title: SIRT1 affects DNA methylation of polycomb group protein target genes, a hotspot of the epigenetic shift observed in ageing.
abstract: BACKGROUND
  SIRT1 is likely to play a role in the extension in healthspan induced by dietary restriction. Actions of SIRT1 are pleiotropic, and effects on healthspan may include effects on DNA methylation. Polycomb group protein target genes (PCGTs) are suppressed by epigenetic mechanisms in stem cells, partly through the actions of the polycomb repressive complexes (PRCs), and have been shown previously to correspond with loci particularly susceptible to age-related changes in DNA methylation. We hypothesised that SIRT1 would affect DNA methylation particularly at PCGTs. To map the sites in the genome where SIRT1 affects DNA methylation, we altered SIRT1 expression in human intestinal (Caco-2) and vascular endothelial (HuVEC) cells by transient transfection with an expression construct or with siRNA. DNA was enriched for the methylated fraction then sequenced (HuVEC) or hybridised to a human promoter microarray (Caco-2).
  
  
  RESULTS
  The profile of genes where SIRT1 manipulation affected DNA methylation was enriched for PCGTs in both cell lines, thus supporting our hypothesis. SIRT1 knockdown affected the mRNA for none of seven PRC components nor for DNMT1 or DNMT3b. We thus find no evidence that SIRT1 affects DNA methylation at PCGTs by affecting the expression of these gene transcripts. EZH2, a component of PRC2 that can affect DNA methylation through association with DNA methyltransferases (DNMTs), did not co-immunoprecipitate with SIRT1, and SIRT1 knockdown did not affect the expression of EZH2 protein. Thus, it is unlikely that the effects of SIRT1 on DNA methylation at PCGTs are mediated through direct intermolecular association with EZH2 or through effects in its expression.
  
  
  CONCLUSIONS
  SIRT1 affects DNA methylation across the genome, but particularly at PCGTs. Although the mechanism through which SIRT1 has these effects is yet to be uncovered, this action is likely to contribute to extended healthspan, for example under conditions of dietary restriction.
authors.affiliation: Institute for Cell and Molecular Biosciences, Human Nutrition Research Centre, Newcastle University Medical School, Newcastle upon Tyne, NE2 4HH, UK. luisa.wakeling@ncl.ac.uk.
authors.forename: Luisa A
authors.initials: LA
authors.lastname: Wakeling
authors.affiliation: Institute for Cell and Molecular Biosciences, Human Nutrition Research Centre, Newcastle University Medical School, Newcastle upon Tyne, NE2 4HH, UK. Laura.Ions@nuth.nhs.uk.
authors.forename: Laura J
authors.initials: LJ
authors.lastname: Ions
authors.affiliation: Institute for Cell and Molecular Biosciences, Human Nutrition Research Centre, Newcastle University Medical School, Newcastle upon Tyne, NE2 4HH, UK. s.m.escolme@ncl.ac.uk.
authors.forename: Suzanne M
authors.initials: SM
authors.lastname: Escolme
authors.affiliation: Faculty of Medical Sciences, Newcastle University Medical School, Newcastle upon Tyne, NE2 4HH, UK. simon.cockell@ncl.ac.uk.
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Cockell
authors.affiliation: Institute for Cell and Molecular Biosciences, Human Nutrition Research Centre, Newcastle University Medical School, Newcastle upon Tyne, NE2 4HH, UK. t.su2@newcastle.ac.uk.
authors.forename: Tianhong
authors.initials: T
authors.lastname: Su
authors.affiliation: Institute for Cell and Molecular Biosciences, Human Nutrition Research Centre, Newcastle University Medical School, Newcastle upon Tyne, NE2 4HH, UK. m.dey@ncl.ac.uk.
authors.forename: Madhurima
authors.initials: M
authors.lastname: Dey
authors.affiliation: Institute for Cell and Molecular Biosciences, Human Nutrition Research Centre, Newcastle University Medical School, Newcastle upon Tyne, NE2 4HH, UK. emilyhampton5@gmail.com.
authors.forename: Emily V
authors.initials: EV
authors.lastname: Hampton
authors.affiliation: Unilever R&D, Colworth Discover, Colworth Science Park, Sharnbrook, Bedfordshire, MK44 1LQ, UK. gail.jenkins@unilever.com.
authors.forename: Gail
authors.initials: G
authors.lastname: Jenkins
authors.affiliation: Unilever R&D, Colworth Discover, Colworth Science Park, Sharnbrook, Bedfordshire, MK44 1LQ, UK. linda.wainwright@unilever.com.
authors.forename: Linda J
authors.initials: LJ
authors.lastname: Wainwright
authors.affiliation: Institute of Health and Society, Human Nutrition Research Centre, Newcastle University Medical School, Newcastle upon Tyne, NE2 4HH, UK. jill.mckay@ncl.ac.uk.
authors.forename: Jill A
authors.initials: JA
authors.lastname: McKay
authors.affiliation: Institute for Cell and Molecular Biosciences, Human Nutrition Research Centre, Newcastle University Medical School, Newcastle upon Tyne, NE2 4HH, UK. dianne.ford@ncl.ac.uk.
authors.forename: Dianne
authors.initials: D
authors.lastname: Ford
mesh_terms: D000375:Aging / Q000235:genetics*; D018938:Caco-2 Cells; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics; D019175:DNA Methylation / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics; D000071221:Enhancer of Zeste Homolog 2 Protein; D044127:Epigenesis, Genetic; D005786:Gene Expression Regulation / Q000235:genetics; D006801:Humans; D063151:Polycomb Repressive Complex 2 / Q000096:biosynthesis / Q000235:genetics*; D063146:Polycomb-Group Proteins / Q000096:biosynthesis / Q000235:genetics*; D011401:Promoter Regions, Genetic; D056564:Sirtuin 1 / Q000096:biosynthesis / Q000235:genetics*; D000097584:DNA Methyltransferase 3B
chemical_list: D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D004268:DNA-Binding Proteins; D000071221:Enhancer of Zeste Homolog 2 Protein; D063151:Polycomb Repressive Complex 2; D063146:Polycomb-Group Proteins; D056564:Sirtuin 1; D000097584:DNA Methyltransferase 3B; C000619380:DNMT1 protein, human; C498409:EZH2 protein, human; C447939:SIRT1 protein, human