胚胎早期发育中父源CenH3介导的着丝粒识别与重塑的动力学机制研究

Traditional theory holds the point that the spermatozoon just delivers haploid DNA to next generation, and the zygote development is determined by maternal factors. Recently, increasing evidences show that the spermatozoon not only transmits DNA, but also delivers protein factors, RNA and epigenetic signatures to the egg that may be crucial for the paternal chromatin remodeling and zygote development. For instance, the cetromere identifier, CenH3 (also named CenpA or Cid) is always retained on the sperm chromatin during spermatogenesis until it goes into egg. However, the incorporation of CenH3 to centromere seems sequence-independent.Therefore, we proposed the hypothesis that CenH3 is retained on sperm chromatin as centromere ID and is required for sperm centromere recognition, centromeric chromatin remodeling and kinetochore assembly after fertilization, which eventually guarantee the accomplishment of the first mitosis and zygotic development. To validate the hypothesis, we will use drosophila as model organism to generate some CenH3 mutants by using our gene targeting method. By combining immunology and living molecular image technology, we will explore the dynamic mechanism of paternal centromere recognition and remodeling mediated by CenH3 during embryonic development. This will make us better understand human reproduction, embryonic development, and chromatin remodeling/reprogramming and male sterility.

传统遗传学理论认为,有性生殖生物中精子主要提供父源DNA给子代，而受精卵早期发育都由母源调控。然而近来发现，附着在精子染色质上的蛋白质因子、RNA或表观遗传信息可能伴随精子一起进入受精卵，而且可能对受精卵父源染色质重建及胚胎发育成败起关键作用。着丝粒蛋白CenH3在精子包装成熟过程中并没有完全丢失，而母源CenH3对父源着丝粒DNA识别不具有序列特异性,因而我们推测：精子保留的父源CenH3可能作为着丝粒身份标识，在进入受精卵后引导父源着丝粒定位以及动粒的形成，保障受精卵第一次有丝分裂正常进行。为验证此假设，本项目将以遗传背景较为清晰的果蝇作为模式材料，运用基因打靶技术构建各种突变CenH3,通过运用免疫与活体分子定位成像技术分析父源着丝粒识别与重塑的动力学机制。这将为哺乳动物包括人类在内的生殖、胚胎发育、染色质重塑、雄性不育等提供直接的理论及实践依据。

传统的遗传学理论一直认为有性生殖中精子主要是提供一半的基因组给子代，而受精卵早期发育大都由母源调控。然而近来也发现，附着在精子染色质上的蛋白质、RNA或其它表观遗传信息可能伴随精子一起进入受精卵，且可能对受精卵父源染色质重建及胚胎发育成败起关键作用。而染色体着丝粒蛋白CenH3在精子细胞发育成熟的过程中并没有完全丢失，因而推测：精子细胞保留的CenH3可能作是一种保守的父源着丝粒身份，受精卵发育过程中起重要的作用。为了验证CenH3的生物学功能和可能的表观遗传分子机制，该项目以遗传背景较为清晰的模式生物果蝇作为研究材料，运用基因打靶技术首先构建了丝粒蛋白CenH3敲除突变体果蝇（loss-of-function），然后通过转基因建立了该基因补偿体系（gain-of-function）。通过这一突变及补偿体系，系统地建立了各种CenH3突变体果蝇。系统地分析了这些CenH3的突变是如何影响后代胚胎发育的过程，分析这些突变CenH3果蝇的存活率、雄性精子发育过程中的CenH3活体定位成像，分析了这些突变的生物学功能及可能参于的父源着丝粒识别与重塑分子机制。项目在丝腺中构建了CenH3介导的LacI/GFP-lacO定位技术，完成了在丝腺组织中CenH3介导的LacI/GFP-lacO定位图像；且开发了酵母三杂交技术分析了CenH3-Cal-CenpC三者对着丝粒定位之间的关系，并通过生化分离技术从果蝇中分离了CenH3-Cal蛋白质复合物，通过质谱技术鉴定了与CenH3相互作用所有蛋白质成份，发现一些新CenH3互作成份。这些都为更深层次研究精子CenH3介导着丝粒重塑过程的分子表观机制研究提供可能。且将为哺乳动物包括人类在内的生殖、胚胎发育、染色质重塑、雄性不育等提供直接的理论及实践依据。