Researchers at Stanford and the University of California, San Francisco (UCSF) have devised a new method to harvest more information from the genomes of archaic humans, such as Neanderthals and Denisovans, to potentially reveal the physical consequences of genomic differences between us and them. The team published its study (“The cis-regulatory effects of modern human-specific variants”) in eLife, focused on sequences related to gene expression.

“The Neanderthal and Denisovan genomes enabled the discovery of sequences that differ between modern and archaic humans, the majority of which are noncoding. However, our understanding of the regulatory consequences of these differences remains limited, in part due to the decay of regulatory marks in ancient samples. Here, we used a massively parallel reporter assay in embryonic stem cells, neural progenitor cells, and bone osteoblasts to investigate the regulatory effects of the 14,042 single-nucleotide modern human-specific variants,” write the investigators.

“Overall, 1791 (13%) of sequences containing these variants showed active regulatory activity, and 407 (23%) of these drove differential expression between human groups. Differentially active sequences were associated with divergent transcription factor binding motifs, and with genes enriched for vocal tract and brain anatomy and function. This work provides insight into the regulatory function of variants that emerged along the modern human lineage and the recent evolution of human gene expression.”

Starting with 14,042 genetic variants unique to modern humans, the researchers found 407 that specifically contribute to differences in gene expression between modern and archaic humans. In further analysis, they determined that the differences were more likely to be associated with the vocal tract and the cerebellum, which is the part of our brain that receives sensory information and controls voluntary movement, including walking, coordination, balance and speech.

“It just seems so implausible that you could make a call like, ‘I think the voice box evolved,’ from the information we have,” said Dmitri Petrov, PhD, the Michelle and Kevin Douglas Professor in the School of Humanities and Sciences, who is co-senior author of the paper with David Gokhman, PhD, postdoctoral Stanford research fellow, department of biology, and Nadav Ahituv, PhD, a professor of bioengineering at UCSF. “The predictions are almost science fiction. If five years ago, somebody told me that this would be possible, I would not have put much money on it.”

The path to modern humans

With such a large number of variants to examine, the researchers relied on a massively parallel reporter assay to test which sequences actually affect gene regulation. Their version of this technique, which was developed by Ahituv, involves packaging the DNA sequence variant into a reporter gene inside a virus. That virus is then put into a cell. If that variant affects gene expression, the reporter gene produces a barcoded molecule that identifies what DNA sequence it came from. The barcode allows the researchers to scan the products of a large number of variants at once.

Essentially, the whole process imitates an abridged version of how each variant would play out in a cell in real life and reports the results.

Lana Harshman, a graduate student at UCSF and co-lead author of the paper, infected three types of cells with the team’s variant packages. These cells were related to the brain, skeleton, and early development, all subjects most likely to reveal evolutionary differences between us and our most recent ancestors. Carly Weiss, PhD, a postdoctoral scholar in the Petrov lab and co-lead author of the paper, analyzed the results of these experiments.

In studying the 407 sequences that represented a change in expression in modern humans compared to our predecessors, genes that affect the cerebellum and the voice box, pharynx, larynx, and vocal cords seem to be overrepresented.

“This would suggest some kind of rapid evolution of those organs or some kind of a path that is specific to modern humans,” said Gokhman. The next step, he added, would be trying to understand more about these sequences and the roles they played in the evolution of modern humans.

Even with those unknowns, this technique by itself is a significant advance for evolutionary research, said Petrov.

“This goes beyond the sequencing of the DNA from the Neanderthal and Denisovan bones. This begins to put meaning on those differences,” said Petrov. “It’s an important conceptual step from just the sequence–no tissue, no cells–to biological information and will enable many future studies.”