The effects of DNASE1L3 or DNASE1 deficiency on cell-free DNA (cfDNA) methylation were explored in plasma of mice deficient in these nucleases and in DNASE1L3-deficient humans. Compared to wild-type cfDNA, cfDNA in DNASE1L3-deficient mice was significantly hypomethylated, while cfDNA in DNASE1-deficient mice was hypermethylated. The cfDNA hypomethylation in DNASE1L3-deficient mice was due to increased fragmentation and representation from open chromatin regions (OCRs) and CpG islands (CGIs). These findings were absent in DNASE1-deficient mice, demonstrating the preference of DNASE1 to cleave in hypomethylated OCRs and CGIs. We also observed a substantial decrease of fragment ends at methylated CpGs in the absence of DNASE1L3, thereby demonstrating that DNASE1L3 prefers to cleave at methylated CpGs. Furthermore, we found that methylation levels of cfDNA varied by fragment size in a periodic pattern, with cfDNA of specific sizes being more hypomethylated and enriched for OCRs and CGIs. These findings were confirmed in DNASE1L3-deficient human cfDNA. Thus, we have found that nuclease-mediated cfDNA fragmentation markedly affects cfDNA methylation level on a genome-wide scale. This work provides a foundational understanding of the relationship between methylation, nuclease biology, and cfDNA fragmentation.

中文翻译：

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核酸酶缺陷改变浆细胞游离 DNA 甲基化谱

在这些核酸酶缺陷的小鼠和 DNASE1L3 缺陷人类的血浆中探索了 DNASE1L3 或 DNASE1 缺陷对无细胞 DNA (cfDNA) 甲基化的影响。与野生型 cfDNA 相比，DNASE1L3 缺陷小鼠中的 cfDNA 显着低甲基化，而 DNASE1 缺陷小鼠中的 cfDNA 高甲基化。DNASE1L3 缺陷小鼠的 cfDNA 低甲基化是由于开放染色质区域 (OCR) 和 CpG 岛 (CGI) 的碎片化和代表性增加。这些发现在 DNASE1 缺陷小鼠中不存在，表明 DNASE1 倾向于在低甲基化 OCR 和 CGI​​ 中切割。我们还观察到在没有 DNASE1L3 的情况下甲基化 CpG 处的片段末端显着减少，从而证明 DNASE1L3 更喜欢在甲基化 CpG 处切割。此外，我们发现 cfDNA 的甲基化水平随片段大小呈周期性变化，特定大小的 cfDNA 甲基化程度更高，并且富含 OCR 和 CGI​​。这些发现在 DNASE1L3 缺陷型人类 cfDNA 中得到证实。因此，我们发现核酸酶介导的 cfDNA 片段化显着影响全基因组范围内的 cfDNA 甲基化水平。这项工作提供了对甲基化、核酸酶生物学和 cfDNA 片段化之间关系的基本理解。