All sequences included in MPRA library.

Interbreeding between anatomically modern humans and archaic hominins has contributed to the genomes of present-day human populations. However, our understanding of the specific gene regulatory consequences of Neanderthal, and particularly, Denisovan introgression is limited. Here, we used a massively parallel reporter assay to investigate the regulatory effects of 25,869 high-confidence introgressed SNPs segregating in present-day individuals of Papuan genetic ancestry in immune cell types. Overall, 8.22% of Denisovan and 8.58% of Neanderthal sequences showed active regulatory activity, and 9.22% of these displayed differential activity between archaic and modern alleles. We found no association between introgressed allele frequency on activity regardless of introgression source, but introgressed Denisovan alleles at higher frequencies were less likely to be differentially active than expected, suggesting introgression is under some degree of selective constraint. Both activity and differentially activity were associated with distance to the nearest transcription start site, while differential activity was additionally associated with differential transcription factor binding. Genes predicted to be regulated by differentially active sequences included IFIH1 and TNFAIP3, key immune genes and known examples of archaic introgression. Overall, this work provides experimental validation of regulatory activity for thousands of archaic variants in populations with the highest levels of Denisovan ancestry worldwide, revealing how human evolutionary history actively shapes present-day genetic diversity and immune function.解剖学现代人类（anatomically modern humans）与古老人属（archaic hominins）之间的基因渗入（introgression），已对当代人类群体的基因组产生了遗传贡献。然而，我们对于尼安德特人（Neanderthal），尤其是丹尼索瓦人（Denisovan）的基因渗入所引发的特定基因调控效应的认知仍较为有限。本研究采用大规模平行报告基因检测（massively parallel reporter assay），针对免疫细胞类型中、巴布亚遗传血统当代个体内呈现多态性的25869个高可信度渗入型单核苷酸多态性（single nucleotide polymorphism, SNP）位点，探究其调控效应。整体而言，8.22%的丹尼索瓦人序列与8.58%的尼安德特人序列展现出活跃的调控活性，其中9.22%的序列在古人类等位基因与现代等位基因间呈现出活性差异。我们未发现无论基因渗入来源如何，渗入等位基因频率与调控活性之间存在关联；但高频的渗入型丹尼索瓦人等位基因出现活性差异的概率低于预期，这提示基因渗入过程受到了一定程度的选择约束。调控活性与活性差异均与最近的转录起始位点（transcription start site）的距离相关，而活性差异还与差异转录因子结合存在关联。被预测受活性差异序列调控的基因包括IFIH1与TNFAIP3，二者均为关键免疫基因，同时也是已知的古人类基因渗入案例。总体而言，本研究为全球丹尼索瓦人血统占比最高的群体中的数千个古人类变异位点的调控活性提供了实验验证，揭示了人类进化历史如何主动塑造当代遗传多样性与免疫功能。