Paternal poly(ADP-ribose) metabolism modulates retention of inheritable sperm histones and early embryonic gene expression [PJ34_sperm_MNaseb]. Mus musculus

To achieve the extreme nuclear condensation necessary for sperm function, most histones are replaced with protamines during spermiogenesis in mammals. Mature sperm retain only a small fraction of nucleosomes, which are, in part, enriched on gene regulatory sequences, and recent findings suggest that these retained histones provide epigenetic information that regulates expression of a subset of genes involved in embryo development after fertilization. We addressed this tantalizing hypothesis by analyzing two mouse models exhibiting abnormal histone positioning in mature sperm due to impaired poly(ADP-ribose) (PAR) metabolism during spermiogenesis and identified altered sperm histone retention in specific gene loci genome-wide using MNase digestion-based enrichment of mononucleosomal DNA. We then set out to determine the extent to which expression of these genes was altered in embryos generated with these sperm. For control sperm, most genes showed some degree of histone association, unexpectedly suggesting that histone retention in sperm genes is not an all-or-none phenomenon and that a small number of histones may remain associated with genes throughout the genome. The amount of retained histones, however, was altered in many loci when PAR metabolism was impaired. To ascertain whether sperm histone association and embryonic gene expression are linked, the transcriptome of individual 2-cell embryos derived from such sperm was determined using microarrays and RNA sequencing. Strikingly, a moderate but statistically significant portion of the genes that were differentially expressed in these embryos also showed different histone retention in the corresponding gene loci in sperm of their fathers. These findings provide new evidence for the existence of a linkage between sperm histone retention and gene expression in the embryo. Overall design: Mnase sensitivity of sperm DNA, indicating nucleosomal, not protamine, packaging was altered in mice by manipulating poly(ADP-ribose) metabolism in adult males using a specific PARP inhibitor for 6 weeks. Abnormal sperm nucleosomal organization of males analyzed by these tiling arrays was compared with differential gene expression in 2 cell embryos fathered by these males analyzed by separate gene expression arrays and RNA sequencing.

为实现精子功能所必需的极致核浓缩，哺乳动物精子发生（spermiogenesis）过程中，绝大多数组蛋白（histones）会被精蛋白（protamines）替换。成熟精子仅保留少量核小体（nucleosomes），此类核小体部分富集于基因调控序列区域；近期研究表明，这些留存的组蛋白可提供表观遗传（epigenetic）信息，调控受精后胚胎发育相关的部分基因表达。

我们针对这一引人入胜的假说展开研究：通过分析两种因精子发生过程中多聚ADP核糖（poly(ADP-ribose), PAR）代谢受损，导致成熟精子组蛋白定位异常的小鼠模型，利用基于微球菌核酸酶（MNase）消化的单核小体DNA富集技术，在全基因组范围内鉴定出特定基因位点的精子组蛋白留存情况发生改变的区域。

随后我们进一步探究利用这些异常精子产生的胚胎中，此类基因的表达改变程度。针对对照精子，多数基因均表现出不同程度的组蛋白结合，这一结果意外表明，精子基因中的组蛋白留存并非全或无现象，基因组中所有基因可能均会保留少量组蛋白。然而，当PAR代谢受损时，众多基因位点的留存组蛋白数量发生显著改变。

为明确精子组蛋白结合与胚胎基因表达之间是否存在关联，我们利用基因芯片（microarrays）与RNA测序（RNA sequencing）技术，对源自上述异常精子的单个2细胞胚胎的转录组进行了检测。值得注意的是，在这些胚胎中出现差异表达的基因中，有比例适中且具有统计学显著性的一部分，其在父代精子的对应基因位点上的组蛋白留存情况也存在差异。

上述研究结果为精子组蛋白留存与胚胎基因表达之间存在关联这一观点提供了新的实验证据。

整体实验设计：通过向成年雄性小鼠施加特异性多聚ADP核糖聚合酶（PARP）抑制剂6周，干预其精子发生过程中的PAR代谢，进而改变精子DNA的微球菌核酸酶敏感性——该敏感性可反映核小体而非精蛋白的包装状态。我们通过平铺式基因芯片阵列（tiling arrays）技术分析了雄性小鼠异常的精子核小体组织，并将其与通过独立基因芯片及RNA测序分析得到的、由这些雄性小鼠繁育的2细胞胚胎的差异基因表达情况进行了对比。