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 甲基化是最明确的表观遗传标记，但其靶向基因组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 结合因子发挥作用的顺式调控 DNA 序列决定。与这一发现一致的是，我们最近表明，罕见单核苷酸变异（SNV）对 CTCF 结合位点的破坏可能是先天性异常患者顺式连接 DNA 甲基化变化的基础。这些数据提出了这样的假设：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 以及源自 ENCODE ChIP-Seq 数据的 133 个预测 TFBS 基序，我们观察到多个结合位点破坏之间的关联。由稀有 SNV 和局部 CpG 处的极端 DNA 甲基化值（在较小程度上）引起的 TF。虽然这些变化中的大多数仅影响单个 CpG，但 24% 的变化与破坏的 TFBS ±1kb 范围内的多个异常 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 位点发生更大的 DNA 甲基化变化。总而言之，这些发现表明 TFBS 上的罕见 SNV 对 TF-DNA 结合产生负面影响，这可能导致局部 DNA 甲基化谱发生改变。此外，随后整合心脏组织的 DNA 甲基化和 RNA-Seq 图谱，使我们能够观察到罕见的 SNV 指导的 DNA 甲基化与附近基因的异常表达之间的关联。

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