Anna V. Lioznova

BACKGROUND: DNA methylation is involved in the regulation of gene expression. Although bisulfite-sequencing based methods profile DNA methylation at a single CpG resolution, methylation levels are usually averaged over genomic regions in the downstream bioinformatic analysis. RESULTS: We demonstrate that on the genome level a single CpG methylation can serve as a more accurate predictor of gene expression than an average promoter / gene body methylation. We define CpG traffic lights (CpG TL) as CpG dinucleotides with a significant correlation between methylation and expression of a gene nearby. CpG TL are enriched in all regulatory regions. Among all promoters, CpG TL are especially enriched in poised ones, suggesting involvement of DNA methylation in their regulation. Yet, binding of only a handful of transcription factors, such as NRF1, ETS, STAT and IRF-family members, could be regulated by direct methylation of transcription factor binding sites (TFBS) or its close proximity. For the majority of TF, an alternative scenario is more likely: methylation and inactivation of the whole regulatory element indirectly represses functional TF binding with a CpG TL being a reliable marker of such inactivation. CONCLUSIONS: CpG TL provide a promising insight into mechanisms of enhancer activity and gene regulation linking methylation of single CpG to gene expression. CpG TL methylation can be used as reliable markers of enhancer activity and gene expression in applications, e.g. in clinic where measuring DNA methylation is easier compared to directly measuring gene expression due to more stable nature of DNA.

Acute myeloid leukemia (AML) is a rapidly progressing heterogeneous disease with a high mortality rate, which is characterized by hyperproliferation of atypical immature myeloid cells. The number of AML patients is expected to increase in the near future, due to the old-age-associated nature of AML and increased longevity in the human population. RUNX1 and CEBPA, key transcription factors (TFs) of hematopoiesis, are frequently and independently mutated in AML. RUNX1 and CEBPA can bind TET2 demethylase and attract it to their binding sites (TFBS) in cell lines, leading to DNA demethylation of the regions nearby. Since TET2 does not have a DNA-binding domain, TFs are crucial for its guidance to target genomic locations. In this paper, we show that RUNX1 and CEBPA mutations in AML patients affect the methylation of important regulatory sites that resulted in the silencing of several RUNX1 and CEBPA target genes, most likely in a TET2-dependent manner. We demonstrated that hypermethylation of TFBS in AML cells with RUNX1 mutations was associated with resistance to anticancer chemotherapy. Demethylation therapy restored expression of the RUNX1 target gene, BIK, and increased sensitivity of AML cells to chemotherapy. If our results are confirmed, mutations in RUNX1 could be an indication for prescribing the combination of cytotoxic and demethylation therapies.

Abstract DNA methylation is involved in regulation of gene expression. Although modern methods profile DNA methylation at single CpG sites, methylation levels are usually averaged over genomic regions in the downstream analyses. In this study we demonstrate that single CpG methylation can serve as a more accurate predictor of gene expression compared to average promoter / gene body methylation. CpG positions with significant correlation between methylation and expression of a gene nearby (named CpG traffic lights) are evolutionary conserved and enriched for exact TSS positions and active enhancers. Among all promoter types, CpG traffic lights are especially enriched in poised promoters. Genes that harbor CpG traffic lights are associated with development and signal transduction. Methylation levels of individual CpG traffic lights vary between cell types dramatically with the increased frequency of intermediate methylation levels, indicating cell population heterogeneity in CpG methylation levels. Being in line with the concept of the inherited stochastic epigenetic variation, methylation of such CpG positions might contribute to transcriptional regulation. Alternatively, one can hypothesize that traffic lights are markers of absent gene expression resulting from inactivation of their regulatory elements. The CpG traffic lights provide a promising insight into mechanisms of enhancer activity and gene regulation linking methylation of single CpG to expression.

Supplementary materials. Figure S1: SCC of the CpG TL located in various gene regions; Figure S2: Distribution of CpG TLs along the genome; Figure S3: TFBS; Table S1: Number of significant SCC between average methylation of genomic region and gene expression; Table S2: Number of significant SCC between CpG methylation and gene expression; Table S3: Number of significant SCC between gene expression and average methylation of the genome region; Table S4: Most enriched with CpG TLs categories of enhancers; Table S5: Names of the cell samples in the study; Table S6: Enhancers = H3K27ac+H3K4me1-H3K4me3; Table S7: Expression data source; Table S8: Methylation data source. (PDF 2562 kb)

DNA methylation is one of the most important mechanisms closely involved in the epigenetic regulation of gene expression. However, the relationship between DNA methylation and expression is not completely understood. There are reported examples of changes in DNA methylation being the cause of gene expression, and vice versa - examples of changes in gene expression to entail changes in methylation. Earlier, we introduced the concept of CpG traffic lights - individual CpG sites methylation levels of which significantly correlate with the expression of a gene nearby - and showed their important role in the regulation of enhancers. In this work, we show that the levels of CpG traffic lights methylation are heterogeneous in the cell population, and suggest that this feature is caused by their dynamic demethylation. The 5hmC enrichment and TET2 localization sites in CpG traffic lights confirm our hypothesis. In order to find out whether methylation of CpG sites is the cause or the consequence of the gene expression, we applied the assessment of the causation direction method developed by Jonas Peters and co-authors. We determined that in CpG sites where changes in methylation are predicted to be the cause of gene expression changes (M→E CpG sites), methylation levels are more stable in different cells and active demethylation occurs less often than in CpG sites where presumably changes in gene expression cause changes in methylation (E→M CpG sites). We also show a greater share of M→E CpG sites in the promoter regions than in the bodies of the genes where E→M CpG sites are more common. Based on these observations, we believe that methylation of M→E CpG sites is stable and functions as an "on/off" switch. On the contrary methylation of E→M sites is dynamic, variable between cells in a cell population, and is caused primarily by active demethylation.