Abstract
DNA methylation on CpG sites is the most common epigenetic modification. Recently, methylation in a non-CpG context was found to occur widely on genomic DNA. Moreover, methylation of non-CpG sites is a highly controlled process, and its level may vary during cellular development. To study non-CpG methylation effects on DNA/protein interactions, we have chosen three human transcription factors (TFs): glucocorticoid receptor (GR), brain and muscle ARNT-like 1 (BMAL1) - circadian locomotor output cycles kaput (CLOCK) and estrogen receptor (ER) with methylated or unmethylated DNA binding sequences, using single-molecule and isothermal titration calorimetry assays. The results demonstrated that these TFs interact with methylated DNA with different effects compared with their cognate DNA sequences. The effects of non-CpG methylation on transcriptional regulation were validated by cell-based luciferase assay at protein level. The mechanisms of non-CpG methylation influencing DNA-protein interactions were investigated by crystallographic analyses and molecular dynamics simulation. With BisChIP-seq assays in HEK-293T cells, we found that GR can recognize highly methylated sites within chromatin in cells. Therefore, we conclude that non-CpG methylation of DNA can provide a mechanism for regulating gene expression through directly affecting the binding of TFs.
Highlights
DNA methylation on CpG sites is the most common epigenetic modification
We focus on the detailed interactions of three important human transcription factors (TFs): glucocorticoid receptor (GR), brain and muscle ARNT-like 1 (BMAL1) - circadian locomotor output cycles kaput (CLOCK), and estrogen receptor (ER) with their methylated or unmethylated DNA binding sequences, to elucidate general principles
The results demonstrated that these TFs represent three possible mechanisms for regulating gene expression by DNA methylation respectively: GR represents the TF with binding affinity enhanced by the methylation; whereas BMAL1-CLOCK represents the TF with binding affinity weakened by the methylation; and there is yet another group of TFs, such as ER without significant affinity change towards the methylated DNA
Summary
DNA methylation on CpG sites is the most common epigenetic modification. Recently, methylation in a non-CpG context was found to occur widely on genomic DNA. To study non-CpG methylation effects on DNA/protein interactions, we have chosen three human transcription factors (TFs): glucocorticoid receptor (GR), brain and muscle ARNT-like 1 (BMAL1) - circadian locomotor output cycles kaput (CLOCK) and estrogen receptor (ER) with methylated or unmethylated DNA binding sequences, using single-molecule and isothermal titration calorimetry assays. We focus on the detailed interactions of three important human TFs: glucocorticoid receptor (GR), brain and muscle ARNT-like 1 (BMAL1) - circadian locomotor output cycles kaput (CLOCK), and estrogen receptor (ER) with their methylated or unmethylated DNA binding sequences, to elucidate general principles www.nature.com/scientificreports/. (d) Dissociation constants KD of the GRDBD, BCDBD and ERDBD interacting with their methylated or unmethylated DNA binding targets measured by ITC (insert and Supplementary Fig. S4). The final KD is the average of three independent titrations and error bars denote the standard deviation. (e) Association constant kon was calculated by koff/KD and error bars denote the standard deviation from three individual experiments
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