Loops of DNA Equipped Ancient Life To Become Complex
Myriam Wares for Quanta Magazine
For evolutionary biologists, what most distinguishes the marine creatures called cnidarians and ctenophores is not the peculiar spelling of their names, nor the fact that they tend to be beautiful, but that they are among the oldest known groups of complex animals. Cnidarians (the “c” is silent) include jellyfish, corals and sea anemones, and their colorful tendrils and fronds adorn oceans all over the globe. Ctenophores (another silent “c”), otherwise known as comb jellies, are mostly translucent and gelatinous, and they glide through the marine world like ectoplasm.
Thought to have arisen between about 740 million and 520 million years ago, both phyla of marine invertebrates were among the first multicellular animals to evolve several different tissue types. Their appearance ramped up the complexity of the animal world: They are more similar to us in terms of size and organization than they are to the single-celled organisms that came before them. The evolutionary debut of cnidarians and ctenophores thus represented one of the most significant steps in the journey from primordial slime to humans.
What made it possible?
The peculiar thing is that many of the genes identified in these oceanic creatures are also found in the unicellular species that preceded them. It wasn’t fundamentally different genes that kick-started the evolution of complex animals during (or before) the Cambrian period that began 540 million years ago, but something else. In a new study published in Nature, researchers think they have figured out what that was.
The findings build on the idea that it’s not a question of what genes are present, but of how they are structured and used — that is, which genes are expressed, or converted into functional proteins, and how they are regulated. “At the origin of animals, there wasn’t so much gene innovation,” said Arnau Sebé-Pedrós of the Center for Genomic Regulation (CRG) in Barcelona, who led the study. Instead, “an important feature was the capacity to modularly regulate these genes in different combinations in space and time.”
Sebé-Pedrós and his colleagues believe that what made these new patterns of gene regulation and expression possible in the earliest complex animals was a rather bizarre gambit that all multicellular creatures now employ. It involves pulling loops of DNA out of the tangled mass of the chromosomes, and letting the loops undergo topologically complicated contortions to put one part of the chromosome in contact with other parts far away. The origin of animal complexity might literally have been loopy.
Arnau Sebé-Pedrós (left) and Iana Kim from the Center for Genomic Regulation (CRG) in Barcelona have found that chromatin looping seems to have been a significant early step in metazoan evolution.
Courtesy of Iana Kim and Arnau Sebé-Pedrós
Drive to Specialize
It’s often implied that the big deal in the origin of complex animals like us, known as metazoans, was the switch from unicellular to multicellular life. But getting cells to work together might not actually be that hard. Some single-celled slime molds gather into collective blobs in times of stress, and even many bacteria collaborate in swarms, for example when forming the resilient colonies called biofilms. It’s thought that a transition from single-celled to multicellular might have happened many times over the course of evolution.
The really big deal was that cells became able to differentiate, to specialize. To make the various tissues of metazoans, some cells must become muscle, some skin, some nerves and so on. Cnidarians might seem a long way from bilaterally symmetrical vertebrates like us, yet they still have distinct cell types, although not as many as we do. (Recent studies using the technique of single-cell RNA sequencing have revealed that jellyfish and corals have more distinct cell types than we thought.)
With a few exceptions, all cells in a given organism have the same genome: the same set of DNA in every cell. But those in different tissues use different sets of genes to create their different functions and properties such as shape and elasticity. “To evolve true multicellularity with stable, differentiated cell types, the challenge was to differentially activate or silence the existing set of genes in different cells,” said Sebé-Pedrós’ CRG colleague Iana Kim.
“You don’t actually need that much to change to give a cell a specialty,” said the cell biologist Tessa Popay of the Salk Institute in La Jolla, California, who was not involved in the new work. What matters is “expressing the right genes in the right place at the right time.”
“More complex organisms are thought to have more regulatory sequences in their genome,” said Marieke Oudelaar, an expert in gene regulation at the Max Planck Institute for Multidisciplinary Sciences in Göttingen, Germany, who was also not involved with the study. Regulatory sequences are segments of DNA that don’t code for genes but do control gene expression. In addition to having more of these regulatory sequences, complex organisms also tend to have much bigger genomes, and it’s hard to figure out how these regulatory regions are organized.
Gene regulation is vital in all organisms; even bacteria don’t want all their genes to be active all the time. Usually, in single-celled organisms, a set of proteins called transcription factors (TFs) bind to DNA just “upstream” of a gene, at a site called a promoter, and will activate or suppress transcription of that gene into its respective mRNA, which is the first step in translating the code in the gene into a protein.
The starlet sea anemone Nematostella vectensis, a species of cnidarian, is thought to be a surviving representative of some of the earliest complex animals that evolved.
Marine Biological Laboratory and BioQuest Studios
But in multicellular, multi-tissue organisms, gene regulation is a more complex process. In any given cell type, certain genes must work together as a module, and a given gene might be involved in several modules in different cell types. Take, for example, the gene that encodes the protein called tubulin. This protein is needed for the formation of cilia — hairlike protrusions that cells can use to move — as well as in the assembly of the mitotic spindle that organizes chromosomes during cell division, and for the transport of neurotransmitters in neurons.
“To be expressed in all these cell types, the tubulin gene would either require a unique TF for each cell or a specific combination of TFs,” Kim said. The first option would result in loads of specialized TFs for each gene in each cell type. Instead, metazoan TFs are less specialized in themselves but can act in pairs or groups — that is, combinatorially, as a module. This way, many different outcomes in different cell types can come from just a few components, much as our eyes can see the whole gamut of colors by using combinations of just three types of wavelength-sensitive cone cells.
However, there’s another problem: how to get all those TFs close to the genes they regulate. “These TFs need to bind in proximity to the gene to regulate it,” Kim said. But the TF binding site — the promoter — only has limited space. “So a gene can only participate in a limited number of modules if regulated only by a proximal region,” Kim said.
The solution is to include more distant parts of the chromosome in the regulatory machinery. DNA sequences that collaborate with promoters to regulate genes from afar are called enhancers and have been known about for several decades. At first they posed a puzzle: Why place a regulatory unit so far away from the gene it regulates (in some cases, hundreds of thousands of DNA base pairs away)? “They provide additional landing spots for TFs, enabling more complex TF code,” Kim said. “This flexibility greatly expands the regulatory potential of the genome. Enhancers make it possible to reuse the same gene in many contexts, contributing to cell-type diversity and functional complexity without expanding the gene count.”
Enhancers make more complex gene regulation possible, but they seem to pose another problem: How can a site far away from a gene have any effect on it? The answer is that cells have ways to close the distance and bring the enhancer in contact with the promoter and gene sequence that it augments. It’s easy to imagine a DNA sequence splayed out in linear fashion, but the double helix is wound around disk-shaped protein units called histones to form a three-dimensional mass called chromatin. There are ways to put distal parts of the chromosome in proximity — if the enhancer can be pulled out of the mass of chromatin and brought close to the promoter and gene on a loop.
Chromatin loops are prevalent in the ctenophore Mnemiopsis leidyi, or sea walnut — around 60 percent of the chromatin contacts are on loops that bring enhancers and promoters together.
Bruno C. Vellutini/Creative Commons
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.
Tessa Popay at the Salk Institute in La Jolla, California says the study findings are supported by other work in mammalian systems.
Dillon Parkford and the Salk Institute Postdoctoral Office
“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.”
