Loops of DNA Equipped Ancient Life To Become Complex

The overall structure of a chromosome can be divided first into large territories, then into more specialized compartments, and then into topologically associating domains, known as TADs. These TADs are often thought of as functional or regulatory “neighborhoods” that put related DNA sequences together. Loops are smaller structures within them that do the finer-scale work of bringing separated parts of the sequence together.

But loops don’t just form via some random fluctuation in chromatin shape; their creation is orchestrated and requires energy. In advanced metazoans like us, a loop of chromatin is constricted at its base by a hoop-shaped protein called cohesin, which acts a bit like the knot of a lasso. The chromatin strand can pass through the hoop until it hits a protein called CTCF that’s bound to DNA and acts as a stopper. In short, distal regulation via chromatin loops is a complicated and costly business, and we can only suppose that the benefits it offered for new regulatory options were worth the effort. It can, for example, greatly enhance the potential for combinatorial complexity. By bringing enhancers to different parts of the chromosome, the loops can not only allow a single enhancer to help regulate more than one gene but also allow a gene to be regulated by more than one enhancer.

In the Loop

Sebé-Pedrós, Kim and their colleagues have now found that chromatin looping seems to have been a significant step in metazoan evolution, one that distinguishes the cnidarians and ctenophores — as well as sponges and placozoans, which were also in the study — from their closest unicellular relatives still living today. The latter are simple eukaryotes with similarly challenging names: ichthyosporeans (which can be parasitic to fish and other marine animals), filastereans (amoebalike organisms with a complex life cycle that includes multicellular aggregation) and choanoflagellates (which can swim and are generally regarded as the closest living relatives of animals).

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

The researchers found that cnidarians, ctenophores and placozoans (simple, flat animals with just a few cell types) possess a more complex genome architecture than the unicellular animals do, including chromatin loops that bring promoters and enhancers together. Even small genomes, such as those of ctenophores, can hold thousands of such loops, while single-celled organisms show no looping. They also observed these loops coalescing into structures like TADs. These mechanisms for finely tuned and modular gene expression seem to be necessary for more complex body plans and cell specialization, and are a key aspect of how our genomes work.

So, it seems these regulatory innovations may have allowed many kinds of multicellular creatures to arise from a set of genes that don’t appear to have differed that much from those of their evolutionary forebears.

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

Rules of Regulation

 It’s not yet known quite how cnidarians and ctenophores create chromatin loops to add this extra layer of regulatory complexity to cell-type-specific gene regulation. They probably use cohesin hoops, as our cells do, but they don’t have the CTCF proteins to control where loops start and stop. Sebé-Pedrós thinks that other proteins in the same family might do the same job.

Nor do they know exactly what role the enhancers played in early metazoans. Some researchers think that enhancers might encode RNA molecules that get 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 basically just places for additional TFs, and that more well-defined insulation of chromatin domains to modularize gene activity came later, possibly with the evolution of bilateral animals.

“I think this is a very interesting hypothesis,” Oudelaar said. But she cautioned that “while there is certainly nothing that speaks against it at the moment, there is also no concrete evidence for it yet beyond correlations [between looping and organismal complexity].”

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

A big challenge is that we don’t know how much early cnidarians and ctenophores look like the species living today, according to Iñaki Ruiz-Trillo, an evolutionary biologist at Pompeu Fabra University in Barcelona. “These lineages have evolved for millions of years, so you cannot take them as a proxy,” he said.

In any event, no one thinks that chromatin looping was the only thing that enabled the rise of complex animals. There was, for example, some genetic novelty too, Sebé-Pedrós said.

And the genomes of these organisms expanded considerably relative to unicellular organisms, even if the number of protein-coding genes did not. The evolutionary changes, he said, were probably due to a combination of factors, and “it’s very difficult to know which aspect triggered the other.”

A first step, Tanay said, is to figure out the logical rules or grammar that govern the regulatory combinations. Looping only really works when TFs abandon the specificity of effect that they show in bacteria and embrace the “fuzziness” of interaction that allows them to work combinatorially. It is not known whether this happened before looping arose. “This is a really exciting question, but we do not have an answer to it,” Sebé-Pedrós said. He says that he and his colleagues are hoping to deduce the molecular rules of regulation in these early metazoans and their unicellular 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 unleashed animal complexity, there’s a puzzling implication: That complexity would seem to have been latent, in a sense, in the genomes of their unicellular ancestors — before evolution had even thought of metazoans, so to speak. It’s not at all obvious why that should have been so; evolution has no universal direction, no foresight. “To me this is a fascinating question,” Ruiz-Trillo said.

To push it even further: Could another burst of regulatory novelty create, from genes that exist today, yet another shift in what living organisms can be? After all, as Tanay said, “Evolution is always full of surprises.”