A First Look At A Neanderthal Face

Reconstructing an Ancestral Face

What does it take to imagine the face of someone you have never seen? A picture of a relative is the most intuitive reference, but what if you could do it with a fingerbone and a handful of teeth?

Thanks to DNA methylation, a group of scientists from Spain and Israel recently reconstructed the facial anatomy of Denisovans, a group closely related to the Neanderthals and whose ancestors split from the lineage of Modern Humans approximately 600,000 to 744,000 years ago. The only Denisovan pieces for the scientists to begin with were a manual phalanx, a lower jawbone, and several teeth. The scientists, working with the pioneer paleogeneticist Svante Pääbo, were able to put the puzzle together by using the DNA methylation profiles detected in Denisovans, Modern Humans, Neanderthals, and chimpanzees.[1,2] They studied the methylation maps of genetic regions that change facial features and managed to predict the appearance of our ancient ancestor.

DNA Methylation Impacts Far and Wide

Why is DNA methylation so effective in reconstructing skeletal morphology? How does it help study an extinct group of humans we know so little about, and have few samples of? Simply, because DNA methylation carries information essential to reconstructing the expression of the genes governing bodily tissues.

DNA methylation is among the most extensively studied epigenetic marks.[2] In eukaryotes, DNA methylation pervasively occurs at CpG dinucleotides.[3] The patterns of DNA methylation at promoters, gene bodies, and enhancers play vital roles in regulating gene expression. Given that precise gene regulation is important for many fundamental biological processes, DNA methylation is essential throughout organismal development and the maintenance of homeostasis.

For example, researchers have found out that babies who received more cuddles from their caregivers presented different DNA methylation profiles from those who didn’t receive as much physical contact. This changed DNA methylation profile further indicated that the babies who got less cuddles might have experienced a less favorable developmental progress.[4] Additionally, a dynamic DNA methylation pattern was revealed during sweet orange fruit ripening.[5] Researchers found that a global increase in DNA methylation contributed to the repression of genes no longer needed and the activation of genes significant for the orange ripening process.

From human to fruits, including everything from behavior to physical changes, DNA methylation has quintessential roles in almost every aspect of our daily life. Perhaps it’s not totally a surprise, after all, that we were able to reconstruct the facial anatomy with the abundant information embedded in DNA methylation.

Bisulfite Sequencing Reveals DNA Methylation Profiles

Bisulfite sequencing is the most popular method to obtain DNA methylation measurements. Using the classic, gold-standard bisulfite conversion reaction, unmethylated cytosine residues in DNA are converted to uracils while the methylated ones resist that change, remaining as cytosines. Hence, methylated cytosines are recognized as “C” in next-generation sequencing (NGS) readouts.

Thus far, researchers have implemented a myriad of bisulfite sequencing methods to uncover DNA methylation information for diverse biological questions. These methods include whole-genome bisulfite sequencing (WGBS) and reduced representation bisulfite sequencing (RRBS). WGBS helps scientists obtain a comprehensive profile of DNA methylation levels across the entire genome. RRBS enriches the CpG-rich regions in the genome, and thus reduces the number of required sequencing reads to achieve a comparable read depth to WGBS. As a result, RRBS is more cost-effective compared to WGBS in sequencing, and is ideal for scientists to screen genome-wide DNA methylation in large-scale studies.

Another NGS-based method to measure DNA methylation is targeted bisulfite sequencing. Scientists design PCR primers for specific gene regions of interest to generate libraries with. This allows them to investigate DNA methylation levels in these specific areas and examine the dynamics. Targeted bisulfite sequencing is a great option for data validation against other methods as well as screening on selected gene regions in large sample cohorts.

NGS-Based DNA Methylation Study is Powerful

Conventionally, DNA methylation is measured via microarray-based methods and many datasets have been generated from these platforms. While robust to use and easy to compare data from different datasets, array-based methods have several limitations. First, the coverage of CpG sites in the genome of interest is largely limited. An example is that the MethylationEpic array covers only ~3% of the total CpG sites in human genome. Second, little flexibility exists in terms of sample species as one array is only compatible with one specific species.

NGS-based methods, on the other hand, have much higher coverages and are compatible with any species. Recently, researchers have applied WGBS to identify the mouse liver methylation heterogeneity among single cells.[6] Averagely, 21.6 million CpG sites per bulk sample and 2.2 million CpG sites per single cell sample were covered. Moreover, scientists have used RRBS to map the DNA methylation in samples from chicken, opossum, and platypus.[7] Their data were the first to demonstrate the participation of DNA methylation in the X chromosome inactivation in marsupial mammals.

Furthermore, RRBS was also used to discover DNA methylation biomarkers for Barrett’s esophagus. This syndrome is an important risk factor for the development of esophageal cancer.[8] Once they identified a CpG patch as the biomarker from the RRBS data of 46 biopsy samples, the researchers relied on targeted bisulfite sequencing for validation and expansion of the enrolled CpG sites in fresh and archived clinical samples.

NGS-based methods for measuring DNA methylation has expanded our knowledge of the multifaceted roles of DNA methylation. As the methodologies keep improving, the power of NGS-based DNA methylation study will undoubtedly be further harnessed to generate more informative pieces to help assemble the puzzles in epigenetics and beyond.

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  1. Gokhman, D.; Mishol, N.; de Manuel, M.; de Juan, D.; Shuqrun, J.; Meshorer, E.; Marques-Bonet, T.; Rak, Y.; Carmel, L. Reconstructing Denisovan Anatomy Using DNA Methylation Maps. Cell 2019, 179 (1), 180-192.e110. DOI: 10.1016/j.cell.2019.08.035.
  2. Gokhman, D.; Lavi, E.; Prüfer, K.; Fraga, M. F.; Riancho, J. A.; Kelso, J.; Pääbo, S.; Meshorer, E.; Carmel, L. Reconstructing the DNA methylation maps of the Neandertal and the Denisovan. Science 2014, 344 (6183), 523-527. DOI: 10.1126/science.1250368.
  3. Smith, Z. D.; Meissner, A. DNA methylation: roles in mammalian development. Nat Rev Genet 2013, 14 (3), 204-220. DOI: 10.1038/nrg3354.
  4. Greenberg, M. V. C.; Bourc'his, D. The diverse roles of DNA methylation in mammalian development and disease. Nat Rev Mol Cell Biol 2019, 20 (10), 590-607. DOI: 10.1038/s41580-019-0159-6.
  5. Moore, S. R.; McEwen, L. M.; Quirt, J.; Morin, A.; Mah, S. M.; Barr, R. G.; Boyce, W. T.; Kobor, M. S. Epigenetic correlates of neonatal contact in humans. Dev Psychopathol 2017, 29 (5), 1517-1538. DOI: 10.1017/S0954579417001213.
  6. Huang, H.; Liu, R.; Niu, Q.; Tang, K.; Zhang, B.; Zhang, H.; Chen, K.; Zhu, J. K.; Lang, Z. Global increase in DNA methylation during orange fruit development and ripening. Proc Natl Acad Sci U S A 2019, 116 (4), 1430-1436. DOI: 10.1073/pnas.1815441116.
  7. Gravina, S.; Dong, X.; Yu, B.; Vijg, J. Single-cell genome-wide bisulfite sequencing uncovers extensive heterogeneity in the mouse liver methylome. Genome Biol 2016, 17 (1), 150. DOI: 10.1186/s13059-016-1011-3.
  8. Waters, S. A.; Livernois, A. M.; Patel, H.; O'Meally, D.; Craig, J. M.; Marshall Graves, J. A.; Suter, C. M.; Waters, P. D. Landscape of DNA Methylation on the Marsupial X. Mol Biol Evol 2018, 35 (2), 431-439. DOI: 10.1093/molbev/msx297.
  9. Moinova, H. R.; LaFramboise, T.; Lutterbaugh, J. D.; Chandar, A. K.; Dumot, J.; Faulx, A.; Brock, W.; De la Cruz Cabrera, O.; Guda, K.; Barnholtz-Sloan, J. S.; et al. Identifying DNA methylation biomarkers for non-endoscopic detection of Barrett's esophagus. Sci Transl Med 2018, 10 (424). DOI: 10.1126/scitranslmed.aao5848.

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