A lifelong exposure to a Western-style diet, but not aging, alters global DNA methylation in mouse colon

Choi, Sang-Woon,Tammen, Stephanie A,Liu, Zhenhua,Friso, Simonetta The Korean Nutrition Society 2015 Nutrition Research and Practice Vol.9 No.4

BACKGROUND/OBJECTIVES: Previous studies have indicated that when compared to young mice, old mice have lower global DNA methylation and higher p16 promoter methylation in colonic mucosa, which is a common finding in colon cancer. It is also known that a Western-style diet (WSD) high in fat and calories, and low in calcium, vitamin D, fiber, methionine and choline (based on the AIN 76A diet) is tumorigenic in colons of mice. Because DNA methylation is modifiable by diet, we investigate whether a WSD disrupts DNA methylation patterns, creating a tumorigenic environment. SUBJECTVIES/METHODS: We investigated the effects of a WSD and aging on global and p16 promoter DNA methylation in the colon. Two month old male C57BL/6 mice were fed either a WSD or a control diet (AIN76A) for 6, 12 or 17 months. Global DNA methylation, p16 promoter methylation and p16 expression were determined by LC/MS, methyl-specific PCR and real time RT-PCR, respectively. RESULTS: The WSD group demonstrated significantly decreased global DNA methylation compared with the control at 17 months (4.05 vs 4.31%, P = 0.019). While both diets did not change global DNA methylation over time, mice fed the WSD had lower global methylation relative to controls when comparing all animals (4.13 vs 4.30%, P = 0.0005). There was an increase in p16 promoter methylation from 6 to 17 months in both diet groups (P < 0.05) but no differences were observed between diet groups. Expression of p16 increased with age in both control and WSD groups. CONCLUSIONS: In this model a WSD reduces global DNA methylation, whereas aging itself has no affect. Although the epigenetic effect of aging was not strong enough to alter global DNA methylation, changes in promoter-specific methylation and gene expression occurred with aging regardless of diet, demonstrating the complexity of epigenetic patterns.

A lifelong exposure to a Western-style diet, but not aging, alters global DNA methylation in mouse colon

Sang-Woon Choi,Stephanie A Tammen,Zhenhua Liu,Simonetta Friso 대한지역사회영양학회 2015 Nutrition Research and Practice Vol.5 No.6

BACKGROUND/OBJECTIVES: Previous studies have indicated that when compared to young mice, old mice have lower global DNA methylation and higher p16 promoter methylation in colonic mucosa, which is a common finding in colon cancer. It is also known that a Western-style diet (WSD) high in fat and calories, and low in calcium, vitamin D, fiber, methionine and choline (based on the AIN 76A diet) is tumorigenic in colons of mice. Because DNA methylation is modifiable by diet, we investigate whether a WSD disrupts DNA methylation patterns, creating a tumorigenic environment. SUBJECTVIES/METHODS: We investigated the effects of a WSD and aging on global and p16 promoter DNA methylation in the colon. Two month old male C57BL/6 mice were fed either a WSD or a control diet (AIN76A) for 6, 12 or 17 months. Global DNA methylation, p16 promoter methylation and p16 expression were determined by LC/MS, methyl-specific PCR and real time RT-PCR, respectively. RESULTS: The WSD group demonstrated significantly decreased global DNA methylation compared with the control at 17 months (4.05 vs 4.31%, P = 0.019). While both diets did not change global DNA methylation over time, mice fed the WSD had lower global methylation relative to controls when comparing all animals (4.13 vs 4.30%, P = 0.0005). There was an increase in p16 promoter methylation from 6 to 17 months in both diet groups (P < 0.05) but no differences were observed between diet groups. Expression of p16 increased with age in both control and WSD groups. CONCLUSIONS: In this model a WSD reduces global DNA methylation, whereas aging itself has no affect. Although the epigenetic effect of aging was not strong enough to alter global DNA methylation, changes in promoter-specific methylation and gene expression occurred with aging regardless of diet, demonstrating the complexity of epigenetic patterns.

A lifelong exposure to a Western-style diet, but not aging, alters global DNA methylation in mouse colon

Sang-Woon Choi,Stephanie A Tammen,Zhenhua Liu,Simonetta Friso 한국영양학회 2015 Nutrition Research and Practice Vol.9 No.4

BACKGROUND/OBJECTIVES: Previous studies have indicated that when compared to young mice, old mice have lower global DNA methylation and higher p16 promoter methylation in colonic mucosa, which is a common finding in colon cancer. It is also known that a Western-style diet (WSD) high in fat and calories, and low in calcium, vitamin D, fiber, methionine and choline (based on the AIN 76A diet) is tumorigenic in colons of mice. Because DNA methylation is modifiable by diet, we investigate whether a WSD disrupts DNA methylation patterns, creating a tumorigenic environment. SUBJECTVIES/METHODS: We investigated the effects of a WSD and aging on global and p16 promoter DNA methylation in the colon. Two month old male C57BL/6 mice were fed either a WSD or a control diet (AIN76A) for 6, 12 or 17 months. Global DNA methylation, p16 promoter methylation and p16 expression were determined by LC/MS, methyl-specific PCR and real time RT-PCR, respectively. RESULTS: The WSD group demonstrated significantly decreased global DNA methylation compared with the control at 17 months (4.05 vs 4.31%, P = 0.019). While both diets did not change global DNA methylation over time, mice fed the WSD had lower global methylation relative to controls when comparing all animals (4.13 vs 4.30%, P = 0.0005). There was an increase in p16 promoter methylation from 6 to 17 months in both diet groups (P < 0.05) but no differences were observed between diet groups. Expression of p16 increased with age in both control and WSD groups. CONCLUSIONS: In this model a WSD reduces global DNA methylation, whereas aging itself has no affect. Although the epigenetic effect of aging was not strong enough to alter global DNA methylation, changes in promoter-specific methylation and gene expression occurred with aging regardless of diet, demonstrating the complexity of epigenetic patterns.

Aging Alters Hepatic DNA Hydroxymethylation, as Measured by Liquid Chromatography/Mass Spectrometry

Stephanie A. Tammen,Gregory G. Dolnikowski,Lynne M. Ausman,Zhenhua Liu,김경철,Simonetta Filippi,최상운 대한암예방학회 2014 Journal of cancer prevention Vol.19 No.4

Background:Aging is one of the most important risk factors for cancer. It appears that aberrant epigenetic changes might be a commondriver of aging and cancer. Among them are changes in DNA methylation and DNA hydroxymethylation. The 5’ carbon of cytosinesin CpG dinucleotides of DNA can be either methylated or hydroxymethylated. Like 5’-methylcytosine, changes in 5’-hydroxymethylcytosinemay occur due to aging, potentially leading to downstream changes in transcription and cancer development. Methods:We set up a method to measure 5’-methyl-2’-deoxycytidine and 5’-hydroxymethyl-2’-deoxycytidine in DNA using liquidchromatography/mass spectrometry (LC/MS-MS) and used this method to measure the percentage of total cytosine that was eithermethylated or hydroxymethylated in the liver tissues of young and old C57Bl/6 male mice. The DNA was enzymatically hydrolyzed bysequential digestion with nuclease P1, phosphodiesterase I and alkaline phosphatase. The isotopomers [15N3]-2’-deoxycytidine and(methyl-d3, ring-6-d1)-5-methyl-2’-deoxycytidine were added to the DNA hydrolysates as internal standards. DNA methylation andhydroxymethylation were calculated as a percentage of total deoxycytidine in genomic DNA. Results:Within day variations for DNA methylation and hydroxymethylation were 3.45% and 8.40%, while day to day variations were6.14% and 17.68%, respectively. Using this method it was determined that hepatic DNA of old mice had increased levels ofhydroxymethylation relative to young (0.32 ± 0.02% vs. 0.24 ± 0.01%, P= 0.02), with no significant changes in 5’-methylcytosine. Conclusions:DNA hydroxymethylation measured by LC/MS-MS method can be a novel biomarker of aging. It will be useful to investigatethe potential role of DNA hydroxymethylation in the development and prevention of age-associated cancer.

Iron Supplementation Reverses the Reduction of Hydroxymethylcytosine in Hepatic DNA Associated With Chronic Alcohol Consumption in Rats

Stephanie A. Tammen,박정은,신필경,Simonetta Friso,정자용,최상운 대한암예방학회 2016 Journal of cancer prevention Vol.21 No.4

Background: Alcohol is known to affect two epigenetic phenomena, DNA methylation and DNA hydroxymethylation, and iron is a cofactor of ten-eleven translocation (TET) enzymes that catalyze the conversion from methylcytosine to hydroxymethylcytosine. In the present study we aimed to determine the effects of alcohol on DNA hydroxymethylation and further effects of iron on alcohol associated epigenetic changes. Methods: Twenty-four male Sprague-Dawley rats were fed either Lieber-DeCarli alcohol diet (36% calories from ethanol) or Lieber-DeCarli control diet along with or without iron supplementation (0.6% carbonyl iron) for 8 weeks. Hepatic non-heme iron concentrations were measured by colorimetric assays. Protein levels of hepatic ferritin and transferrin receptor were determined by Western blotting. Methylcytosine, hydroxymethylcytosine and unmodified cytosine in DNA were simultaneously measured by liquid chromatography/mass spectrometry method. Results: Iron supplementation significantly increased hepatic non-heme iron contents (P < 0.05) but alcohol alone did not. However, both alcohol and iron significantly increased hepatic ferritin levels and decreased hepatic transferrin receptor levels (P < 0.05). Alcohol reduced hepatic DNA hydroxymethylation (0.21% ± 0.04% vs. 0.33% ± 0.04%, P = 0.01) compared to control, while iron supplementation to alcohol diet did not change DNA hydroxymethylation. There was no significant difference in methylcytosine levels, while unmodified cytosine levels were significantly increased in alcohol-fed groups compared to control (95.61% ± 0.08% vs. 95.26% ± 0.12%, P = 0.03), suggesting that alcohol further increases the conversion from hydroxymethylcytosine to unmodified cytosine. Conclusions: Chronic alcohol consumption alters global DNA hydroxymethylation in the liver but iron supplementation reverses the epigenetic effect of alcohol.

Genome-wide hepatic DNA methylation changes in high-fat diet-induced obese mice

AhRam Yoon,Stephanie A. Tammen,Soyoung Park,Sung Nim Han,Sang-Woon Choi 한국영양학회 2017 Nutrition Research and Practice Vol.11 No.2

BACKGROUND/OBJECTIVES: A high-fat diet (HFD) induces obesity, which is a major risk factor for cardiovascular disease and cancer, while a calorie-restricted diet can extend life span by reducing the risk of these diseases. It is known that health effects of diet are partially conveyed through epigenetic mechanism including DNA methylation. In this study, we investigated the genome-wide hepatic DNA methylation to identify the epigenetic effects of HFD-induced obesity. MATERIALS AND METHODS: Seven-week-old male C57BL/6 mice were fed control diet (CD), calorie-restricted control diet (CRCD), or HFD for 16 weeks (after one week of acclimation to the control diet). Food intake, body weight, and liver weight were measured. Hepatic triacylglycerol and cholesterol levels were determined using enzymatic colorimetric methods. Changes in genome-wide DNA methylation were determined by a DNA methylation microarray method combined with methylated DNA immunoprecipitation. The level of transcription of individual genes was measured by real-time PCR. RESULTS: The DNA methylation statuses of genes in biological networks related to lipid metabolism and hepatic steatosis were influenced by HFD-induced obesity. In HFD group, a proinflammatory Casp1 (Caspase 1) gene had hypomethylated CpG sites at the 1.5-kb upstream region of its transcription start site (TSS), and its mRNA level was higher compared with that in CD group. Additionally, an energy metabolism-associated gene Ndufb9 (NADH dehydrogenase 1 beta subcomplex 9) in HFD group had hypermethylated CpG sites at the 2.6-kb downstream region of its TSS, and its mRNA level was lower compared with that in CRCD group. CONCLUSIONS: HFD alters DNA methylation profiles in genes associated with liver lipid metabolism and hepatic steatosis. The methylation statuses of Casp1 and Ndufb9 were particularly influenced by the HFD. The expression of these genes in HFD differed significantly compared with CD and CRCD, respectively, suggesting that the expressions of Casp1 and Ndufb9 in liver were regulated by their methylation statuses.