Solving a century-old mystery in cell division

It has been a biological mystery for over 100 years and stimulated fierce debate amongst scientists. How do cells package long, tangled strands of chromosomes into tightly compact structures before cell division. A remarkable new study published in Science has revealed the answer. Professor Bill Earnshaw explains how he tackled this problem and why collaboration was the key to success.

We know quite a lot about the different stages of cell division. Why is this aspect so important?

DNA helix wound into chromosome — Credit: istock.com/ttsz

When trying to divide and produce two daughters, one problem the cell has is there is such a huge length of DNA encoding our genetic blueprint. Before the cells divide, the DNA is packaged into long tangled strands called chromatin. These must be duplicated prior to division so that each of the daughter cells can receive a full complement of identical genetic material. It is a huge challenge to divide these long tangled chromosome strands correctly.

So the cell has to figure out a way to shorten the chromosomes in order to create structures that are the right sort of size to line-up in the middle of the cell and be partitioned equally between the two daughter cells. If you work out the numbers for this compaction, the DNA packaged into a mitotic chromosome is roughly 10,000 times shorter than it would normally be. One way of thinking about this is if the cell nucleus was the size of a tennis ball, the length of the DNA in the largest chromosome would span 40 tennis courts!

Scientists have struggled with this problem since the 1880s when they first looked at chromosomes under the microscope. Why has it been so difficult to find an answer?

It's really one of those examples where you can't see the forest for the trees. At the light microscope level, you can see the chromosomes and you can sometimes get a feeling for how parts of them are organised. But when you look at chromosomes under an electron microscope the chromatin is so jam-packed together that you can’t see the component molecular parts that make it up. No one has ever been able to see the path of the DNA winding through that structure.

What theories developed over the years as scientists sought the answer to this question?

The first thing that scientists saw when they looked at chromosomes under the microscope were the two sister chromatids - the two copies of the DNA - together with the proteins that package them up. They first thought that they looked like spirals and some very sophisticated scientific articles from the 1930s describe this.

Then when electron microscopes became available, scientists thought that the chromosomes looked more like disorderly spaghetti. So the idea developed that somehow all this DNA was jammed together basically randomly - like string dumped into a box - and that there was no real order to chromosomes. Other studies, included ones I carried out myself, showed that the chromosomes looked like they were made of loops.

If you imagine DNA as a rope, all three of these things would work. You could shorten it by making it into loops, coil it or simply shove it into a bag randomly. Scientist had argued amongst themselves for decades over which of these hypotheses were right and what we have actually shown is how they were all correct!

The work involved in this discovery drew on the expertise from other labs around the world. Why was the collaboration essential to its success?

The project was born when Kumiko Samejima, a postdoc in my lab, developed a cell line where she could get the cells to all enter mitosis at the same time. In biochemical experiments where you look at an average of what is going on in all cells of a population, that synchrony meant we could see every step along the pathway of chromosome formation. She also made genetic knockouts to target some of the critical proteins, called condensins, which are involved in chromosome structure.

Job Dekker (University of Massachusetts and Howard Hughes Medical Institute) developed a technology, called Hi-C. In Hi-C cells are treated to crosslink proteins to DNA. Often one clump of proteins will be crosslinked to several pieces of DNA that were near one another in the cell. When Job’s postdoc Johan Gibcus now joined the ends of the pieces of DNA together, isolated just the junctions and sequenced the DNA he could make a map of what regions of chromosomes are near one another in the mitotic chromosome. The results revealed that the chromosomal DNA is organised into random loops of 60,000 – 90,000 base pairs.

At this point, we had 1000 billion base pairs worth of DNA sequence and complicated maps of what sequences were near each other but we could not yet see the big picture of how it was all organised. To make sense of all this we needed to develop a mathematical model to explain how the loops were organised. That’s where Leonid Mirny and his Ph.D. student Anton Golobdorodko (MIT) came in. They are geniuses at mathematical modelling. People tend to think that the mathematical modelling is quite fast but it's actually pretty slow - the calculations are so complicated and so huge that to run them on a powerful computer can take a week. So when you want to try lots of different parameters then you end up with months of work.

Was there a particular point in time when you realised that you had struck upon the answer?

After a few months of slowly getting up to speed, Johan completed his first experiments and all of a sudden we got these emails. “Oh my god I can't believe what I'm seeing”. The results were unlike anything they had ever seen before.

Looking at the profiles, we could see that even though chromosomes are made of loops there must be helical coiling involved too. This was one of the original theories that was debated by scientists over the years dating back to 1880. Before I got the email from Johan I was quite convinced that the chromosome structure had to involve loops but I was very sceptical about helical coiling.

We knew from that moment that this was going to end up in an important paper. I set up my lab in 1980, and this is one of the landmark papers of my whole career. It’s been a really fun time to give talks because everyone has been really impressed by our findings. Our paper has 99 references and we have tried hard to acknowledge the work done before us that enabled us to make these advances. Our approach was not to prove that anyone was wrong in terms of the original theories they developed on chromosome packing, but actually we tried to show which aspects of each theory was right.

The structure makes even more sense when you understand the role of the condensin proteins in forming the loops

Condensed chromosome with a helical arrangement of loops

One way to think about the structure is imagine a spiral staircase with a huge loop floating out from each step. But there seemed to be a fatal flaw. If you just make simple loops along this staircase you can’t fit all the DNA in and make a structure as compact as chromosomes are in cells.

The wonderful insight that Anton’s modelling showed is that actually that the big loops are subdivided into smaller loops. Our genetic experiments revealed that a protein called condensin II, forms the spiral staircase. The modelling revealed that another more abundant form called condensin I, makes loops within the loops. Our paper is the first to propose this structure that allows the chromosomes to package efficiently.

Do you think that the way you approach this problem and the collaboration you built, was different from what others were trying to do?

To study this type of complex problem, you make progress when you bring together the right kind of multidisciplinary team that understand each other, enjoy working together, and have absolute trust in one another. Kumiko had the cell biology expertise in Edinburgh and at the other end of the spectrum, we had Leonid and Anton who love developing hugely complicated mathematical models. Then in the middle, we had Job and Johan who had the genomics technology but who also understood the physics and cell biology. They were a lynchpin because they could understand and bridge discussions between the two camps. With any of these experts missing this discovery would not have happened. And the best possible icing on the cake – we all really like one another! I will always treasure our fantastic afternoon-long Skype meetings with 6 different screens up on the computer bouncing ideas around and exploring what to try next.

What are the next steps for your research?

We want to look at what happens at the end of mitosis. The chromosomes don’t just unravel - it’s much more complicated than that. Gene expression is regulated by clusters of loops but they are different types of loops from the mitotic loops. It is not about tight packaging: instead it’s about organising loops around common important functions. We are relishing getting down to work tackling the new approaches that we are going to have to develop together to understand this process.