-adenosyl-L-methionine (SAM) to the 5-position of cytosines, principally in the CpG dinucleotides (Law and Jacobsen, 2010). As the most abundant DNMT, DNMT1 plays an important role in maintaining the established methylation pattern during DNA replication (Flores and Amdam, 2011). It has been reported that DNMT1 has acrucial effect on global genomic methylation. For example, the inactivation of DNMT1 causes DNA demethylation, and homozygous null deletions of DNMT1 result in an 80% genomic loss of DNA methylationin mouse (Leiet al., 1996; Takebayashiet al., 2007). In human cells,DNMT1transcription steadily declines throughout the aging process (Lopatinaet al., 2002). These findings suggest that in vertebrates, reduced genome-wide methylation during aging can be attributed to a decreased abundance ofDNMT1.

Invertebrates display a wide diversity of DNA methylation patterns (Suzukiet al., 2007). For example, with the lack of essential DNMTs, 5-methylcytosine could not be detected in the nematode wormCaenorhabditis elegansat any time during development or aging (Simpsonet al., 1986). In comparison, the fruit flyDrosophila melanogasterlacks most of the classical DNMTs and displays limited cytosine methylation (Hunget al., 1999; Lykoet al., 2000). At the same time, the honey beeApis melliferabears a fully functional set of DNMTs, and DNA methylation is widespread across its genome (Elangoet al., 2009).

Although bivalve organisms comprise more than 30000 species and constitute the second largest group of mollusks, only limited research on DNA methylation patterns has been conducted in this taxonomic group (Gavery and Roberts, 2010; Riviereet al., 2013). A recent study has revealed changes in the DNA methylation during the early life of oysters and the importance of DNA methylation for proper larval development (Riviereet al., 2013). Such observations might indicate time-dependent patterns of DNA methylation in mollusks and the evolution of 5 mC during the process of aging. In this study, we examined the changes in DNA methylation andDNMT1transcript abundance in the marine bivalveChlamys farreriat 1 to 4 years of age. Detailed information on the changing patterns in different tissues was also collected. This study will aid us in obtaining a better understanding of the role of DNA methylation in the aging process of bivalve mollusks.

2 Materials and Methods

2.1 Sample Collection

Zhikong scallop individuals (1, 2, 3 and 4 years of age) were provided by a shellfish farm in Rongcheng (Shandong Province, China). A total of 24 individuals (6 of each age) were randomly collected and then acclimated at 15℃in filtered seawater for one week. Then, six tissues, including mantle, gill, gonad, kidney, adductor muscle and hepatopancreas, were dissected, immediately frozen in liquid nitrogen and kept at -80℃.

2.2 Global DNA Methylation Analysis

Genomic DNA was extracted using a standard phenol-chloroform protocol. RNase A was added to avoid RNA contamination. DNA concentration and purity was determined by the NanoVue Plus UV spectrophotometer (GE Healthcare). DNA methylation was quantified using the Methylamp? Global DNA Methylation Quantification Kit from Epigentek (Brooklyn, NY) following the manufacturer’s instructions. The methylated fraction of the DNA is recognized by 5-methylcytosine antibody and quantified through an ELISA-like reaction. For each sample, methylation analysis was performed in triplicate. The methylation percentage of each sample was calculated according to the slope of the standard curve. The standard curve was generated by plotting the OD values of a dilution series made from a 100% methylated DNA standard that was supplied in the kit.

2.3 Total RNA Extraction

The total RNA was extracted using the method described by Huet al. (2006). Genomic DNA contamination in RNA samples was removed by DNase I treatment. The RNA concentration and purity was determined using the NanoVue Plus UV spectrophotometer (GE Healthcare), and the RNA integrity was verified by agarose gel electrophoresis.

2.4 Analysis ofDNMT1Transcript Abundance

The transcript abundance ofDNMT1was detected using quantitative reverse transcription PCR (qRT-PCR). Firststrand cDNA was synthesized from 500 ng total RNA using oligo (dT)18and MMLV reverse transcriptase (Promega, Madison, WI, USA). A control reaction without reverse transcriptase was performed to preclude the DNA contamination. The amplification mixture contained 2 μL of diluted cDNA (1：50), 4 μL of primers (2 μmol L-1each) and 10 μL of SYBR Green Real-time PCR Master Mix (TOYOBO, Osaka, Japan). All of the PCR reactions were performed in duplicate and run on a 7500 Real-Time PCR System (Applied Biosystems, CA, USA), using the following program： initial denaturation at 95℃ for 10 min, followed by 40 cycles of 95℃ for 15 s and 60℃ for 1 min. Here, β-actin (ACTB), elongation factor 1 beta (EF1β) andribosomal protein L16 (RPL16) were chosen as internal reference genes (IRGs) (Table 1). PCR efficiencies and optimal Ct values were estimated using the online software real-time PCR Miner (Zhao and Fernald, 2005). TheDNMT1transcripts were quantified relative to the three IRGs using the algorithm proposed by Hellemanset al. (2007).

Table 1 List of primers used for qRT-PCR

2.5 Statistical Analysis

All of the data were subjected to one-way ANOVA using SPSS 16.0 (Norusis, 2008). Fisher’s least significant difference (LSD) test was applied when the ANOVA indicated a significant (P＜ 0.05) difference. The relationship between the data from different assays was determined using the Pearson correlation coefficient (R).

3 Results and Discussion

3.1 Age Effect on DNA Methylation

To roughly estimate the change in the DNA methylation during scallop aging, the entire soft tissue was subjected to global DNA methylation analysis. Based on the results (Fig.1a), the DNA methylation ratio ranged from 0.1% to 0.5% across all of the samples, which was much lower than the values obtained in the vertebrates, such as human, mice and zebrafish (Vuceticet al., 2010; Liuet al., 2011; Fanget al., 2013). One-way ANOVA showed that age was significantly associated with the DNA methylation fraction (P＜ 0.001). It appeared that with the increase in the age of the scallops, DNA methylation declined, which is similar to the findings in most of the vertebrates and in thein vitromodels (Kresset al., 2001, 2006; Rodriguezet al., 2008; Mazin, 2009; Bollatiet al., 2009). We also noticed that the 1-year-old scallops had a significantly higher DNA methylation fraction than the other three age groups, and a dramatic drop in DNA methylation was detected when the scallops entered their second year. Afterward, the DNA methylation declined gradually, and a significant decrease was found only between the 2- and 4-year-old individuals (P＜ 0.01). Although the above results were based on all of the soft tissue, they indicated an age effect on the DNA methylation in scallop and provided some clues on the age-dependent changes of DNA methylation in marine bivalves.

3.2 Effect of the Tissue Type on the DNA Methylation

DNA methylation could be different among the tissues, which leads to an inaccurate estimation of the age effect based on the results from all of the soft tissue. Therefore, we further tested the DNA methylation fraction in six tissues (mantle, gill, gonad, kidney, adductor muscle and hepatopancreas) of 2-year-old scallop. According to oneway ANOVA, the DNA methylation fractions were significantly different among the tissue types (P＜ 0.001). As shown in Fig.1b, the kidney and adductor muscle had the highest (approximately 0.3%) DNA methylation ratio, followed by gonad and hepatopancreas (approximately 0.2%), and the lowest DNA methylation ratio was found in mantle and gill (approximately 0.1%). The observed tissue difference in the DNA methylation is similar to the finding in mammals (Romanov and Vanyushin, 1981; Gama-Sosaet al., 1983; Maegawaet al., 2010), which suggests that the tissue type should be considered when examining the effect of age on DNA methylation.

Fig.1 Effects of age (a) and tissue type (b) on DNA methylation. One-way ANOVA followed by Fisher’s LSD test was used for the comparisons. The vertical bars represent the mean ± S.E. (n= 6). The values marked with different letters differed significantly from one another (P＜ 0.05).

3.3 Tissue-Specific Effect of Age on DNA Methylation

Consistent with the observation on the entire soft tissue, a decline in the DNA methylation fraction with age was also observed across all of the six tissues (Fig.2a). However, a significant difference among the ages was detected only in mantle, kidney and hepatopancreas. Based on the previous studies, all of these three tissues participate in excreting and depurating metals and other toxic materials in marine bivalves (Carmichael and Fowler, 1981; Cembellaet al., 1994; Arévaloet al., 1998; Blancoet al., 2002; Suzukiet al., 2005). Considering the depuration function, the damage from toxic residue and the burden of oxidative metabolism in these three tissues, their aging rate could be higher than that of the others, which would result in a significant decrease in the methylation ratio.

In comparison with the change in the DNA methylation fraction among the different age-related groups in the mantle (＜0.1%), a higher drop (up to 0.3%) was found in the hepatopancreas and kidney. The higher drop in DNA methylation rate in the two tissues was possibly caused by the relatively low toxin effect (Cembellaet al., 1994; Bauderet al., 2001; Blancoet al., 2002) and faster cell self-renewal rate in the mantle compared with kidney and hepatopancreas, which could help to slow down the decline in the methylation rate. In addition, research showed that the promoter methylation of some stress-responding genes, which are involved in cellular responses to environmental stresses, are mediated by DNMT1 and DNMT3B together (Yinget al., 2005). DNMT3 can also contribute to the methylation pattern change in the mantle and should be verified in the future.

3.4 Tissue-Specific Effect of Age onDNMT1Transcription

Inhibition ofDNMT1could lead to reduced methylation levels in various animals, such as frog, mouse, and human (Stancheva and Meehan, 2000; Sadoet al., 2000; Rheeet al., 2002; Gaudetet al., 2003), which would indicate that DNMT1 plays a critical role in maintaining the global DNA methylation level. In this study,DNMT1transcription also declined with age in all six tissues (Fig.2b), which is similar to the findings in various mammals, including mouse and human (Vertinoet al., 1994; Hamataniet al., 2004; Kimet al., 2009; Liuet al., 2009). The 1-year-old scallops contained moreDNMT1transcripts than the other age groups across all of the tissues, which is consistent with the trend in the DNA methylation levels. A significant difference among the ages was detected in the mantle and hepatopancreas, with the 4-year-old scallops containing remarkably lower abundance ofDNMT1mRNA than the 1-year-old scallops. There was a similar reduction tendency in the global DNA methylation and inDNMT1transcription of gill, gonad and muscle of 4-year-old scallops, although the reduction was not significant. In addition, based on the data from different tissues of all four groups, the DNA methylation ratio was significantly and positively (R= 0.72;P＜ 0.001) correlated with the relative abundance ofDNMT1transcripts (Fig.3), which is similar to related results (R= 0.746) in human cells (Leiet al., 2009). Thus, DNMT1 is likely to participate in maintaining DNA methylation in scallops in a similar way as in other organisms. In addition, the agedependent decrease in DNA methylation can be attributed to the down-regulation ofDNMT1during the aging process of scallop.

Fig.2 Changes in the DNA methylation ratio (a) and relative abundance ofDNMT1transcripts in six tissues of scallop at 1-4 years of age (b). One-way ANOVA followed by Fisher’s LSD test was used for the comparisons. The vertical bars represent the mean ± S.E. (n= 6). The values marked with different letters differed significantly from one another (P＜ 0.05).

Fig.3 Correlation between the relative percentages (%) of DNA methylation andDNMT1mRNA abundance (n= 24).

In summary, we found that both the age and tissue type had strong effects on DNA methylation inChlamys farreri. A significant decrease in DNA methylation with age was observed in the mantle, kidney and hepatopancreas. Agreeing with the results of DNA methylation,DNMT1transcription in mantle and hepatopancreas also declined with age. In addition, theDNMT1transcript abundance was significantly correlated with the DNA methylation ratio, which suggests the important role of DNMT1 in maintaining DNA methylation in scallop. This study can contribute to a better understanding of aging-related DNA methylation changes in bivalve mollusks.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (31130054), the National HighTechnology Research and Development Program of China (2012AA10A401), and Doctoral Fund of Ministry of Education of China (20120132130002).

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(Edited by Qiu Yantao)

(Received October 10, 2013; revised January 19, 2014; accepted March 23, 2015)

? Ocean University of China, Science Press and Spring-Verlag Berlin Heidelberg 2015

* Corresponding author. Tel： 0086-532-82031960 E-mail： zmbao@ouc.edu.cn