中国科学院昆明动物研究所机构知识库

人类区别于非人灵长类最主要的特征就是扩增的大脑容量和高度发达的认知能力。研究者们将可能影响这一人类显著变化的遗传基础总结为四个方面，即基因编码区的变化，基因调控区的变化，基因复制和基因丢失。对人和非人灵长类基因组的比较发现，两者基因组序列上的平均差异并不如想象中的那么显著。因此，除了蛋白序列变化之外，基因的调控和基因复制产生的新功能基因可能对人类大脑的进化也发挥着重要作用。为了解析由基因复制而产生的新功能基因对人类大脑进化的作用，我们采用慢病毒感染技术成功构建了人类特异的基因复制片段srGAP2C转基因食蟹猴模型。我们共获得四只存活的阳性食蟹猴以及三只胚胎期80天阳性食蟹猴样本。我们采用转录组学、影像学、细胞形态学和免疫组织化学等手段对srGAP2C转基因食蟹猴进行了初步分析，探讨人类特异的基因复制片段对人类大脑发育和功能的影响及其对人类特异大脑表型的贡献。同时，为了揭秘因蛋白质序列变化而引起的人类特异性表型如语言等的出现，我们尝试利用CRISPR/Cas9技术对猕猴基因组进行精确编辑，在猕猴Foxp2基因序列上引入两个人类特异的突变(T303N和N325S)，以期构建Foxp2人源化的基因位点替代猕猴模型。我们检测了不同实验条件下CRISPR/Cas9对猕猴胚胎基因位点替换效率的影响度。实验结果显示仅仅只抑制非同源末端连接修复并不能有效提高同源重组修复的效率，为进一步改进利用CRISP/Cas9进行基因组的精确编辑提供了有价值的基础数据。为了探究DNA甲基化在人类大脑进化中的作用，我们采用亚硫酸氢盐测序法，重点检测了人类(Homo sapiens)、黑猩猩(Pan troglodytes)、长臂猿(Hylobatidae)和猕猴(Macaca malatta)大脑前额叶区小头症基因MCPH1、ASPM、CDK5RAP2、CENPJ启动子区DNA的甲基化模式。我们发现了CENPJ在人类大脑中特异的低甲基化模式(p<0.01)；体外实验证明，CENPJ启动子区DNA CpG的甲基化确实影响了基因表达，这与CENPJ在人与非人灵长类成体和胚胎大脑发育中较大的表达差异相符合。研究结果提示，人类特异的表观遗传学变化在人类大脑容量扩增和高级认知能力起源中具有重要作用。

The most distinctive characteristics that set humans apart from other primates are the enlarged brain and concurrent highly developed cognitive competence. Reachers summarized the genetic alterations leading to human dramatic changes are gene coding-region changes, gene regulatory-region changes, gene duplication and gene loss. The comparative genomics analysis shows that the mean difference between human and non-human primates are not remarkable. Therefor, despit the protein sequence changes, gene regulatory changes and neofunctional genes via gene duplication may play essential roles in human brain evolution.To better understand the roles of neofunctional genes in human brain evolution, we contructed the transgenic monkey model of gene srGAP2C which is a human-specific gene duplication fragment (GDF) through lentiviral infection. We have four alive positive cynomolgus monkeys and three positive embryonic cynomolgus monkey samples aged 80 gestation-days. We have intergrated various methodologies like transcriptome analysis, imageology, cytomorphology and immunohistochemistry to explore the roles of human specific GDF in the formation and function process of human specific brain traits. Meanwhile, we ultilized CRISPR/Cas9 to introduce two human specific mutations (T303N and N325S) into the macaque DNA sequence of Foxp2 precisely thus constructing the Foxp2 humanized replacement monkey models in order to uncover the mechanism underlie the alterations of protein sequence leading to the human specific trait such as the emergence of language. We have tested the efficiency of macaque embryo genomic loci replacement with different CRISPR/Cas9 conditions. The results exhibite a whorthy basic data to adapt the CRIPR/Cas9 to precise genome editing by showing that inhibiting the non-homologous end joining (NHEJ) only could not enhance the homologous direct repair (HDR) effectively. To investigate the role of DNA methylation during human brain evolution, we compared the methylation patterns of four brain size regulatory genes including ASPM, CDK5RAP2, CENPJ, and MCPH1 across the prefrontal cortex of human(Homo sapiens), chimpanzee(Pan troglodytes), gibbon(Hylobatidae) and macaque(Macaca malatta). As a result, a significant hypomethylation(p-value<0.01) in the promoter region of gene CENPJ was specifically found in the human brain. Further in vitro methylation assays exhibited that the methylation status of the CENPJ promoter have an impact on its gene expression consistent with the significant difference in CENPJ expression in the human and nonhuman primate brains throughout the major stages of fetal and adult brains. The hypomethylation and high expression of gene CENPJ in the human central nervous system hinted us that a human-specific and likely-heritable-epigenetic modification may contribute to the dramatically enlarged brain and the highly developed cognitive competence.