Modern humans are known to have interbred with Neanderthals [1], Denisovans [2] and possibly other archaic hominins [3]. While genetically similar populations of Neanderthals are thought to have contributed approximately 2% to non-African genomes, Denisovan introgression has been observed to be more variable [4]. Particularly, Denisovan ancestry accounts for up to 5% of the genomes of the Indigenous peoples of Island Southeast Asia and Australia [4, 5]. In addition, these components exhibit a deep divergence from the reference Altai Denisovan genome, providing strong evidence for the occurrence of multiple Denisovan introgression events across time and space [6, 7].

At the genomic level these introgressed archaic alleles are mostly observed outside protein-coding sequences, distributed over non-functional and regulatory regions [8, 9]. Enhancers, in particular, are amongst the top targeted elements of archaic hominin introgression [10, 11]. Here, archaic alleles are thought to drive phenotypic differences by altering gene pre- and post-transcriptional regulatory processes [11]. Furthermore, both Neanderthal and Denisovan variants seem to preferentially affect enhancers in a tissue-specific manner, with highly pleiotropic elements being depleted of archaic variation [12]. However, beyond general agreement that introgressed archaic DNA has mainly been deleterious and actively removed from coding sequences and conserved non-coding elements [8, 12–14], the actual phenotypic consequences of this variation are not well understood. Several lines of evidence highlight associations between archaic DNA and risk for disease traits, including autoimmune diseases [8, 15, 16], or with traits of possible evolutionary advantage for early non-Africans [17–20]. For example, Neanderthal variants within immune genes and immune-related cis-regulatory elements (CREs) have been associated with differential responses to viral infections among present-day Europeans [21, 22].

Unfortunately, two main factors limit our interpretation of the biology of these alleles. First, we still lack a detailed characterisation of global levels of human genetic diversity, vital to identifying differential archaic hominin contributions across modern populations [23, 24]. Because Denisovan DNA is not present in the genomes of modern Europeans, which make up the vast diversity of biomedical genetics cohorts, it has been impossible to link them to phenotype, unlike segregating Neanderthal variants [16, 25]. Second, and more challenging, most of these alleles lie within non-coding sequences where, despite their acknowledged contributions to human evolutionary history [26, 27], an understanding of their actual biological functions remains elusive.

To gain insights into the consequences of archaic introgression in Papuans, we have analysed a previously published dataset of 56 present-day genomes sampled across the island of New Guinea [6]. By comparing the distribution of archaic single nucleotide polymorphisms (aSNPs) and non-archaic SNPs (naSNPs) segregating within these populations, across multiple genomic elements and cell types, we find that aSNPs are enriched within functional cis-regulatory elements (CREs) and transcribed regions, particularly those active within immune-related cells. We also observe that the presence of archaic alleles within these elements can lead to substantial disruption of transcription factor binding sites (TFBSs), with a consistent Denisovan-specific signal affecting immune-related processes. We finally validate these results through experimental testing of multiple Denisovan aSNPs, and find they are associated with significant transcriptional changes in reporter gene experiments...