HighlightsA method is presented for 5hmC detection and analysis using Infinium 450K BeadChips.The oxBS-450K method can discriminate between 5mC and 5hmC in human gDNA5hmC levels were quantified genome-wide in 3 distinct biological samples.The reported 5hmC signal was validated using mass spectrometry and pyrosequencing.The effects of differing amounts of input DNA on final 5hmC call rate are discussed.

The Infinium 450K Methylation array is an established tool for measuring methylation. However, the bisulfite (BS) reaction commonly used with the 450K array cannot distinguish between 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). The oxidative-bisulfite assay disambiguates 5mC and 5hmC. We describe the use of oxBS in conjunction with the 450K array (oxBS-array) to analyse 5hmC/5mC in cerebellum DNA. The “methylation” level derived by the BS reaction is the combined level of 5mC and 5hmC at a given base, while the oxBS reaction gives the level of 5mC alone. The level of 5hmC is derived by subtracting the oxBS level from the BS level. Here we present an analysis method that distinguishes genuine positive levels of 5hmC at levels as low as 3%. We performed four replicates of the same sample of cerebellum and found a high level of reproducibility (average r for BS = 98.3, and average r for oxBS = 96.8). In total, 114,734 probes showed a significant positive measurement for 5hmC. The range at which we were able to distinguish 5hmC occupancy was between 3% and 42%. In order to investigate the effects of multiple replicates on 5hmC detection we also simulated fewer replicates and found that decreasing the number of replicates to two reduced the number of positive probes identified by > 50%. We validated our results using qPCR in conjunction with glucosylation of 5hmC sites followed by MspI digestion and we found good concordance with the array estimates (r = 0.94). This experiment provides a map of 5hmC in the cerebellum and a robust dataset for use as a standard in future 5hmC analyses. We also provide a novel method for validating the presence of 5hmC at low levels, and highlight some of the pitfalls associated with measuring 5hmC and 5mC.

Controlling the functional dynamics of DNAwithin living cells is essential in biomedical research. Epigenetic modifications such as DNAmethylation play akey role in this endeavour.D NA methylation can be controlled by genetic means.Y et there are few chemical tools available for the spatial and temporal modulation of this modification. Herein, we present as mall-molecule approach to modulate DNAm ethylation with light. The strategy uses aphoto-tuneable version of ac linically used drug (5-aza-2'-deoxycytidine) to alter the catalytic activity of DNAmethyltransferases,the enzymes that methylate DNA. After uptake by cells,t he photo-regulated molecule can be light-controlled to reduce genome-wide DNA methylation levels in proliferating cells.T he chemical tool complements genetic,b iochemical, and pharmacological approaches to study the role of DNAm ethylation in biology and medicine.The methylation of DNAatposition 5ofcytosine residues is chemically av ery simple but biologically one of the most important modifications of DNA. It influences many biological processes in humans such as the regulation of cell function, cellular reprogramming,a nd organismal development. [1][2][3][4][5][6][7] Biological effects of higher methylation levels at promoters are mediated by lowering the transcription of genes either by blocking the binding of transcription factors or by recruiting unique methyl-recognizing proteins that lower gene expression. Altered levels of methylation are also associated with several diseases [8][9][10][11] including cancer. [8,[12][13][14][15][16] Driven by the growing importance of DNAmethylation in biomedical research, there is as trong interest to experimentally lower or increase methylation levels [17][18][19][20][21][22][23] to study,f or example,t he role of epigenetic reprogramming in tissue development or regenerative medicine. [24,25] Optical control is of particular relevance given the high spatial and temporal resolution of light. Often, the approach is implemented with photosensitive small molecules of tuneable bioactivity. [26][27][28][29][30][31] These can be used without the need for genetic engineering of cells leading to powerful applications within cell biology. [32] Yet, despite the importance of DNAm ethylation in biology, no light-tuneable small-molecule tool has been developed to manipulate methylation levels in cells.Herein, we present ap hoto-mediated small-molecule strategy that modulates methylation in light-exposed cells. At the approachsc entre is an inhibitor that interferes with DNAmethyltransferases (DNMTs), the enzymes responsible for DNAm ethylation [33] including the maintenance DNA methyltransferase 1( DNMT1). [34] Thei nhibitorsb ioactivity becomes tuneable with light by the chemical derivatization with ap hotocage.A ss chematically illustrated in Figure 1a, the attached photocage renders the inhibitor biologically inactive.However, light exposure cleaves off the photocage to restore the original inhibitory effect (Figure 1a). Thep hotocaged molecule is hence expected...

Controlling the functional dynamics of DNAwithin living cells is essential in biomedical research. Epigenetic modifications such as DNAmethylation play akey role in this endeavour.D NA methylation can be controlled by genetic means.Y et there are few chemical tools available for the spatial and temporal modulation of this modification. Herein, we present as mall-molecule approach to modulate DNAm ethylation with light. The strategy uses aphoto-tuneable version of ac linically used drug (5-aza-2'-deoxycytidine) to alter the catalytic activity of DNAmethyltransferases,the enzymes that methylate DNA. After uptake by cells,t he photo-regulated molecule can be light-controlled to reduce genome-wide DNA methylation levels in proliferating cells.T he chemical tool complements genetic,b iochemical, and pharmacological approaches to study the role of DNAm ethylation in biology and medicine.The methylation of DNAatposition 5ofcytosine residues is chemically av ery simple but biologically one of the most important modifications of DNA. It influences many biological processes in humans such as the regulation of cell function, cellular reprogramming,a nd organismal development. [1][2][3][4][5][6][7] Biological effects of higher methylation levels at promoters are mediated by lowering the transcription of genes either by blocking the binding of transcription factors or by recruiting unique methyl-recognizing proteins that lower gene expression. Altered levels of methylation are also associated with several diseases [8][9][10][11] including cancer. [8,[12][13][14][15][16] Driven by the growing importance of DNAmethylation in biomedical research, there is as trong interest to experimentally lower or increase methylation levels [17][18][19][20][21][22][23] to study,f or example,t he role of epigenetic reprogramming in tissue development or regenerative medicine. [24,25] Optical control is of particular relevance given the high spatial and temporal resolution of light. Often, the approach is implemented with photosensitive small molecules of tuneable bioactivity. [26][27][28][29][30][31] These can be used without the need for genetic engineering of cells leading to powerful applications within cell biology. [32] Yet, despite the importance of DNAm ethylation in biology, no light-tuneable small-molecule tool has been developed to manipulate methylation levels in cells.Herein, we present ap hoto-mediated small-molecule strategy that modulates methylation in light-exposed cells. At the approachsc entre is an inhibitor that interferes with DNAmethyltransferases (DNMTs), the enzymes responsible for DNAm ethylation [33] including the maintenance DNA methyltransferase 1( DNMT1). [34] Thei nhibitorsb ioactivity becomes tuneable with light by the chemical derivatization with ap hotocage.A ss chematically illustrated in Figure 1a, the attached photocage renders the inhibitor biologically inactive.However, light exposure cleaves off the photocage to restore the original inhibitory effect (Figure 1a). Thep hotocaged molecule is hence expected...

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