绵羊Callipyge沉默子元件的互作蛋白鉴定

The Callipyge Phenotype is a hereditary muscular hypertrophy of sheep, demonstrating a unique mode of inheritance referred to as “ploar overdominance”. Its causative mutation was charactrized to be located to an 12 bp silencer within the intergenic region of DLK1-GTL2. However, the proteins interacting with the silencer has not been identified yet. In this study, enCHIP techinology combined with mass spectrometry are intend to be used for “fishing” the DNA binding proteins interacting with the silencer in sheep satellite cells. The binding ability of the screened DNA binding proteins through mass spectrometry and probe against with Callipyge allele is to be assessed through EMSA experiments. Morever, influence of the screened proteins on genes around the DLK1-GTL2 imprinted region was validated by overexpression and interference experiments. The validated binding proteins through in vitro experiments was to be used for generation of skeletal muscle conditional knockout transgenic mice so as to verify the binding ability in vivo and check the influence on muscular phenotype. The implementation of this project will be expected to identify DNA binding proteins of Callipyge silencer, and it has great significance on perfection of “polar overdominance” molecular hypothesis and reveal of molecular mechalism of sheep muscle growth. Hence, this project has very important academic value.

Callipyge表型是绵羊的一种遗传性肌肉肥大，呈现“极性超显性”遗传模式，其致因突变位于DLK1-GTL2基因间一个12bp沉默子元件上，但是该沉默子元件的互作蛋白一直未被鉴定。本项目拟通过enChIP技术联合质谱测序在绵羊骨骼肌卫星细胞中钓取与该沉默子元件互作的DNA结合蛋白；对质谱筛选到的DNA结合蛋白，通过EMSA体外验证其与Callipyge两种等位基因探针的结合能力，并通过超表达与干扰实验验证其对DLK1-GTL2印记区基因表达的影响；对被验证的互作蛋白，构建其骨骼肌条件性敲除小鼠模型，体内验证其与Callipyge沉默子的互作及对DLK1-GTL2区域基因表达及肌肉表型的影响。本项目的实施将有望鉴定到与Callipyge沉默子元件互作的DNA结合蛋白，对完善“极性超显性”分子假说具有重要意义，也有利于深度揭示绵羊肌肉生长的分子机制，因此本项目具有十分重要的学术价值。

Callipyge表型是绵羊的一种遗传性肌肉肥大，呈现“极性超显性”遗传模式。围绕Callipyge表型形成的分子机制，有两个方面的问题需要回答：1)Callipyge顺式调控元件是怎样发挥功能的？对此，我们利用CRISPR/Cas9技术成功构建了Callipyge元件缺失的C2C12细胞系，为顺式调控的机制解析奠定了基础。2)Callipyge突变的靶基因PEG11在肌肉肥大中发挥怎样的功能？因为体内野生型PEG11受到多个miRNA抑制而无法高效表达，对此，我们构建了miRNA结合位点突变的PEG11条件性超表达转基因小鼠。利用该小鼠，我们在三种Cre模型中开展了研究：1）体外肌肉注射AAV9-Cre腺病毒，PEG11可以被成功诱导表达，而且在注射病毒后1周、3周和7周表现出典型的肌肉肥大表型，初步复制了绵羊Callipyge表型；2）与MCK-Cre小鼠杂交，PEG11与Cre双阳性小鼠表现出心肌肥大表型，小鼠在出生后9天左右死亡；3）与Myf5-Cre小鼠杂交，PEG11与Cre双阳性小鼠表现出肺脏无法扩张，出生后呼吸困难，推测膈肌发育异常。因此，PEG11条件性超表达转基因小鼠初步确认了PEG11是造成绵羊遗传性肌肉肥大的靶基因。本项目研究结果为下一步深入研究Callipyge表型形成的分子机制提供了良好的细胞和小鼠模型，对完善“极性超显性”分子假说具有重要意义，也有望深度揭示绵羊肌肉生长的分子机制。