Although DNA methylation is the best characterized epigenetic mark, the mechanism by which it is targeted to specific regions in the genome remains unclear. Recent studies have revealed that local DNA methylation profiles might be dictated by cis-regulatory DNA sequences that mainly operate via DNA-binding factors. Consistent with this finding, we have recently shown that disruption of CTCF-binding sites by rare single nucleotide variants (SNVs) can underlie cis-linked DNA methylation changes in patients with congenital anomalies. These data raise the hypothesis that rare genetic variation at transcription factor binding sites (TFBSs) might contribute to local DNA methylation patterning.

In this work, by combining blood genome-wide DNA methylation profiles, whole genome sequencing-derived SNVs from 247 unrelated individuals along with 133 predicted TFBS motifs derived from ENCODE ChIP-Seq data, we observed an association between the disruption of binding sites for multiple TFs by rare SNVs and extreme DNA methylation values at both local and, to a lesser extent, distant CpGs. While the majority of these changes affected only single CpGs, 24% were associated with multiple outlier CpGs within ±1kb of the disrupted TFBS. Interestingly, disruption of functionally constrained sites within TF motifs lead to larger DNA methylation changes at nearby CpG sites. Altogether, these findings suggest that rare SNVs at TFBS negatively influence TF-DNA binding, which can lead to an altered local DNA methylation profile. Furthermore, subsequent integration of DNA methylation and RNA-Seq profiles from cardiac tissues enabled us to observe an association between rare SNV-directed DNA methylation and outlier expression of nearby genes.

In conclusion, our findings not only provide insights into the effect of rare genetic variation at TFBS on shaping local DNA methylation and its consequences on genome regulation, but also provide a rationale to incorporate DNA methylation data to interpret the functional role of rare variants.

尽管 DNA 甲基化是表征最好的表观遗传标记，但其靶向基因组中特定区域的机制仍不清楚。最近的研究表明，局部 DNA 甲基化谱可能由主要通过 DNA 结合因子起作用的顺式调节 DNA 序列决定。与这一发现一致，我们最近表明，罕见的单核苷酸变异 (SNV) 对 CTCF 结合位点的破坏可以成为先天性异常患者顺式连锁DNA 甲基化变化的基础。这些数据提出了一个假设，即转录因子结合位点 (TFBS) 的罕见遗传变异可能有助于局部 DNA 甲基化模式。

在这项工作中，通过结合血液全基因组 DNA 甲基化谱、来自 247 个无关个体的全基因组测序衍生的 SNV 以及来自 ENCODE ChIP-Seq 数据的 133 个预测的 TFBS 基序，我们观察到多个结合位点的破坏之间的关联。由罕见 SNV 和极端 DNA 甲基化值引起的 TF，在局部和在较小程度上，远距离 CpG。虽然这些变化中的大部分只影响单个 CpG，但 24% 与被破坏的 TFBS 的 ±1kb 内的多个异常 CpG 相关。有趣的是，TF 基序内功能受限位点的破坏会导致附近 CpG 位点的 DNA 甲基化变化更大。总之，这些发现表明 TFBS 中罕见的 SNV 会对 TF-DNA 结合产生负面影响，这可能导致局部 DNA 甲基化谱的改变。此外，

总之，我们的研究结果不仅提供了对 TFBS 罕见遗传变异对塑造局部 DNA 甲基化的影响及其对基因组调控的影响的见解，而且还提供了结合 DNA 甲基化数据来解释罕见变异的功能作用的基本原理。