The John Curtin School of Medical Research, The Australian National University, Canberra, ACT, 2601, Australia.
Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, 4072, Australia.
Genome Biol. 2019 Jan 31;20(1):23. doi: 10.1186/s13059-019-1633-3.
Altering the biochemical makeup of chromatin by the incorporation of histone variants during development represents a key mechanism in regulating gene expression. The histone variant H2A.B, H2A.B.3 in mice, appeared late in evolution and is most highly expressed in the testis. In the mouse, it is encoded by three different genes. H2A.B expression is spatially and temporally regulated during spermatogenesis being most highly expressed in the haploid round spermatid stage. Active genes gain H2A.B where it directly interacts with polymerase II and RNA processing factors within splicing speckles. However, the importance of H2A.B for gene expression and fertility are unknown.
Here, we report the first mouse knockout of this histone variant and its effects on fertility, nuclear organization, and gene expression. In view of the controversy related to the generation of off-target mutations by gene editing approaches, we test the specificity of TALENs by disrupting the H2A.B multi-copy gene family using only one pair of TALENs. We show that TALENs do display a high level of specificity since no off-target mutations are detected by bioinformatics analyses of exome sequences obtained from three consecutive generations of knockout mice and by Sanger DNA sequencing. Male H2A.B.3 knockout mice are subfertile and display an increase in the proportion of abnormal sperm and clogged seminiferous tubules. Significantly, a loss of proper RNA Pol II targeting to distinct transcription-splicing territories and changes to pre-mRNA splicing are observed.
We have produced the first H2A.B knockout mouse using the TALEN approach.
在发育过程中通过组蛋白变体的掺入来改变染色质的生化组成是调节基因表达的关键机制。组蛋白变体 H2A.B、小鼠中的 H2A.B.3 在进化后期出现，在睾丸中表达水平最高。在小鼠中，它由三个不同的基因编码。H2A.B 在精子发生过程中的时空表达受到调节，在单倍体圆形精子阶段表达水平最高。活性基因获得 H2A.B，它直接与聚合酶 II 和剪接斑点内的 RNA 加工因子相互作用。然而，H2A.B 对于基因表达和生育力的重要性尚不清楚。
在这里，我们报告了该组蛋白变体的第一个小鼠敲除及其对生育力、核组织和基因表达的影响。鉴于基因编辑方法产生脱靶突变的争议，我们通过仅使用一对 TALEN 来破坏 H2A.B 多拷贝基因家族来测试 TALEN 的特异性。我们表明 TALEN 确实显示出高度的特异性，因为通过对来自连续三代敲除小鼠的外显子序列进行生物信息学分析以及 Sanger DNA 测序，没有检测到脱靶突变。雄性 H2A.B.3 敲除小鼠的繁殖力降低，并显示出精子异常比例增加和曲细精管堵塞。重要的是，观察到 RNA Pol II 向不同转录剪接区域的适当靶向丧失以及前体 mRNA 剪接的变化。
我们使用 TALEN 方法产生了第一个 H2A.B 敲除小鼠。
