Table_1_Evolution and Comprehensive Analysis of DNaseI Hypersensitive Sites in Regulatory Regions of Primate Brain-Related Genes.XLSX

How the human brain differs from those of non-human primates is largely unknown and the complex drivers underlying such differences at the genomic level remain unclear. In this study, we selected 243 brain-related genes, based on Gene Ontology, and identified 184,113 DNaseI hypersensitive sites (DHSs) within their regulatory regions. To performed comprehensive evolutionary analyses, we set strict filtering criteria for alignment quality and filtered 39,132 DHSs for inclusion in the investigation and found that 2,397 (~6%) exhibited evidence of accelerated evolution (aceDHSs), which was a much higher proportion that DHSs genome-wide. Target genes predicted to be regulated by brain-aceDHSs were functionally enriched for brain development and exhibited differential expression between human and chimpanzee. Alignments indicated 61 potential human-specific transcription factor binding sites in brain-aceDHSs, including for CTCF, FOXH1, and FOXQ1. Furthermore, based on GWAS, Hi-C, and eQTL data, 16 GWAS SNPs, and 82 eQTL SNPs were in brain-aceDHSs that regulate genes related to brain development or disease. Among these brain-aceDHSs, we confirmed that one enhanced the expression of GPR133, using CRISPR-Cas9 and western blotting. The GPR133 gene is associated with glioblastoma, indicating that SNPs within DHSs could be related to brain disorders. These findings suggest that brain-related gene regulatory regions are under adaptive evolution and contribute to the differential expression profiles among primates, providing new insights into the genetic basis of brain phenotypes or disorders between humans and other primates.

人类大脑与非人灵长类大脑的差异在很大程度上仍未被阐明，而这类差异在基因组层面的复杂驱动机制至今尚不明确。本研究基于基因本体论（Gene Ontology）筛选得到243个大脑相关基因，并在其调控区域内鉴定出184113个DNaseI超敏感位点（DNaseI hypersensitive sites, DHSs）。为开展全面的进化分析，我们制定了严格的比对质量过滤标准，最终筛选出39132个DHSs用于本研究，其中2397个（约6%）被证实存在加速进化特征，这类位点被称为加速进化DHSs（accelerated evolution DHSs，简称aceDHSs），其占比远高于全基因组范围内DHSs的整体比例。经预测，受大脑aceDHSs调控的靶基因在大脑发育功能上显著富集，且在人类与黑猩猩之间存在表达差异。比对分析显示，大脑aceDHSs中存在61个潜在的人类特异性转录因子结合位点，涵盖CTCF、FOXH1以及FOXQ1等转录因子。此外，基于全基因组关联研究（Genome-Wide Association Study, GWAS）、染色体构象捕获技术（Hi-C）以及表达数量性状基因座（expression quantitative trait locus, eQTL）数据，我们发现有16个GWAS单核苷酸多态性（single nucleotide polymorphism, SNPs）以及82个eQTL SNPs位于调控大脑发育或疾病相关基因的大脑aceDHSs区域内。在这些大脑aceDHSs中，我们通过CRISPR-Cas9技术与蛋白质免疫印迹（western blotting）实验验证了其中一个位点可增强GPR133基因的表达。GPR133基因与胶质母细胞瘤相关，这表明DHSs区域内的SNPs可能与脑部疾病存在关联。本研究结果表明，大脑相关基因的调控区域处于适应性进化过程中，并对灵长类动物间的表达差异谱具有贡献，为理解人类与其他灵长类动物在大脑表型或脑部疾病相关的遗传基础提供了全新视角。