Abstract
Epigenetic marks operate at multiple chromosomal levels to regulate gene expression, from direct covalent modification of DNA to three-dimensional chromosomal structure. Research has shown that 5-methylcytosine (5-mC) and its oxidized form, 5-hydroxymethylcytosine (5-hmC), are stable epigenetic marks with distinct genomic distributions and separate regulatory functions. In addition, recent data indicate that 5-hmC plays a critical regulatory role in the mammalian brain, emphasizing the importance of considering this alternative DNA modification in the context of neuroepigenetics. Traditional bisulfite (BS) treatment-based methods to measure the methylome are not able to distinguish between 5-mC and 5-hmC, meaning much of the existing literature does not differentiate these two DNA modifications. Recently developed methods, including Tet-assisted bisulfite treatment and oxidative bisulfite treatment, allow for differentiation of 5-hmC and/or 5-mC levels at base-pair resolution when combined with next-generation sequencing or methylation arrays. Despite these technological advances, there remains a lack of clarity regarding the appropriate statistical methods for integration of 5-mC and 5-hmC data. As a result, it can be difficult to determine the effects of an experimental treatment on 5-mC and 5-hmC dynamics. Here, we propose a statistical approach involving mixed effects to simultaneously model paired 5-mC and 5-hmC data as repeated measures. We tested this approach using publicly available BS/oxidative bisulfite-450K array data and showed that our new approach detected far more CpG probes with paired changes in 5-mC and 5-hmC by Alzheimer’s disease status (n = 14,183 probes) compared with the overlapping differential probes generated from separate models for each epigenetic mark (n = 68). Of note, all 68 of the overlapping probe IDs from the separate models were also significant in our new modeling approach, supporting the sensitivity of our new analysis method. Using the proposed approach, it will be possible to determine the effects of an experimental treatment on both 5-mC and 5-hmC at the base-pair level.
Highlights
EpigeneticsEpigenetic marks operate at four major levels—DNA modifications, histone modifications, noncoding RNAs, and three-dimensional chromatin structure (Chen et al, 2017b)
These data were similar to the results reported in the dataset’s original publication, where the authors identified only one differentially methylated probe (DMP) 2 differentially methylated regions, and one differentially hydroxymethylated region (Smith et al, 2019)
Since there was so little significance in the separate models, it was difficult to compare the traditional method with our novel approach, so we repeated the separate modeling approach with a more lenient p-value < 0.001 cutoff
Summary
EpigeneticsEpigenetic marks operate at four major levels—DNA modifications, histone modifications, noncoding RNAs, and three-dimensional chromatin structure (Chen et al, 2017b). An abundance of research shows associations between 5-mC and gene expression and suggests that this epigenetic mark plays a key role in transcriptional control (Moore et al, 2013). In addition to 5-mC, there are three further oxidized DNA modifications—5hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC), and 5-carboxylcytosine (5-caC) (Shen et al, 2014). These alternative DNA modifications are formed when 5-mC is successively oxidized by the ten-eleven translocase (Tet) family of proteins (Shen et al, 2014). 5-hmC appears to play an important role in the central nervous system, where it is present at much higher levels than embryonic stem cells and other somatic tissues (Globisch et al, 2010; Szwagierczak et al, 2010; Nestor et al, 2012; Cheng et al, 2015)
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