Abstract：
Genomic integrity is critical for sexual reproduction, ensuring correct transmission of parental genetic information to the descendant. To preserve genomic integrity, germ cells have evolved multiple DNA repair mechanisms, together termed as DNA damage response. The RNA N6-methyladenosine is the most abundant mRNA modification in eukaryotic cells, which plays important roles in DNA damage response, and YTH N6-methyladenosine RNA binding protein 2 (YTHDF2) is a well-acknowledged N6-methyladenosine reader protein regulating the mRNA decay and stress response. Despite this, the correlation between YTHDF2 and DNA damage response in germ cells, if any, remains enigmatic. Here, by employing a Ythdf2-conditional knockout mouse model as well as a Ythdf2-null GC-1 mouse spermatogonial cell line, we explored the role and the underlying mechanism for YTHDF2 in spermatogonial DNA damage response. We identified that, despite no evident testicular morphological abnormalities under the normal circumstance, conditional mutation of Ythdf2 in adult male mice sensitized germ cells, including spermatogonia, to etoposide-induced DNA damage. Consistently, Ythdf2-KO GC-1 cells displayed increased sensitivity and apoptosis in response to DNA damage, accompanied by the decreased SET domain bifurcated 1 (SETDB1, a histone methyltransferase) and H3K9me3 levels. The Setdb1 knockdown in GC-1 cells generated a similar phenotype, but its overexpression in Ythdf2-null GC-1 cells alleviated the sensitivity and apoptosis in response to DNA damage. Taken together, these results demonstrate that the N6-methyladenosine reader YTHDF2 promotes DNA damage repair by positively regulating the histone methyltransferase SETDB1 in spermatogonia, which provides novel insights into the mechanisms underlying spermatogonial genome integrity maintenance and therefore contributes to safe reproduction.
摘要：
基因组完整性对有性生殖至关重要,确保父母遗传信息正确传递给后代。为了保持基因组的完整性，生殖细胞已经进化出多种DNA修复机制,一起称为DNA损伤反应。RNAN6-甲基腺苷（m6A）是真核细胞中最丰富的mRNA修饰，在DNA损伤反应中起重要作用。YTHDF2是公认的调节mRNA衰变和应激反应的m6A阅读蛋白。尽管如此,YTHDF2与生殖细胞DNA损伤反应的相关性,如果有的话,仍然是个谜.这里,通过使用Ythdf2条件敲除（cKO）小鼠模型以及Ythdf2空GC-1小鼠精原细胞系，我们探讨了YTHDF2在精原DNA损伤反应中的作用和潜在机制。我们发现，尽管在正常情况下没有明显的睾丸形态异常，Ythdf2在成年雄性小鼠致敏生殖细胞中的条件突变，包括精原细胞,依托泊苷诱导的DNA损伤。始终如一，Ythdf2-KOGC-1细胞对DNA损伤的敏感性和凋亡增加,伴随着SETDB1（组蛋白甲基转移酶）和H3K9me3水平的降低。GC-1细胞中的Settdb1敲低产生了相似的表型，但是其在Ythdf2-nullGC-1细胞中的过表达减轻了对DNA损伤的敏感性和凋亡。一起来看,这些结果表明，m6A阅读器YTHDF2通过正向调节精原细胞中的组蛋白甲基转移酶SETDB1促进DNA损伤修复，这提供了对精原基因组完整性维持的潜在机制的新见解，因此有助于安全繁殖。
