Svante Pääbo

Understanding the genetic changes that drove the evolution of human-specific traits is a central goal in biology. Changes in gene expression are thought to be a major source of phenotypic innovations ^1,2 , but identifying the underlying changes in cis-regulatory elements (CREs) along the human lineage has been difficult: functional data from great apes are scarce and unobtainable for extinct hominins, and pinpointing causal substitutions requires knowing which sequences determine CRE activity. Here, we used AlphaGenome ³ , a sequence-to-function deep-learning model, to predict the cis-regulatory effects of ∼17 million single-nucleotide substitutions specific to the human lineage and traced their consequences for gene expression evolution across 305 tissues and cell types. The 71,345 predicted cis-regulatory gains lie mostly outside human accelerated regions ^4,5 and arose either through creation of new CREs or, more often, through co-option of CREs ancestrally accessible in other tissues. They are also enriched for distal enhancers and for target gene functions characteristic of the corresponding tissues, suggesting that they contributed to the evolution of tissue-specific biology in humans. Genes linked to more gains were more likely to be upregulated in humans, and in silico reversion to ancestral alleles traced such upregulations to individual substitutions creating transcription factor binding motifs. Comparison with Neandertal and Denisovan genomes revealed that nearly all gains now fixed in humans predate the split from archaic hominins, yet a few modern-human-specific gains contribute substantially to gene upregulation. Our study provides a genome-wide, tissue-resolved map of cis-regulatory changes and their contribution to gene expression evolution along the human lineage.

The Teshik-Tash 1 child whose remains were found in Uzbekistan represents the southeastern-most extent of the known Neandertal range, providing an important link with the better studied Caucasus and Altai Mountain ranges. However, due to poor DNA preservation, studying the genetics of Teshik Tash 1 has remained elusive. Here we present analyses of the nuclear DNA from the Teshik-Tash 1, from extracts that are highly contaminated with present-day human DNA. To achieve this, we developed a new computational method, admixslug , that jointly models contamination and population relationships, in order to infer the relationship of a target individual from which only low-quality nuclear DNA is available, to high-quality archaic human genomes. After validating admixslug , we show that Teshik-Tash 1 is genetically more similar to later Neandertals from Western Eurasia than to older Neandertals from the Altai Mountains. We estimate that Teshik-Tash 1 split from the Western Eurasian lineage between 80,000 and 100,000 years ago. Despite the geographical proximity of Teshik-Tash 1 to the Denisovan range, we find no evidence for Denisovan ancestry in his genome. Our results demonstrate that admixslug enables the study of archaic human specimens in cases where DNA preservation was previously considered too poor for population genetic analyses.

Archaeological, osteological and genetic evidence suggests that Neanderthals lived in small groups ^1,2 ; however, less is known about whether these groups were part of isolated communities or belonged to larger, well-connected populations ³ . The dense concentration of broadly contemporaneous Neanderthal sites in the Meuse Basin, Belgium ⁴ , provides a rare opportunity to study regional populations at high resolution. Here we generated genetic data from 27 Neanderthals who lived less than approximately 52,500 years ago from ten archaeological sites in Belgium and France, including a high-coverage genome from a 45,000-year-old individual from Goyet, Belgium. We show that most of these individuals are more closely related to one another than to other contemporaneous late Neanderthals in Europe. Further, some of these individuals carry DNA from a Neanderthal lineage predating the split of late Neanderthals. Although these Neanderthals overlapped temporally with early modern humans in northwestern Europe from around 47,000 years ago, we find no evidence of recent gene flow from modern humans. They also do not show the genetic signatures of mating among close relatives found in Altai Neanderthals, suggesting that they lived in larger or better-connected groups. Moreover, genetic load did not accumulate over time, arguing against progressive genetic deterioration as a driver of Neanderthal extinction.

N -acetyltransferase 1 (NAT1) is an enzyme that acetylates certain drugs and carcinogens and is involved in folate metabolism. Some NAT1 variants are associated with susceptibility to cancer and birth defects. We show that while nearly all present-day humans carry a valine residue at position 149 and a serine residue at position 214, some carry the ancestral amino acids isoleucine and alanine at these positions because of gene flow from Neanderthals. Neither substitution affects enzymatic activity. However, replacing the serine residue, a known phosphorylation site, with a phosphomimetic aspartate reduces NAT1 activity by ~60%. Phosphorylation at this site may allow NAT1 to be down-regulated during pregnancy to reduce birth defect risk, while maintaining high activity in adults for xenobiotic clearance. This regulatory site is a molecular feature that differs between modern humans and Neanderthals.

We present a genome sequenced to ~37-fold genomic coverage from an approximately 110,000-y-old male Neandertal from Denisova Cave in the Altai Mountains and analyze it together with previously published Neandertal genomes of high quality. We show that he belonged to a population more closely related to a ~120,000-y-old Neandertal from Denisova Cave than to Neandertals in Europe or to a ~80,000-y-old Neandertal from Chagyrskaya Cave in the Altai Mountains. Both Neandertals from Denisova Cave show evidence of gene flow from Denisovans, a pattern not seen in later Neandertals from the Altai region or from Western Europe. The extent of chromosomal regions of homozygosity in Neandertals from the Altai region between 120,000 and 80,000 y ago indicates that they lived in smaller and more isolated groups than later Neandertals in Europe (54,000 to 40,000 y ago). We estimate the extent of allele frequency differentiation among Neandertal populations and find that the older Eastern Neandertals in the Altai region and younger Western Neandertals in Europe were as differentiated as the most differentiated present-day human populations worldwide.

Gene regulatory changes are considered major drivers of evolutionary innovations, including the cerebellum’s expansion during human evolution, yet they remain largely unexplored. In this study, we combined single-nucleus measurements of gene expression and chromatin accessibility from six mammals (human, bonobo, macaque, marmoset, mouse, and opossum) to uncover conserved and diverged regulatory networks in cerebellum development. We identified core regulators of cell identity and developed sequence-based models that revealed conserved regulatory codes. By predicting chromatin accessibility across 240 mammalian species, we reconstructed the evolutionary histories of human cis-regulatory elements, identifying sets associated with positive selection and gene expression changes, including the recent gain of THRB expression in cerebellar progenitor cells. Collectively, our work reveals the shared and mammalian lineage-specific regulatory programs governing cerebellum development.

The selection of cells that have acquired a desired gene edit is often done by the introduction of additional genes that confer drug resistance or encode fluorophores. However, such marker genes can have unintended physiological effects and are not compatible with editing of single nucleotides. Here, we present SNIPE, a method that allows the marker-free selection of edited cells based on single nucleotide differences to unedited cells. SNIPE drastically enriches for cells, which have been precisely edited (median 7-fold). We validate the approach for 42 different edits using Cas9 or Cas12a in different cell types and species. We use it to enrich for combinations of substitutions that change missense mutations carried by all people today back to the ancestral state seen in Neandertals and Denisovans. We also show that it can be used to kill cultured tumor cells with aberrant genotypes and to repair heterozygous tumorigenic mutations.

Adenylosuccinate lyase (ADSL), an enzyme that is crucial for purine biosynthesis, carries an amino acid substitution that is present in almost all humans today but absent in Neandertals and Denisovans. This substitution reduces the stability of the enzyme, but what functional consequences it has are unknown. Here, we show that when introduced into mice, this substitution causes substrates of the enzyme to accumulate in amounts that correlate negatively with ADSL expression levels. In the brain, where the expression of the enzyme is low, the substitution results in particularly high substrate levels. When the behavior of the mice is analyzed, female mice expressing the modern human-like version of ADSL access water more efficiently for drinking than their wild-type littermates. In addition to the amino acid substitution, a haplotype in the ADSL gene occurs at a carrier frequency of >97% in present-day humans and exhibits evidence of positive selection. It is associated with less ADSL expression as well as with increased concentrations of succinyladenosine, one of the substrates of the enzyme, in cerebrospinal fluid. Thus, two genetic changes have reduced ADSL activity in human tissues since modern and archaic humans separated, affecting purine biosynthesis, particularly in the brain.

The enzyme AMPD1 is expressed in skeletal muscle and is involved in ATP production. All available Neandertal genomes carry a lysine-to-isoleucine substitution at position 287 in AMPD1. This variant, which occurs at an allele frequency of 0–8% outside Africa, was introduced to modern humans by gene flow from Neandertals. Here, we show that the catalytic activity of the purified Neandertal AMPD1 is ~25% lower than the ancestral enzyme, and when introduced in mice, it reduces AMPD activity in muscle extracts by ~80%. Among present-day Europeans, another AMPD1 variant encoding a stop codon occurs at an allele frequency of 9–14%. Individuals heterozygous for this variant are less likely to be top-performing athletes in various sports, but otherwise reduced AMPD1 activity is well tolerated in present-day humans. While being conserved among vertebrates, AMPD1 seems to have become less functionally important among Neandertals and modern humans.

Denisova Cave in southern Siberia is the only site known to have been occupied by Denisovans, Neanderthals and modern humans. The cave consists of three chambers (Main, East and South), with the archaeological assemblages and remains of hominins, fauna and flora recovered from Main and East Chambers being the most thoroughly investigated to date. Here we report the results of analyses of the Palaeolithic artefacts, faunal remains and hominin and mammalian mitochondrial (mt) DNA recovered from renewed excavations in South Chamber. We construct a calendar-year time scale for the stratified Pleistocene deposits from optical dating of the sediments. The timing of hominin occupation and major turnovers in the mtDNA of Denisovans and large mammals largely accords with the patterns detected in Main and East Chambers. Time gaps in those sequences are partly filled by the South Chamber data and the sediment DNA record of Denisovans after 80,000 years ago is more than doubled in size. We combine the sediment dating and DNA records for all three chambers to reveal the whole-of-cave history of this unique site and the climatic conditions experienced by hominins and fauna over the past 300,000 years, including potential changes in habitat suitability for Denisovans and Neanderthals.