PaleoHi-C

PaleoHi-C is a specialized version of the Hi-C technique, used to study the three-dimensional architecture of the genome in ancient DNA samples, such as the remains of extinct species or organisms preserved under specific conditions. This technique has revolutionized the field of paleogenomics by allowing detailed analysis of the structural organization of the genome in DNA samples that have been exposed to natural degradation for thousands of years.
Development of PaleoHi-C
The Hi-C method was initially developed to analyze DNA interactions within the cell nucleus of living cells. In Hi-C, DNA regions that are physically close in the cell, but may be distant in the genome sequence, are ligated and sequenced to create a three-dimensional genome contact map. This approach has been instrumental in studying genome organization in modern species, but faced significant challenges when dealing with ancient DNA.
The development of PaleoHi-C emerged as a solution to these challenges. Ancient DNA, especially from extinct species, tends to be highly fragmented due to the degradation of genetic material over time. PaleoHi-C adapts the Hi-C protocol to work with fragmented and degraded DNA samples, allowing not only to obtain the genetic sequence, but also to reconstruct the three-dimensional organization of chromosomes.

Method and Procedure

The PaleoHi-C process follows several key steps that allow the reconstruction of genomic structure in ancient samples:
1. Collection of ancient DNA samples:

DNA samples generally come from well-preserved biological remains, such as bones, teeth, or skin, that have been preserved in special conditions, such as permafrost.
Once the sample is obtained, it is stabilized to avoid further degradation and DNA is extracted.

2. DNA fragmentation and ligation:

DNA fragments into small pieces due to its natural decomposition.
Next, a proximity ligation process is used, which involves temporarily joining DNA regions that are physically close but may be distant in linear sequence.
This ligation makes it possible to capture interactions between DNA regions that are in contact in the cell, even if they are not adjacent in sequence.

3. DNA sequencing and creation of contact maps:

Once the DNA has been fragmented and nearby regions have been joined, the sequences are analyzed by high-throughput sequencing.
These data are processed to generate a contact map showing how the different DNA regions interact in 3D within the cell nucleus. This map provides information about the spatial organization of chromosomes in the cell.

4. Analysis of genome architecture:

With the contact maps obtained, key three-dimensional structures of the genome can be identified, such as chromosomal territories, active and inactive chromatin compartments (A and B), chromosomal loops, and superdomains.
These analyses provide a deep insight into how DNA was organized within the nucleus of the cells of the extinct species.

Applications and Main Results

1. Preservation of Genome Architecture:

In one of the most remarkable early studies using PaleoHi-C, researchers were able to study the skin of a woolly mammoth (Mammuthus primigenius) found in Siberian permafrost. Although the DNA was highly fragmented, the results showed that the three-dimensional architecture of the genome was well preserved.
The chromosomal structures observed included chromosomal territories, A/B compartments, chromatin loops, Barr bodies and superdomains, all of which are key elements in the genomic organization of cells.

2.Differences in Gene Activation:

PaleoHi-C also made it possible to analyze the differences in gene activity between the woolly mammoth and its closest relative, the Asian elephant. Several genes were identified whose regulations were different in both animals, such as Edaradd (related to hair follicles) and Egfr (involved in skin homeostasis).
This discovery not only provided insight into the mammoth's evolutionary adaptations to its cold environment, but also helped to understand how changes in genome architecture can influence specific physical characteristics.

3.Generation of Reference Genomes for Extinct Species:

Using PaleoHi-C in combination with a novel algorithm for reference-assisted genome assembly, the scientists were able to generate a mammoth reference genome with complete chromosome scaffolds, which opens up the possibility of reconstructing complete genomes of extinct species.
This approach could also be applied to other extinct species, opening the door to new studies on the evolution and biology of prehistoric species.

Preservation of Genomic Architecture under Extreme Conditions: The "Chromoglass" Hypothesis

One of the most fascinating findings of the study was the proposal that mammoth DNA may have been preserved in a state called chromoglass. This glassy state of chromatin occurs when DNA molecules are trapped in a state of low mobility, allowing the three-dimensional architecture of the genome to remain intact despite DNA fragmentation.

It is suggested that this phenomenon could have been induced by the low temperatures of the permafrost, which acted as a natural preservation mechanism, similar to the freeze-drying process (sublimation of water) that occurs in food.

Future Applications and Potential

PaleoHi-C has the potential to revolutionize the study of genetics and evolutionary biology, making it possible to reconstruct not only complete genomes of extinct species, but also to study the structural and epigenetic features of their genomes. As more data are obtained from PaleoHi-C, it will be possible to perform genome assemblies of extinct species de novo, without the need for a reference genome.

In addition, this technique could be useful in historical genomics research, applied to samples of Egyptian mummies or prehistoric animals preserved in cold conditions, opening new avenues for the study of molecular biology and evolution.

References

Sandoval-Velasco, M., Dudchenko, O., Rodríguez, J. A., Estrada, C. P., Dehasque, M., Fontsere, C., ... & Aiden, E. L. (2024). Three-dimensional genome architecture persists in a 52,000-year-old woolly mammoth skin sample. Cell, 187(14), 3541-3562.[1]