CpG Traffic Lights are Involved in Active DNA Demethylation

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Abstract

DNA methylation is one of the most important mechanisms involved in the epigenetic regulation of gene expression. However, the relationship between DNA methylation and expression is still not fully understood. There are examples where changes in DNA methylation level cause changes in gene expression, and vice versa ‒ changes in expression entail changes in the methylation level. Earlier, we introduced the concept of CpG traffic lights, individual CpG sites whose methylation significantly correlates with expression, and showed their important role in enhancer regulation. Here, we showed that the methylation levels of CpG traffic lights are heterogeneous in the cell population and suggested that this is due to their dynamic demethylation. The observed enrichment of CpG traffic lights with 5-hydroxymethylcytosine (5hmC) and TET2 (Tet methylcytosine dioxygenase 2) localization sites has now confirmed our hypothesis. In order to find out whether the methylation of CpG sites is a cause or a consequence of the expression of the corresponding gene, we applied the method of causal inference. As a result, among the CpG sites, we distinguished those for which methylation was the cause of expression changes and those for which expression changes caused methylation changes. CpG sites of the first type were characterized by more stable methylation levels in different cells and less pronounced demethylation compared to CpG sites of the second type. It was also shown that the proportion of CpG sites whose methylation affected expression was greater in promoter regions than in the gene body, for which methylation was likely to be a consequence of expression. Based on these observations, we can assume that the methylation levels of CpG sites, which determine the expression of the gene associated with them, are stable and work on the principle of a “switch”. Conversely, for expression-dependent CpG sites, methylation levels are dynamic and vary between cells in the population, primarily due to active demethylation.

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A. V. Lioznova

Research Center of Biotechnology of the Russian Academy of Sciences

Author for correspondence.
Email: ju.medvedeva@gmail.com
Russian Federation, Moscow, 119071

Yu. A. Medvedeva

Research Center of Biotechnology of the Russian Academy of Sciences

Email: ju.medvedeva@gmail.com
Russian Federation, Moscow, 119071

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3. Fig. 1. Distribution of methylation and expression of genes associated with CpG traffic lights and background positions with a negative Spearman correlation coefficient. The tone reflects the density of points on a logarithmic scale. The distribution is shown for random background CpG positions in the promoter (the number of positions is equal to the number of CpG traffic lights) (a); CpG traffic lights in the promoter (b); random background CpG positions in the body of the gene (c); CpG traffic lights in the body of the gene [+500, TTS] (d). Panel (e) shows the distribution of the proportion of CpG traffic lights and background CpG positions depending on the level of 5hmC in the cerebellar sample.; Panel (e) shows the quantiles of the distribution (e). Panels (g) and (h) show the number of CpG positions colocalized with TET2 signal peaks in two different samples, respectively: neurons differentiated on day 12 (NPC cells) and macrophages derived from monocytes. Here and further in the figures: the error bars correspond to the 5% and 95% percentiles of 50 samples of background positions; for background positions, the median of 50 samples is shown; background. positions — background positions; corr — Spearman correlation coefficient.

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4. Fig. 2. Analysis of the cause-effect relationships and the distribution of 5hmC depending on the direction of communication. The left panel shows the results of a quantitative analysis of CpG positions, for which the presence of a causal relationship has been revealed. The left column shows the total number of positions, and the column on the right shows the number of positions depending on the direction of communication. The graphs show the number of positions with a causal relationship coefficient for CpG traffic lights and background CpG positions throughout the genome (a) and their refinement — the number of positions depending on the direction of communication (b). Species (c) and (d) show positions in the promoter region [-1000, TSS+500], while species (e) and (e) show positions in the gene body [TSS+500, TTS]. The right panel shows the distribution of 5hmC: (g) — the distribution of the proportion of CpG traffic lights depending on the level of 5hmC for both directions of causation; (h) — quantiles of the distribution in the form (g); (i) — a distribution similar to (g), only for background CpG positions; (k) — quantiles of the distribution in the panel (i).

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5. Fig. 3. The proposed mechanism of functioning of CpG traffic lights. The left panel shows CpG traffic lights, in which changes in the level of methylation (Me) cause changes in the expression of the associated gene. Such CpG traffic lights are more common in promoters, their methylation is stable and they work according to the “on/off” principle. The right panel shows CpG traffic lights, for which the level of methylation depends on the expression of the gene. Their methylation varies between cells and is dynamic, which is caused by active demethylation. Demethylation occurs under the action of methylcytosine dioxygenase TEST2, which is attracted by transcription factors (TF).

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