Molecular features driving cellular and regulatory complexity of human brain evolution [ATAC-seq]

Genomic changes acquired in human evolution contribute to the unique abilities of human brain. However, characterizing the molecular underpinnings of human-specific traits is a multifaceted challenge due to the cellular heterogeneity of human brain and complex regulation of gene expression. Here, we performed single-nuclei RNA-sequencing (snRNA-seq) and single-nuclei ATAC-seq (snATAC-seq) in human, chimpanzee, and rhesus macaque brain tissue (brodmann area 23, posterior cingulate cortex). Human-specific changes were distinct among neuronal subtypes indicating that human brain evolution was accompanied by molecular alterations in finer cellular resolution. We also observed more human-specific alterations in epigenome compared to transcriptome. Interestingly, human-specific accessibility changes in neurons were not as concordant with gene expression changes in comparison to other species, partially explaining this discrepancy. These cis-regulatory elements were enriched for immediate early gene motifs, identifying accelerated evolution of activity regulated genes in humans. We also uncovered associations between human evolution and brain disease genes at the cell type level. Together, these results reveal multiple mechanisms for human brain evolution at cell type resolution and establish the first direct evidence for accelerated human-specificity of activity-dependent molecular changes. Brodmann Area 23 was dissected from human, chimpanzee and rhesus macaque (Macaca Mulatta). Each species contained 4 adult, post-mortem samples. Each sample was obtained from a different individual. Droplet-based single-nuclei ATAC-seq libraries were prepared using the Chromium Single Cell ATAC Library kit (1000110, 10x Genomics) according to the manufacturer’s protocols. Libraries were sequenced using an Illumina NovaSeq 6000.

人类演化过程中获得的基因组改变，赋予了人类大脑独有的功能特性。然而，由于人类大脑存在细胞异质性且基因表达调控机制复杂，解析人类特有性状的分子基础是一项多维度挑战。本研究对人类、黑猩猩以及恒河猴的大脑组织（布罗德曼23区，后扣带回皮层）开展了单细胞核RNA测序（single-nuclei RNA-sequencing, snRNA-seq）与单细胞核ATAC测序（single-nuclei ATAC-seq, snATAC-seq）。人类特有改变在神经元亚型间呈现显著差异，表明人类大脑演化伴随着更精细细胞分辨率下的分子层面改变。相较于转录组，我们在表观基因组中观测到更多人类特有的改变。有趣的是，与其他物种相比，人类神经元中特有的染色质开放区域改变与基因表达改变的一致性较低，这在一定程度上解释了该差异。此类顺式调控元件富集于即刻早期基因基序，揭示了人类中活动调控基因的演化加速现象。我们还在细胞类型层面发现了人类演化与脑部疾病基因之间的关联。综上，本研究在细胞类型分辨率下揭示了人类大脑演化的多种机制，并首次为活动依赖型分子改变的人类特异性加速演化提供了直接证据。本研究从人类、黑猩猩以及恒河猴（Macaca Mulatta）中分离获取布罗德曼23区组织；每个物种均包含4例成人死后样本，且每例样本均来自不同个体。基于微滴的单细胞核ATAC测序文库构建参照制造商操作流程，使用Chromium单细胞ATAC文库试剂盒（1000110，10x Genomics）完成；文库使用Illumina NovaSeq 6000测序平台进行测序。