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Effects of Sirt1 on DNA methylation and expression of genes affected by dietary restriction

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Abstract

Changes in DNA methylation across the life course may contribute to the ageing process. We hypothesised that some effects of dietary restriction to extend lifespan and/or mitigate against features of ageing result from changes in DNA methylation, so we determined if genes that respond to dietary restriction also show age-related changes in DNA methylation. In support of our hypothesis, the intersection of lists of genes compiled from published sources that (1) were differentially expressed in response to dietary restriction and (2) showed altered methylation with increased age was greater than expected. We also hypothesised that some effects of Sirt1, which may play a pivotal role in beneficial effects of dietary restriction, are mediated through DNA methylation. We thus measured effects of Sirt1 overexpression and knockdown in a human cell line on DNA methylation and expression of a panel of eight genes that respond to dietary restriction and show altered methylation with age. Six genes were affected at the level of DNA methylation, and for six expressions were affected. In further support of our hypothesis, we observed by DNA microarray analysis that genes showing differential expression in response to Sirt1 knockdown were over-represented in the complied list of genes that respond to dietary restriction. The findings reveal that Sirt1 has effects on DNA methylation across the genome and affects, in particular, the expression of genes that respond to dietary restriction. Sirt1-mediated effects on DNA methylation and, consequently, gene expression may thus be one of the mechanisms underlying the response to dietary restriction.

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Acknowledgments

This work was funded by the BBSRC (studentship to LJI and research grant nos. BBE0074571 and BBF0196371), Strategic Promotion of Ageing Research Capacity (SPARC), MRC (studentships to HJB and JEJH) and the Rank Prize Funds (studentship to SME).

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Authors and Affiliations

  1. Human Nutrition Research Centre, Newcastle University, Newcastle upon Tyne, NE4 5PL, UK

    Laura J Ions, Luisa A Wakeling, Helen J Bosomworth, Joy EJ Hardyman, Suzanne M Escolme, Ruth A Valentine, John C Mathers & Dianne Ford

  2. Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, NE2 4NN, UK

    Laura J Ions, Luisa A Wakeling, Helen J Bosomworth, Joy EJ Hardyman, Suzanne M Escolme, Daniel C Swan, Ruth A Valentine & Dianne Ford

  3. Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne, NE4 5PL, UK

    John C Mathers & Dianne Ford

  4. School of Dental Sciences, Newcastle University, Newcastle upon Tyne, NE2 4BW, UK

    Helen J Bosomworth & Ruth A Valentine

  5. Institute for Cell and Molecular Biosciences, Medical School, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK

    Dianne Ford

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  1. Laura J Ions
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Correspondence to Dianne Ford.

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Laura J Ions and Luisa A Wakeling are both joint first authors, based on equal contribution.

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ESM 1

Primers and annealing temperatures used for RT-qPCR. Sequences are in the 5′–3′ direction, and subscript numerals refer to positions within the sequences deposited under the stated GenBank accession numbers (DOC 38 kb)

ESM 2

Primers and annealing temperatures used for pyrosequencing. PCR products were generated using HotStarTaq Master Mix (Qiagen) from bisulphite-converted DNA using primers (0.25 μM each) and annealing temperatures as listed, including a 5′ biotin label on the sense primer of the nested reaction (antisense for KLF3), as indicated, and the following cycling parameters: 95 °C, 15 min then 95 °C, 15 s; 40–58 °C (annealing), 60 s; 72 °C 60 s—35 cycles for the first-round PCR and 50 cycles for the nested reaction—followed by 72 °C for 5 min. Primers sequences are in the 5′ to 3′ direction, and subscript numbers indicate position relative to the transcription start site (DOC 43 kb)

ESM 3

Complied lists of genes used for the in silico analysis (XLS 1053 kb)

ESM 4

DNA microarray data for effects of Sirt1 knockdown in Caco-2 cells by two different siRNAs (XLS 181 kb)

ESM 5

Comparative effects on TBX3 DNA methylation at the three CpG sites in the region −135 to +1, relative to the start of transcription, of increasing (transgene overexpression) or reducing (siRNA) expression of Sirt1 in Caco-2 cells and HuVECs. Values for the control condition were derived through combining the data for controls for Sirt1 overexpression (plasmid minus Sirt1 transgene insert) and knockdown (control siRNA), which did not differ. Data are stated to the nearest 0.25 %, based on calculation of medians and quartiles from values rounded to the nearest 1 %. Q1 first quartile, Q3 third quartile. **P < 0.01, compared with control; #P < 0.05; ##P < 0.01, compared with SIRT1 overexpression, by Kruskal Wallis followed by Dunn’s multiple comparisons tests (DOC 33 kb)

ESM 6

Comparative effects on relative TBX3 mRNA levels of increasing (transgene overexpression) or reducing (siRNA) expression of Sirt1 in Caco-2 cells and HuVECs. Values for the control condition were derived through combining the data for controls for Sirt1 overexpression (plasmid minus Sirt1 transgene insert) and knockdown (control siRNA), which did not differ. Data are expressed relative to the mean for two reference genes—GAPDH and WNT11—for Caco-2 cells and relative to GAPDH for HuVECs and are normalised to the control condition. *P < 0.05 compared with control by one-way ANOVA followed by Tukey’s post test (DOC 30 kb)

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Ions, L.J., Wakeling, L.A., Bosomworth, H.J. et al. Effects of Sirt1 on DNA methylation and expression of genes affected by dietary restriction. AGE 35, 1835–1849 (2013). https://doi.org/10.1007/s11357-012-9485-8

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