CXXC1 is not essential for normal DNA double-strand break formation and meiotic recombination in mouse. CXXC1 is not essential for normal DNA double-strand break formation and meiotic recombination in mouse

In most mammals, including mice and humans, meiotic recombination is determined by the meiosis specific histone methytransferase PRDM9, which binds to specific DNA sequences and trimethylates histone 3 at lysine-4 and lysine-36 at the adjacent nucleosomes. These actions ensure successful DNA double strand break formation and repair that occur on the proteinaceous structure forming the chromosome axis. The process of hotspot association with the axis after their activation by PRDM9 is poorly understood. Previously, we and others have identified CXXC1, an ortholog of S. cerevisiae Spp1 in mammals, as a PRDM9 interactor. In yeast, Spp1 is a histone methyl reader that links H3K4me3 sites with the recombination machinery, promoting DSB formation. Here, we investigated whether CXXC1 has a similar function in mouse meiosis. We created two Cxxc1 conditional knockout mouse to deplete CXXC1 generally in germ cells, and before the onset of meiosis. Surprisingly, male knockout mice were fertile, and the loss of CXXC1 in spermatocytes had no effect on hotspot trimethylation, double-strand break formation or repair. Our results demonstrate that CXXC1 is not an essential link between PRDM9-activated recombination hotspot sites and DSB machinery and that the hotspot recognition pathway in mouse is independent of CXXC1. Overall design: We performed ChIP-seq with antibodies against single strand DNA binding protein DMC1 in spermatocytes isolated from Cxxc1 heterozygous and Cxxc1 conditional knockout mouse to analyze meiotic DNA double strand formation level.

在包括小鼠与人类在内的多数哺乳动物中，减数分裂重组（meiotic recombination）由减数分裂特异性组蛋白甲基转移酶PRDM9（PRDM9）介导，该酶可结合特定DNA序列，并对邻近核小体（nucleosomes）上的组蛋白H3的赖氨酸4与赖氨酸36位点进行三甲基化修饰。上述作用可确保在构成染色体轴（chromosome axis）的蛋白质结构上顺利发生DNA双链断裂的形成与修复。目前，PRDM9激活重组热点（recombination hotspot）后，热点与染色体轴结合的过程仍未得到充分阐释。此前，我们与其他团队均已鉴定出CXXC1——哺乳动物中酿酒酵母（S. cerevisiae）Spp1的同源蛋白（ortholog）——为PRDM9的互作因子。在酿酒酵母中，Spp1是一种组蛋白甲基阅读蛋白（histone methyl reader），可将H3K4me3位点与重组调控机器相连，进而促进双链断裂（double-strand break, DSB）的形成。本研究旨在探究CXXC1在小鼠减数分裂中是否发挥类似功能。我们构建了两种Cxxc1条件性敲除（conditional knockout）小鼠模型，分别在生殖细胞（germ cells）整体水平以及减数分裂启动（onset of meiosis）前实现CXXC1的敲除。令人意外的是，雄性敲除小鼠仍可正常繁育，且精母细胞（spermatocytes）中CXXC1的缺失对重组热点的三甲基化修饰、双链断裂的形成与修复均无影响。本研究结果表明，CXXC1并非PRDM9激活的重组热点位点与双链断裂作用机器之间的必需连接因子，且小鼠的重组热点识别通路不依赖于CXXC1。实验设计：我们从Cxxc1杂合子（heterozygous）及Cxxc1条件性敲除小鼠中分离精母细胞，使用针对单链DNA结合蛋白（single strand DNA binding protein）DMC1的抗体进行染色质免疫共沉淀测序（ChIP-seq），以分析减数分裂过程中DNA双链断裂的形成水平。