The PIWI-interacting RNA (piRNA) pathway guides the DNA methylation of young, active transposons during germline development in male mice¹. piRNAs tether the PIWI protein MIWI2 (PIWIL4) to the nascent transposon transcript, resulting in DNA methylation through SPOCD1 (refs. ^2,3,4,5). Transposon methylation requires great precision: every copy needs to be methylated but off-target methylation must be avoided. However, the underlying mechanisms that ensure this precision remain unknown. Here, we show that SPOCD1 interacts directly with SPIN1 (SPINDLIN1), a chromatin reader that primarily binds to H3K4me3-K9me3 (ref. ⁶). The prevailing assumption is that all the molecular events required for piRNA-directed DNA methylation occur after the engagement of MIWI2. We find that SPIN1 expression precedes that of both SPOCD1 and MIWI2. Furthermore, we demonstrate that young LINE1 copies, but not old ones, are marked by H3K4me3, H3K9me3 and SPIN1 before the initiation of piRNA-directed DNA methylation. We generated a Spocd1 separation-of-function allele in the mouse that encodes a SPOCD1 variant that no longer interacts with SPIN1. We found that the interaction between SPOCD1 and SPIN1 is essential for spermatogenesis and piRNA-directed DNA methylation of young LINE1 elements. We propose that piRNA-directed LINE1 DNA methylation requires a developmentally timed two-factor authentication process. The first authentication is the recruitment of SPIN1–SPOCD1 to the young LINE1 promoter, and the second is MIWI2 engagement with the nascent transcript. In summary, independent authentication events underpin the precision of piRNA-directed LINE1 DNA methylation.

PIWI 相互作用 RNA （piRNA） 通路指导雄性小鼠种系发育过程中年轻、活跃转座子的 DNA 甲基化¹。piRNA 将 PIWI 蛋白 MIWI2 （PIWIL4） 拴在新生的转座子转录物上，导致 DNA 通过 SPOCD1 甲基化（参考文献。^转座子甲基化需要非常精确：每个拷贝都需要甲基化，但必须避免脱靶甲基化。但是，确保这种精度的底层机制仍然未知。在这里，我们表明 SPOCD1 直接与 SPIN1 （SPINDLIN1） 相互作用，SPIN1 是一种主要与 H3K4me3-K9me3 结合的染色质读取器（参考文献 ⁶）。普遍的假设是 piRNA 导向的 DNA 甲基化所需的所有分子事件都发生在 MIWI2 接合之后。我们发现 SPIN1 的表达先于 SPOCD1 和 MIWI2 的表达。此外，我们证明在 piRNA 定向 DNA 甲基化开始之前，年轻的 LINE1 拷贝而不是旧的 LINE1 拷贝被 H3K4me3、H3K9me3 和 SPIN1 标记。我们在小鼠中生成了一个 Spocd1 功能分离等位基因，该等位基因编码不再与 SPIN1 相互作用的 SPOCD1 变体。我们发现 SPOCD1 和 SPIN1 之间的相互作用对于年轻 LINE1 元件的精子发生和 piRNA 导向的 DNA 甲基化至关重要。我们提出 piRNA 导向的 LINE1 DNA 甲基化需要一个发育定时的双因素认证过程。第一个身份验证是将 SPIN1-SPOCD1 募集到年轻的 LINE1 启动子中，第二个是 MIWI2 与新生转录本的参与。总之，独立的鉴定事件支撑着 piRNA 导向的 LINE1 DNA 甲基化的精确性。