What is Paleoepigenetics?

Ancient Sasquatch Epigenetics

To unravel the complexities surrounding the genome, advanced technologies in genomics and epigenetics are typically necessary. Most of these tests and instruments are designed with high-quality DNA and tissue specimens in mind. Accordingly, scientists preferentially use concentrated and freshly extracted DNA to achieve the best results.

Yet, some researchers do not have the luxury of working with modern DNA. This is why the field of ancient DNA (aDNA) emerged. Evolutionary biologists and anthropologists that study humans and other organisms from earlier time periods must overcome the challenges of working with degraded and damaged DNA. They customize molecular techniques so that DNA from ancient tissues (typically a few hundred to thousands of years old) can be extracted and trace amounts of DNA fragments can be analyzed.

Early aDNA studies were mostly restricted to mitochondrial DNA (mtDNA). One reason for this is because a cell has hundreds of copies of mtDNA. In comparison, only a single unit of nuclear genomic DNA exists within a cell; meaning the chance of loss over time is higher. However, as technology and computational abilities improved, whole genome sequencing of ancient remains became possible. Perhaps the greatest achievement in the field was the release of the draft Neanderthal sequence in May of 2010, which involved extracting DNA from a 38,000-year-old Neanderthal individual.

Meanwhile, research interest in epigenetics continued to grow. For instance, it has been long recognized that the 1.2% DNA nucleotide difference between chimpanzees and us was insufficient to explain our physical differences fully. For more clues about organismal evolution, researchers in comparative genomics started looking more closely at epigenetic regulation of noncoding DNA regions. Genetic regulatory changes (e.g., methylation) can have a major impact on physical appearance and can also evolve faster than permanent changes to coding DNA. Therefore, epigenetic marks on the genome have the potential to tell us about adaptations that occurred more recently in an evolutionary lineage.

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At first, it was not entirely certain if premortem epigenomes could be accurately reconstructed from postmortem epigenomes. A hallmark of aDNA is the deamination of cytosine bases,which is a biochemical decay process. Because this decay changes the DNA code, scientists must determine the original premortem DNA sequence. Fortunately, it was discovered that cytosine nucleotide decay is dependent on its premortem methylation status. This observation is what made the study of paleoepigenetics possible.

Christopher Barrett is a PhD candidate at the University of Kansas who studies Unangan (Aleut) population history and pre-history using ancient genomic data. He also studies bioinformatics methods to reconstruct ancient methylomes and other epigenomic markers with computational methods.

Barrett explained, “In this process of deamination, methylated cytosine bases are structurally and biochemically modified into thymine bases, while unmethylated cytosine bases decay into uracil bases.”

Therefore, the ratios of cytosine (‘C’), thymine (‘T’) and uracil (‘U’) can be examined to infer premortem methylation signatures. However, heavy computational methods are required.

Barrett continued, “Bioinformatics protocols for handling ancient DNA remove uracils during data processing, but leave thymines untouched. We can use these C → T ratios reconstructed from ancient DNA sequences while also leveraging the characteristic damage or decay patterns seen in ancient sequences as a proxy for reconstructing ancient epigenomic signals and other methylation processes.”

A pioneering study by Llamas et al. (2012) used bisulfite sequencing (i.e., a DNA treatment used for analyzing cytosine methylation patterns) and proved that reconstructing ancient epigenomes was possible. They showed that remains from a late Pleistocene Bison priscus (~26,000 years ago) had a similar methylation pattern when compared to fresh bison bones. The researchers concluded that as long as genomic DNA can be recovered from ancient specimens, then cytosine methylation patterns can be determined. Expanding on these findings, Smith et al. (2015) evaluated if bisulfite sequencing could be performed on a larger number of ancient remains in a different archaeological context (i.e., age and geographical location). They were also successful.

Initially, the field of paleoepigenetics was limited to a few ancient individuals. However, the availability of more diverse and higher quality genomes is increasing. This is important because it helps researchers reconstruct ancient DNA methylation patterns better.

Using a comparative genomics approach, Gokhman et al. (2014) identified differentially methylated regions (DMRs) between present-day humans and other hominins (specifically, Neanderthal and Denisovan). A closer look revealed that a few of these DMRs are located in the HOXD gene cluster. HOX genes are well known for playing a crucial role in body plan development. Notably, present-day humans have less robust skeletal features in comparison to Neanderthals and other archaic relatives. DMRs were also discovered in a network of genes related to facial and vocal tract anatomy. Together, these findings helped pinpoint a few molecular determinants of morphological changes that occurred more recently in human evolution.

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Studying DMRs can provide insight into the origin of human diseases. In comparison to genes without DMRs, the genes that do contain them are nearly twice as likely to be linked to diseases. Moreover, it was observed that a high proportion of DMR-containing genes are associated with neurological and psychiatric disorders.

Due to its high prevalence in humans (~1%), schizophrenia has received considerable research interest. Recently, Banjeerie et al. (2018:6) showed that present-day human DMRs are “enriched for association with schizophrenia.” These findings suggest that recent regulatory changes in the human lineage may have caused a pre-deposition for developing schizophrenia.

In addition to studying what is uniquely human, researchers continue to discover places in the genome with intriguing origins. Thousands of years ago, our ancestors interbred with other populations of archaic hominins. As a result, we still retain some of the genes and regulatory patterns from Neanderthals and Denisovans. This occurrence is known as introgression, and molecular signatures indicate that some of these introgressed genes likely gave human ancestors an adaptive advantage in certain environments. One example is the Denisovan TBX15 gene variant.

Dr. David Gokhman is a post-doctoral researcher at Stanford University that is currently interested in genetic regulatory differences between humans and other great apes, including elucidating the evolutionary forces that propelled these differences. He commented on TBX15:

“This gene is involved in the differentiation of brown fat cells, which are heat-producing mitochondria-packed cells, possibly explaining the positive selection in Greenland. Almost all present-day Greenlandic Inuits carry the Denisovan version of this region, which we have shown to be associated with the methylation and expression levels of TBX15,” Gokhman said.

Epigenetic mechanisms are sensitive to different environmental exposures. In the case of paleoepigenetics, adaptations to changing environments in a diversity of populations can be studied throughout time. However, this is where the field is really in its infancy. Finding environmental responsive loci (ERLs) requires more precise knowledge about genome-environment interactions, including where in the genome it occurs and how a gene’s expression is affected. Once elucidated, methylation changes resulting from toxins, natural disasters, traumatic stress (e.g., wars), and diet could be determined.

“I’m very excited about the potential in inferring the environment to which an individual was exposed through their methylation maps. As more and more environmentally responsive loci are identified, this may become a key tool in studying ancient individuals,” Gokhman said.

Although some major studies have been released, a quick glance at available publications shows that paleoepigenetics papers are few. The scarcity is likely due in part to some of the challenges this field faces.

For instance, there are different patterns of epigenetic regulation that occurs in different tissue types—a heart cell will have a different pattern of gene expression than a liver cell. Therefore, tissue types vary in the genetic regulatory information they possess. Because bones and teeth are the primary tissue sources used for extracting aDNA, paleoepigenetics is mainly restricted to studying methylation in only a few cell types.

Gokhman provided additional comments, “Ancient genetics is an exploding field. However, one of the biggest challenges in this field is the lack of cellular context: DNA molecules that are retrieved from ancient samples lack any binding proteins or regulatory layers – they are naked sequences which are very hard to interpret. We have to develop methods to reconstruct other epigenetic layers, such as histone modifications. Same goes for RNA and protein levels.”

Current and near future advances in biotechnology mean new possibilities for paleoepigenetics research is on the horizon. This includes the ability to sequence DNA and detect epigenetics modifications simultaneously.

“DNA methylation is a highly stable modification, with a half-life of tens of thousands of years. This technology will hopefully allow direct, rather than reconstructed, measurements of DNA methylation in ancient samples,” Gokhman said.

Sources Cited

Banjerjee N., Polushina T., Bettella F., et al. (2018). Recently evolved human-specific methylated regions are enriched in schizophrenia signals. BMC Evolutionary Biology, 18:63.

Gockhman D., Agranat-Tamir L., Housman G., et al. (2017a). Extensive regulatory changes in genes affecting vocal and facial anatomy separate modern from archaic humans. bioRxiv, doi:10.1101/106955.

Gokhman D., Lavi E., Prüfer K., et al. (2014). Reconstructing the DNA methylation maps of the Neandertal and the Denisovan. Science, 344(6183):523-27.

Gokhman D., Malul A., Carmel L. (2017b). Inferring past environments from ancient epigenomes. Molecular Biology and Evolution, 34(1):2429-38.

Gokhman D., Meshorer E., Carmel L. (2016). Epigenetics: It’s getting old. Past meets future in paleoepigenetics. Trends in Ecology & Evolution, 31(4):P290-300.

Llamas B., Holland M.L., Chen K., et al. (2012). High-resolution analysis of cytosine methylation in ancient DNA. PLoS ONE, 7(1):e30226.

Thayer Z.M., Non A.L. (2015). Anthropology meets epigenetics: Current and future directions. American Anthropologist, 117(4):722-35.

Racimo F., Gokham D., Fumagalli M., Ko A., et al. (2017). Archaic adaptive introgression in TBX15/WATS2. Molecular Biology and Evolution. 34(3):509-24.

Smith R.W.A., Monroe C., Bolnick D.A. (2015). Detection of cytosine methylation in ancient DNA from five Native American populations using bisulfite sequencing. PLoS ONE, 10(5):e0125344.

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