The signals that cells use to switch genes on have remained almost unchanged across two billion years of evolution, but the ones used to switch genes off vary dramatically from one branch of life to another, according to a new study by researchers at the Centre for Genomic Regulation (CRG), Barcelona Institute of Science and Technology (BIST).

The findings result from the broadest comparative exercise to date of how different life forms regulate their genomes. The researchers carried out the first detailed analysis of chromatin, the protein scaffold that controls how DNA is read, in several major branches of life that have been largely absent from studies to date, including lineages such as discobans, rhizarians, ichtyosporeans, and cryptomonads.

The work helps understand how genomes evolved on Earth and could have implications for medical research into diseases involving faulty gene regulation. It also delivers a new method, developed at the CRG, which can help support international efforts to characterize life on Earth at the molecular level.

“The cell’s instructions for activating genes are essentially the same in a human, a sea anemone and a soil amoeba,” said Arnau Sebé-Pedrós, PhD, ICREA Research Professor and senior author of the team’s published paper in Nature Genetics. “But the instructions for silencing genes and other genomic elements like transposons have been continuously evolving since our last common eukaryotic ancestor. Different branches of life have developed different molecular toolkits to do the same thing.” The team’s report is titled “Diversity and evolution of chromatin regulatory states across eukaryotes.”

DNA is wrapped, inside every cell, around proteins called histones. Small chemical tags attached to these proteins tell the cell which stretches of DNA to read and which to ignore. The chemical tags are ancient, dating back roughly two billion years to a single-celled organism known as the last eukaryotic common ancestor, or LECA, the founder of all complex cellular life, from which every plant, animal, fungus and protist on Earth descends. The system, known as chromatin regulation, is what allows the same genome to produce a liver cell or a neuron, and a leaf or a root. Faults in the regulation of chromatin underpin many human diseases, including cancers.

“Histone post-translational modifications (hPTMs) are central to defining functional chromatin states,” the authors explained further. “These hPTMs are conserved across diverse eukaryotes, with dozens tracing back to the last eukaryotic common ancestor, which we confirmed by histone mass spectrometry.”

The enzymes that add and remove the tags are also broadly shared across plants, animals, fungi and microbial eukaryotes. Until now, however, almost all detailed knowledge of how these tags work has come from a handful of laboratory species such as humans, mice, fruit flies, yeast and the model plant Arabidopsis. The vast majority of life’s diversity has remained unexplored at this level.

The authors’ project began in 2017, when Sebé-Pedrós and David Lara-Astiaso, PhD, were using a technique called iChIP to study chromatin in comb jellies and placozoans, animals not traditionally studied in the lab. The researchers wondered whether the approach could be scaled up for use in other species in the eukaryotic tree of life.

“We wanted to map epigenetic states in scarce cell types in mice and humans,” recalls Lara-Astiaso, now at the Arc Institute in California. “Eventually, we managed to transform that precursor into a general method for mapping genome regulation across the tree of life—more streamlined, more sensitive, and finally able to handle the particularities of very different species.”

The new method, iChIP2 can label chromatin from many species with unique molecular barcodes and read them all in a single experiment. Using the technology helped profile twelve chemical tags, or histone modifications, across twelve phylogenetically diverse species, spanning amoebae, fungi, plants, algae, single-celled predators and animals.

Some organisms had never had their chromatin mapped before. “We initially hoped to build a completely universal protocol, but species differ too much for that,” noted co-first author Cristina Navarrete, PhD. Plants and algae have cell walls that require specialized preparation, for example. Once a lab has extracted chromatin from their favorite species, iChIP2 takes over robustly, and from very small amounts of material.”

The researchers found that the signature of an active gene, marked by the pattern of histone modifications clustered around its start and along its body, was nearly identical in every species the team examined. The signature of a silenced gene was not. The results indicated that different lineages used different combinations of modifications, in different patterns, to keep stretches of DNA silent. “Our analyses revealed highly conserved euchromatin states at active gene promoters and gene bodies,” the investigators stated. “In contrast, we observed diverse configurations of repressive heterochromatin states associated with silenced genes and transposable elements …”

In some species, one modification silenced transposable elements while a different tag silenced unused genes. In others, the same modifications piled up together on the same regions. In the soil amoeba Acanthamoeba, a chemical mark that signals gene activation in animals had been repurposed to switch genes off.

“We’ve established so many new rules from looking at such few species,” said study co-author Sean Montgomery, PhD, “It’s the power of looking at non-model organisms to see how evolution has brought about many differing solutions to the same problems.”

The researchers suggest the diversity reflects an ancient and ongoing conflict between genomes and the parasitic DNA within them, like transposable elements, also known as “jumping genes,” and endogenized viruses. Every genome carries within it stretches of jumping genes, sequences that copy and paste themselves into new locations, sometimes harmlessly, sometimes destructively. In a human genome, they account for roughly half of all DNA. In their paper the team wrote, “The diversity of repressive states across eukaryotes, compared with the highly conserved active states, reflects the history of genomic invasions by parasitic elements in different lineages and could also define the permissiveness of these genomes to future invasions.”

Keeping jumping genes silenced is a matter of survival, but they evolve. Their parasitic nature means they acquire new sequences and sometimes even fragments of the chromatin machinery itself to evade detection.

“If a species loses its repressive mechanisms completely, it can’t tolerate parasitic elements like transposable elements or endogenized viruses. The result is that it’s no longer there. It’s dead,” says Sebé-Pedrós.

Over hundreds of millions of years, the result is host and parasite adapting and a tree of life on which each branch has developed its own bespoke strategy to silence genes. Some of those strategies, the team suggests, were later borrowed for other purposes.

The work lands at an important moment for comparative genomics. International efforts such as the Earth BioGenome Project and the Wellcome Sanger Institute’s Tree of Life programme, with which Sebé-Pedrós is affiliated, are sequencing the genomes of life on Earth at unprecedented speed.

The data generated by the initiatives offer potential new insights into how life has evolved on Earth, but a genome sequence alone says little about how the genome is used. Methods like iChIP2 make it possible to ask how life forms regulate their genomes. “… our results exemplify the potential of biodiversity epigenomic profiling,” the team suggested. “As genome sequencing is rapidly advancing across the tree of life, this approach offers a valuable opportunity to similarly expand our understanding of eukaryotic genome function and regulation.”

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