Ancient life loops equipped with DNA will become complex

The overall structure of a chromosome can be divided first into large territories, then into more specialized compartments, and then into topologically associated domains, called TADs. These TADs are often considered functional or regulatory “neighborhoods” that bring together associated DNA sequences. Loops are smaller structures within them that do the larger-scale work of bringing separate parts of the sequence together.

But loops don’t simply form via random fluctuation in chromatin shape; their creation is orchestrated and requires energy. In advanced metazoans like us, a loop of chromatin is tightened at its base by a hoop-shaped protein called cohesin, which acts much like the knot of a lasso. The chromatin strand can pass through the hoop until it encounters a protein called CTCF which is bound to the DNA and acts as a stopper. In short, distal regulation via chromatin loops is a complex and expensive activity, and we can only assume that the benefits it offers for new regulatory options would be worth the effort. This can, for example, greatly improve the potential for combinatorial complexity. By bringing enhancers to different parts of the chromosome, loops can not only allow a single enhancer to help regulate more than one gene, but also allow a gene to be regulated by multiple enhancers.

In the loop

Sebé-Pedrós, Kim and their colleagues found that chromatin looping appears to have been an important step in metazoan evolution, one that distinguishes cnidarians and ctenophores, as well as sponges and placozoans, which were also part of the study, from their closest single-celled relatives still living today. The latter are simple eukaryotes with equally difficult names: ichthyosporians (which can parasitize fish and other marine animals), filasterates (amoebic organisms with a complex life cycle that includes multicellular aggregation), and choanoflagellates (which can swim and are generally considered the animals’ closest living relatives).

The team used a technique introduced 10 years ago called Micro-C to reveal which parts of chromatin are physically close to each other. The method involves chemically linking nearby regions of chromatin, then cutting the chromatin and observing which sequences in the fragments are linked together. The result is a genome-wide chromatin proximity map, which encodes the three-dimensional organization of the genome. Techniques like this have been around for a while, but Micro-C uses an enzyme that can cut DNA more finely than before. “Micro-C has been a game changer for us, because we are dealing with species with small genomes,” Sebé-Pedrós said. It is therefore crucial to be able to divide it into many tiny fragments.

Researchers have found that cnidarians, ctenophores, and placozoans (simple, flat animals with only a few cell types) have more complex genomic architecture than single-celled animals, including chromatin loops that bring promoters and enhancers together. Even small genomes, such as those of ctenophores, can contain thousands of such loops, whereas single-celled organisms have no loops. They also observed that these loops coalesced into structures such as TADs. These mechanisms of finely tuned and modular gene expression appear to be necessary for more complex body plans and cellular specialization, and are a key aspect of how our genomes function.

So it seems that these regulatory innovations allowed many types of multicellular creatures to arise from a set of genes that do not appear to have been very different from those of their evolutionary ancestors.

“The view that chromatin loops and distal regulatory elements helped enable cell specialization in multicellular organisms is very reasonable,” Popay said. “This is supported by other work in mammalian systems that suggests that chromatin looping, particularly between enhancers and promoters, is important for the expression of certain cell identity genes.”

Regulation rules

It is not yet clear exactly how cnidarians and ctenophores create chromatin loops to add this additional layer of regulatory complexity to cell type-specific gene regulation. They probably use cohesin rings, as our cells do, but they lack the CTCF proteins to control the start and end of the loops. Sebé-Pedrós thinks other proteins in the same family could do the same job.

They also don’t know exactly what role enhancers played in early metazoans. Some researchers think that enhancers might encode RNA molecules that would be transcribed and interact with other molecules on the regulatory “committee” that determines gene activation – just as they do in vertebrates like us. But Sebé-Pedrós and colleagues suspect that enhancers in cnidarians and ctenophores are essentially just places for additional TFs, and that more well-defined isolation of chromatin domains to modularize gene activity came later, perhaps with the evolution of bilaterian animals.

“I think it’s a very interesting hypothesis,” Oudelaar said. But she cautioned that “while there is certainly nothing to argue against this at the moment, there is also no concrete evidence for this beyond the correlations (between the loops and the complexity of the organism).”

Amos Tanay, an expert in genomic regulation at the Weizmann Institute of Science in Rehovot, Israel, agrees. “The idea that long-term regulation facilitates complex multicellularity makes perfect sense, but I will need to see more results from more species to build confidence in this hypothesis,” he said.

A big challenge is that we don’t know how closely early cnidarians and ctenophores resemble species living today, according to Iñaki Ruiz-Trillo, an evolutionary biologist at Pompeu Fabra University in Barcelona. “These lineages evolved over millions of years, so you can’t take them as an approximation,” he said.

Regardless, no one thinks that chromatin looping is the only thing that allowed complex animals to emerge. There was, for example, also some genetic novelty, Sebé-Pedrós said.

And the genomes of these organisms have expanded significantly compared to single-celled organisms, even though the number of protein-coding genes has not increased. Evolutionary changes, he says, are likely due to a combination of factors, and “it’s very difficult to know which aspect triggered the other.”

According to Tanay, a first step is to determine the logical rules or grammar that govern regulatory combinations. Looping only really works when TFs abandon the specificity of effect they exhibit in bacteria and adopt the “fuzziness” of interaction that allows them to function combinatorially. It is unclear whether this occurred before the loop appeared. “It’s a really exciting question, but we don’t have an answer to it,” Sebé-Pedrós said.. He says he and his colleagues hope to deduce the molecular rules regulating these early metazoans and their single-celled precursors. “It will be exciting to compare these regulatory logics across animal evolution,” he said.

And if chromatin looping was indeed a key innovation that unlocked animal complexity, there is a puzzling implication: that complexity would appear to have been latent, in a sense, in the genomes of their single-celled ancestors – before evolution even thought of metazoans, so to speak. Why this should have been so is not at all obvious; evolution has no universal direction, no foresight. “To me, it’s a fascinating question,” Ruiz-Trillo said.

To go even further: could another explosion of new regulations create, based on the genes that exist today, another change in what living organisms can be? After all, as Tanay said, “evolution is always full of surprises.”